computational and spectroscopic insight into the …
TRANSCRIPT
COMPUTATIONAL AND SPECTROSCOPIC INSIGHTS INTO THE CATALYTIC MECHANISM OF BACILLUS SUBTILIS OXALATE DECARBOXYLASE
By
JUSTIN LLOYD GOODSELL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2018
© 2018 Justin Goodsell
To my wife, Alicia Treat
4
ACKNOWLEDGMENTS
First and foremost, I thank my wife, Alicia Treat. Without her support this
undertaking would have been far more difficult.
I thank my advisor, Alex Angerhofer, for his continual support and allowing me
the freedom to explore different aspects of the project. I never thought I'd be exposed
to so many different methodologies and systems during my grad school career. I
deeply appreciate the opportunities I was given to attend and present at different
conferences and the encouragement to take unusual approaches to this predominately
biochemistry project. I appreciate the chance I was given to do both low-level synthetic
chemistry along with computational chemistry and spectroscopy. I will always value the
skills I learned working on this project. I also am grateful for the outreach I have been
able to take part it, especially co-teaching the Chemistry Merit Badge, which was a part
of my past that I did not expect to relive any time soon.
I would like to thank my committee members: Professors Gail Fanucci, Nick
Polfer, Nicole Horenstein and Steve Hagen. I know that each of you has many
commitments between teaching, committees, faculty searches, and running your own
research programs. I appreciate the discussions I have had with you and the different
perspectives you have brought when discussing both my project and science in general.
I thank my previous group-mate Umar Twahir for being willing to discuss issues
with experimental design and project goals both before he graduated and afterwards, as
he continued in EPR at Emory. Your support and guidance has helped more than you
realize. Some of these projects could have taken some very interesting and
unproductive detours.
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I thank Adam Cismesia for both providing motivation and being willing to just take
a walk when nothing seemed to be working.
I would like to thank Andrew Ozarowski, at the National High Magnetic Field
Laboratory (NHMFL) for his assistance in running the high-field spectrometer and his
willingness to put in some crazy hours so I could get all of my spectra recorded. I also
thank Sebastian Stoian for his discussions at the NHMFL regarding rapid freeze quench
design and troubleshooting. His experience was invaluable in not repeating the same
mistakes he made trying to build his own. I also thank Todd Prox and Brian Smith in the
machine shop. Their direction, guidance and suggestions really took the RFQ idea to
the next level. There is no way that project would have moved forward without the help
of talented machinists.
I thank John Graham from UF Physics Cryogenic Services for always facilitating
my helium experiments, even on short notice. Sometimes, on very very short notice.
Funding for this work was provided by the National Science Foundation under
Grant CHE-1213440 and the National Institute of Health Instrumentation Grant NIH S10
RR031603. The high-field EPR spectra were recorded at the NHMFL, which is funded
by the NSF through the Cooperative Agreement no. DMR-1157490, the State of Florida
and the U.S. DOE. Additional funding was provided in the form of a Graduate School
Fellowship, given by the Graduate School at the University of Florida. This fellowship
support offered the time and flexibility to pursue collaborative projects that really
enhanced my ability to think critically about different systems.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 9
LIST OF FIGURES ........................................................................................................ 12
ABSTRACT ................................................................................................................... 19
CHAPTER
1 INTRODUCTION .................................................................................................... 21
Oxalic Acid .............................................................................................................. 21 Naturally Occurring Pathways for Oxalate Degradation .......................................... 21
Oxalate Oxidase ..................................................................................................... 22 Oxalate Decarboxylase ........................................................................................... 23 Electron Paramagnetic Resonance Spectroscopy .................................................. 28
Spin Hamiltonian .............................................................................................. 29 Hyperfine Interaction ........................................................................................ 31
Higher Spin Species and Zero-Field Splitting ................................................... 32 Pulsed EPR ...................................................................................................... 35
Density Functional Theory ...................................................................................... 35
Functionals ....................................................................................................... 36 Basis Sets ........................................................................................................ 37
Research Objectives and Overview ........................................................................ 39
2 OXALATE BINDING MODES, CAVITY SIZE, REDOX AND HYPERFINE SPLITTING CALCULATIONS ................................................................................. 50
Background ............................................................................................................. 50 Computational Protocol ........................................................................................... 51
Cavity Size, Accessibility, and Potential for Bidentate Oxalate Binding Mode ........ 53 Computational Optimization of Oxalate Binding Modes and Localization in
Crystal Structure .................................................................................................. 53
Electrostatic Effects of R92 ..................................................................................... 54
Predicted ENDOR Spectra for Oxalate Binding Orientations .................................. 56 Calculation of Redox Potentials .............................................................................. 63 Conclusions ............................................................................................................ 71
3 MODEL COMPLEXES - IMPACTS OF OXALATE, RIGIDITY AND COORDINATION ENVIRONMENT......................................................................... 95
Introduction ............................................................................................................. 95 Experimental ........................................................................................................... 96
7
Mn(pyim)(oxalate) ............................................................................................ 96
Cd(pyim)(oxalate) ............................................................................................. 96 X-Ray experimental ................................................................................... 96
X-ray of Mn(pyim)oxalate ........................................................................... 97 X-ray of Cd(pyim)oxalate ........................................................................... 97 EPR ........................................................................................................... 98
Manganese (II) Macrocycles ............................................................................ 99 MnLCl/MnL(Otf)2............................................................................................. 100
Discussion ............................................................................................................ 101 Manganese(II)pyim(oxalate)2 ......................................................................... 101 Cadmium (II) pyim (oxalate)2 .......................................................................... 102 Mn(tacud)2 ...................................................................................................... 106 Mn(tacd)2 ........................................................................................................ 107
Mn(tacn)2 ........................................................................................................ 108 MnLCl ............................................................................................................. 111
Conclusions .......................................................................................................... 119
Mn and Cd(pyim)(oxalate)2 ............................................................................. 119 Mn(tacn)2, Mn(tacd)2, Mn(tacud)2 ................................................................... 119 MnLCl ............................................................................................................. 120
4 DESIGN AND IMPLEMENTATION OF A RAPID FREEZE QUENCH FOR SHORT TIMESCALE KINETICS AND REACTIVE SPECIES ............................... 144
Rapid Quenching Techniques ............................................................................... 144 Design of a Portable Rapid Freeze Quench ......................................................... 149
Rotating Plate and LN2 Interface .................................................................... 150 Sample Tube Holder and Scraper .................................................................. 151 Removable Crush Wheel ................................................................................ 153
Full Assembly ................................................................................................. 153 Mixing Station and Flow Control ..................................................................... 154
Emitter Nozzle ................................................................................................ 155 RFQ Mixer Testing ......................................................................................... 155
Conclusions .......................................................................................................... 160
5 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 174
Conclusions .......................................................................................................... 174
Future Directions .................................................................................................. 175 APPENDIX
A DFT FINAL GEOMETRIES AND FREQUENCY TABLES .................................... 178
B SAMPLE CALCULATIONS FOR RFQ AGING TIMES AND REYNOLDS NUMBER .............................................................................................................. 214
LIST OF REFERENCES ............................................................................................. 217
8
BIOGRAPHICAL SKETCH .......................................................................................... 234
9
LIST OF TABLES
Table page 2-1 Simulated Magnetic Parameters for OxDC Mn(III) Signal in Parallel Mode
Under Various Conditions. .................................................................................. 72
2-2 Bond distances, angles and dihedral for monodentate oxalate coordination in the presence of R92. Labels correspond to Figure 2-5. ..................................... 72
2-3 Bond distances, angles and dihedral for monodentate oxalate coordination in the absence of R92. Labels correspond to Figure 2-6. ...................................... 72
2-4 Bond distances, angles and dihedral for bidentate oxalate coordination in the presence of R92. Labels correspond to Figure 2-7. ........................................... 73
2-5 Bond distances, angles and dihedral for bidentate oxalate coordination in the absence of R92. Labels correspond to Figure 2-8. ............................................ 74
2-6 E280Q Magnetic Parameters used for Bidentate Simulation. Hyperfine Splitting from DFT calculations. Other Parameters Derived From High-Field Work ................................................................................................................... 74
2-7 E280Q Magnetic Parameters used for Monodentate Simulation. Hyperfine Splitting from DFT calculations. Other Parameters Derived From High-Field Work ................................................................................................................... 75
2-8 E280Q Adjusted Magnetic Parameters used for Bidentate Simulation Hyperfine Splitting Adjusted Manually Using 4-2 as a Starting Point ................. 76
2-9 Comparison of Calculated Distances from Dipolar Hyperfine Splitting with DFT Optimized Distances. .................................................................................. 76
2-10 PDB Crystallographic Distances and Identifiers for Oxalate Coordination to Divalent Cations. ................................................................................................ 77
2-11 Calculated values for redox cycling. Values are presented in eV unless otherwise labeled. Values labeled Ha are presented in Hartree. ........................ 78
3-1 Crystal Data and Structure Refinement for Mn(pyim)oxalate2 and Cd(pyim)oxalate2. ............................................................................................. 121
3-2 Simulated EPR parameters. All parameters were fit manually using EasySpin. ......................................................................................................... 122
3-3 Simulation parameters for Mn macrocycles ...................................................... 122
10
3-4 Crystallographic distances for coordinating residues in Mn(tacud)2, Mn(tacd)2, and Mn(tacn)2 .................................................................................. 123
3-5 Crystallographic angles for coordinating residues in Mn(tacud)2, Mn(tacd)2, and Mn(tacn)2 ................................................................................................... 124
3-6 Simulated EPR parameters for MnLCl in Perpendicular and Parallel Mode ..... 125
3-7 Simulated EPR Parameters for Various Mn(III) Complexes Determined Using High Field EPR. ................................................................................................ 126
4-1 Timescales of Various Rapid Quench Techniques ........................................... 161
4-2 Reaction Time Added by Aging Hoses using a 4 cm/s ram displacement velocity. Sample Calculations Provided in Appendix B. ................................... 161
A-1 Mn(II) n-terminus water/water coordination - atomic coordinates ..................... 178
A-2 Mn(II) n-terminus water/water coordination - frequency tables ......................... 179
A-3 Mn(III) n-terminus water/water coordination - atomic coordinates .................... 180
A-4 Mn(III) n-terminus water/water coordination - frequency tables ........................ 181
A-5 Mn(II) n-terminus water/formate coordination - atomic coordinates .................. 182
A-6 Mn(II) n-terminus water/formate coordination - frequency tables...................... 183
A-7 Mn(III) n-terminus water/formate coordination - atomic coordinates ................. 184
A-8 Mn(III) n-terminus water/formate coordination - frequency tables ..................... 185
A-9 Mn(II) n-terminus water/acetate coordination - atomic coordinates .................. 186
A-10 Mn(II) n-terminus water/acetate coordination - frequency tables ...................... 187
A-11 Mn(III) n-terminus water/acetate coordination - atomic coordinates ................. 188
A-12 Mn(III) n-terminus water/acetate coordination - frequency tables ..................... 189
A-13 Mn(II) n-terminus water/oxalate coordination - atomic coordinates .................. 190
A-14 Mn(II) n-terminus water/oxalate coordination - frequency tables ...................... 191
A-15 Mn(III) n-terminus water/oxalate coordination - atomic coordinates ................. 192
A-16 Mn(III) n-terminus water/oxalate coordination - frequency tables ..................... 193
A-17 Mn(II) n-terminus bidentate oxalate coordination - atomic coordinates ............ 194
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A-18 Mn(II) n-terminus bidentate oxalate coordination - frequency tables ................ 195
A-19 Mn(III) n-terminus bidentate oxalate coordination - atomic coordinates ........... 196
A-20 Mn(III) n-terminus bidentate oxalate coordination - frequency tables ............... 197
A-21 Mn(II) n-terminus bidentate oxalate coordination with R92- atomic coordinates ....................................................................................................... 198
A-22 Mn(II) n-terminus bidentate oxalate coordination with R92 - frequency tables . 199
A-23 Mn(II) n-terminus monodentate oxalate coordination with R92- atomic coordinates ....................................................................................................... 201
A-24 Mn(II) n-terminus monodentate oxalate coordination with R92 - frequency tables ................................................................................................................ 202
A-25 Mn(II) n-terminus water/water coordination with SMD - frequency tables ......... 204
A-26 Mn(III) n-terminus water/water coordination with SMD - frequency tables ........ 205
A-27 Mn(II) n-terminus formate/water coordination with SMD - frequency tables ..... 206
A-28 Mn(III) n-terminus formate/water coordination with SMD - frequency tables .... 207
A-29 Mn(II) n-terminus acetate/water coordination with SMD - frequency tables ...... 208
A-30 Mn(III) n-terminus acetate/water coordination with SMD - frequency tables ..... 209
A-31 Mn(II) n-terminus monodentate oxalate/water coordination with SMD - frequency tables ............................................................................................... 210
A-32 Mn(III) n-terminus monodentate oxalate/water coordination with SMD - frequency tables ............................................................................................... 211
A-33 Mn(II) n-terminus bidentate oxalate coordination with SMD - frequency tables 212
A-34 Mn(III) n-terminus bidentate oxalate coordination with SMD - frequency tables ................................................................................................................ 213
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LIST OF FIGURES
Figure page 1-1 Oxalic acid .......................................................................................................... 41
1-2 Enzymatic pathways identified for breakdown of oxalic acid. (A) Oxalate Decarboxylase, (B) Oxalate Oxidase and (C) Oxalyl-CoA Decarboxylase ......... 41
1-3 OxOx Quaternary structure. Coordinated manganese ions are represented as purple spheres. PDB 2ETE. .......................................................................... 42
1-4 Current literature mechanism for Oxalate Oxidase ............................................. 43
1-5 OxDC quaternary structure, from PDB 1UW8, monomer is highlighted in blue. OxDC exists crystallographically as a stacked dimer of trimers. ............... 44
1-6 Currently accepted literature mechanism for Oxalate Decarboxylase.30............. 45
1-7 W96 and W274 π-stacking orientation at the interface of two monomers. Distances are in Angstrom and determined from the center of each aromatic residue for H and W. PDB 1UW8. ...................................................................... 46
1-8 Hyperfine Interaction for an S=1/2, I=1/2 system with isotropic g and isotropic hyperfine.. ........................................................................................................... 47
1-9 Simulation of an S=3/2, I=0 system in the absence of additional effects. Resonance positions are highlighted in red. ....................................................... 48
1-10 Simulation of an S=3/2, I=0 system with the addition of a 2 GHz ZFS value. Resonance positions are highlighted in red. ....................................................... 49
2-1 1.95 Å resolution crystal structure of Thermatoga maritima T1287. (A) with coordinated oxalate. (B) RMSD overlay with OxDC active site, (RMSD = 0606A., global RMSD 1.2 Å). Blue - T1287, Green - OxDC ............................... 79
2-2 DFT Optimization of Binding Modes for Oxalate Placed in CAVER Identified Binding Pocket. (A) Oxalate Monodentate with Water, (B) Oxalate Bidentate. ... 79
2-3 (A) mono- and (B) bidentate oxalate geometry optimization with the inclusion of residue R92. ................................................................................................... 80
2-4 (A) mono- and (B) bidentate oxalate geometry optimization overlays in the presence and absence of R92.. .......................................................................... 80
2-5 DFT optimized monodentate oxalate coordination in the presence of R92. ....... 81
2-6 DFT optimized monodentate oxalate coordination in the absence of R92. ......... 81
13
2-7 DFT optimized monodentate oxalate coordination in the presence of R92. ....... 82
2-8 DFT optimized monodentate oxalate coordination in the absence of R92. ......... 82
2-9 Arginine bite angles for oxalate stabilization. (A) Monodentate oxalate with water coordination. (B) Bidentate oxalate ........................................................... 83
2-10 Bidentate oxalate simulation at X-band using output from ORCA hyperfine calculations and previously determined EPR parameters for E280Q. ................ 84
2-11 Monodentate oxalate simulation at X-band using output from ORCA hyperfine calculations and previously determined EPR parameters for E280Q. ............................................................................................................... 85
2-12 Summed simulated spectra at X-band combining various weights of the calculated monodentate spectrum combined with the calculated bidentate spectrum ............................................................................................................. 86
2-13 Bidentate oxalate simulation at X-band beginning with DFT calculated parameters and adjusted for better fit. ................................................................ 87
2-14 Bidentate oxalate simulation overlaid on Q-band WT pH 8.0 ENDOR data. ...... 88
2-15 Bidentate oxalate simulation re-optimized at Q-band overlaid on WT pH 8.0 ENDOR data. ...................................................................................................... 89
2-16 Bidentate oxalate simulation re-optimized at Q-band overlaid on X-band E280Q ENDOR data. X-band data collection parameters 9.735602 GHz,
345 mT, 360ns , 601 points, 5K. ....................................................................... 90
2-17 Thermodynamic cycle for calculation of redox potentials. (g) notation indicates gas phase, (aq) indicates solvated. Roman numeral subtext indicates oxidation state of the metal in each calculation. .................................. 91
2-18 General coordination model for redox calculations. Protein residues are used from PDB file 1UW8. X and Y represent coordinating molecules used in the calculation. ................................................................................................ 91
2-19 DFT optimized structures for different coordination models. Green represents Mn(II) structure, Cyan represents Mn(III) structure. ......................... 92
2-20 DFT optimized structures for different coordination models. Green represents Mn(II) structure, Cyan represents Mn(III) structure. ......................... 93
2-21 DFT optimized structures for Mn(bipy)3 and Mn(acac)3. Green represents Mn(II) structure, Cyan represents Mn(III) structure. ........................................... 94
3-1 Manganese pyim oxalate (A) and cadmium pyim oxalate (B). .......................... 127
14
3-2 Manganese(pyim)oxalate2 crystal packing orientations. (A) Chains aligned along crystallographic c axis and (B) aligned along crystallographic b axis. ..... 127
3-3 Cadmium(pyim)oxalate2 crystal packing orientations. (A) Chains aligned along crystallographic b axis and (B) chains aligned along crystallographic a axis. .................................................................................................................. 128
3-4 Manganese(pyim)oxalate2 X-band EPR signal at 5K (purple dash). 0.3% Mn doped cadmium(pyim)oxalate2 X-band EPR signal at 5K (black trace). ........... 129
3-5 208 GHz High Field EPR of 0.3% Mn doped cadmium(pyim)oxalate2 collected at 5 K. ................................................................................................ 130
3-6 208 GHz EPR collected at variable sample temperature. Population increase is observed at the low-field position with deceasing temperature ..................... 130
3-7 Ligand geometries for Mn(tacn)2 (A), Mn(tacd)2 (B) and Mn(tacud)2 (C). Crystallographic representations are presented below each ligand. ................. 131
3-8 Mn(tacud)2 spectrum collected at 5K and 208GHz, focusing on the g=2 region. Experimental spectrum is presented in black with simulation presented in blue. ............................................................................................. 132
3-9 Mn(tacud)2 spectrum collected at 5K and 208GHz, encompassing all observed features. Experimental spectrum is presented in black with simulation presented in blue. ............................................................................ 133
3-10 Mn(tacd)2 spectrum collected at 5K and 208GHz, focusing on the g=2 region. Experimental spectrum is presented in black with simulation presented in blue. .................................................................................................................. 134
3-11 Mn(tacd)2 spectrum collected at 5K and 208GHz, encompassing all observed features. Experimental spectrum is presented in black with simulation presented in blue. ............................................................................................. 135
3-12 Mn(tacn)2 spectrum collected at 5K and 214.4GHz, focusing on the g=2 region. Experimental spectrum is presented in black with simulation presented in blue. ............................................................................................. 136
3-13 Mn(tacn)2 spectrum collected at 5K and 208GHz, encompassing all observed features. Experimental spectrum is presented in black with simulation presented in blue. ............................................................................................. 137
3-14 Principal Component Analysis based on the bond distances in the complexes for coordinating atoms. ..................................................................................... 138
3-15 Principal Component Analysis based on the angles in the complexes for coordinating atoms. .......................................................................................... 139
15
3-16 Coordination environment of MnLCl ................................................................. 140
3-17 Perpendicular mode EPR spectrum of MnLCl with 20 eq. TBAO. Black line indicates spectrum acquired before exposure to ambient oxygen. Blue line indicates spectrum acquired 2 hours after oxygen exposure. ........................... 140
3-18 Parallel mode EPR spectrum of MnLCl with 20 eq. TBAO. Black line indicates spectrum acquired before exposure to ambient oxygen. Blue line indicates spectrum acquired 2 hours after oxygen exposure. ........................... 141
3-19 High field EPR spectrum of pre oxidized MnLCl at 208 GHz. The oxidized sample did not show enough signal intensity to be reliable, which is common for manganese (III) in solution. ......................................................................... 142
3-20 Simulated parallel mode EPR signal observed for the oxidized complex. Black line represents experimental data, red represents simulation. ................ 143
4-1 Wheel Based RFQ. (A) Full assembly with motors. (B) Closer detail of wheel interface with base visible. Image provided by Professor Gary Gerfen. 162
4-2 RFQ Copper Freeze Plate (A) side and (B) top. ............................................... 162
4-3 (A) RFQ freeze plate mounted in double-walled vacuum dewar. (B) RFQ plate with cold nitrogen line. ............................................................................. 163
4-4 Sample holder, sized for 4 mm O.D. sample tubes. (A) solid assembly and (B) wireframe, showing sample holes. .............................................................. 163
4-5 Sample collection scraper (A) from top and (B) from side, showing holding pins that maintain nitrogen contact. .................................................................. 164
4-6 Scraper interface with sample tube holder (A) wireframe showing mounting and nitrogen contact and (B) top view showing interface with freeze plate. ..... 164
4-7 Removable crushing wheel with handle and mounting bracket. ....................... 165
4-8 (A) RFQ full assembly with insulating box and (B) RFQ full assembly wireframe. ......................................................................................................... 166
4-9 Photographs of completed RFQ. (A) Full assembly, (B) detail zoom of scraper and crush wheel, (C) detail of nitrogen line. ......................................... 167
4-10 Microscope image of the grid-type mixer used in the RFQ experiment. Hole diameter shown in center is 1/16" (159 μm). .................................................... 168
4-11 Prototype reaction for RFQ mixer testing. 500 μM TEMPOL is mixed with varying amounts of an equimolar ascorbic acid solution and diluted to a given final volume. ..................................................................................................... 169
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4-12 RFQ reaction testing. 1 mM TEMPOL is loaded on one side of the syringe ram with 1mM ascorbate on the opposing side to give final concentrations at full mixing of 500 μM. ........................................................................................ 170
4-13 Exponential decay fit on the maximum intensity for each tubing length used in Figure 4-12. Fit equation 1.869∙e(-0.025576∙x). ............................................ 171
4-14 RFQ reaction performed in triplicate using 1mM stock TEMPOL and Ascorbic acid solutions.. .................................................................................................. 172
4-15 Second Order kinetics plot of the RFQ test reaction ......................................... 173
17
LIST OF ABBREVIATIONS
ACN Acetonitrile
AO Atomic Orbital
Arg or R Arginine
13C Carbon-13 Enriched
CGF Contracted Gaussian Function
CW Continuous Wave
DFT Density Functional Theory
ENDOR Electron Nuclear Double Resonance
EPR Electron Paramagnetic Resonance Spectroscopy
eV Electron Volts
G gauss
GHz Gigahertz
Glu or E Glutamate
GTO Gaussian-type Orbital
His or H Histidine
KIE Kinetic Isotope Effect
LDA Local Density Approximation
LHe Liquid Helium
LN2 Liquid Nitrogen
LRET Long Range Electron Transfer
MeOH Methanol
18
MHz Megahertz
MnLCl manganese(II) 1-benzyl-4-acetato-1,4,7-triazacyclononane chloride
mT milli Tesla
mW milliwatt
OFC Oxygen-Free Copper
OxDC Oxalate Decarboxylase
OxOx Oxalate Oxidase
PCET Proton Coupled Electron Transfer
PEEK Polyetheretherketone
PTFE Polytetrafluoroethylene
Pyim 2(1-H pyridyl) imidazole
RFQ Rapid Freeze Quench
STO Slater-type Orbital
TEMPOL 2,2,6,6-tetramethyl-4-hydroxypiperidine-
1-oxyl
Tacd 1,4,7- Triazacyclodecane
Tacn 1,4,7-Triazacyclononane
Tacud 1,4,8- Triazacycloundecane
Trp or W Tryptophan
WT Wild-Type
19
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
COMPUTATIONAL AND SPECTROSCOPIC INSIGHTS INTO THE CATALYTIC
MECHANISM OF BACILLUS SUBTILIS OXALATE DECARBOXYLASE
By
Justin Lloyd Goodsell
May 2018
Chair: Alexander Angerhofer Major: Chemistry
Bacillus subtilis Oxalate Decarboxylase is a manganese-based metalloenzyme
that catalyzes the decomposition of oxalic acid to carbon dioxide and formate. This
enzyme contains two manganese binding domains inside β-barrel folds with identical
coordinating environments, however only one of these sites is proposed to be
catalytically active. The protein is very similar to the plant-derived Oxalate Oxidase, in
both function and active site, however the chemistry performed diverges, with Oxalate
Oxidase producing carbon dioxide and hydrogen peroxide instead. Oxygen is required
for both systems, but OxDC does not consume oxygen upon turnover.
Oxalate binding orientations were studied using both inorganic modeling, EPR
spectroscopy, and Density Functional Theory, indicating that a bidentate mode is
strongly favored in this system. The redox potential of the N-terminus Mn(II) center was
calculated to decrease drastically upon exposure to a coordinating carboxylate, with the
most notable decrease coming from a bidentate oxalate mode. Monodentate oxalate
was shown to provide the smallest stabilizing benefit to the Mn(III) species that is
proposed to drive the reaction. Calculation of the hyperfine splitting constants for 13C
20
coupling to the manganese center allowed for fitting of previously collected ENDOR
spectra, confirming the bidentate mode experimentally.
These results suggest two things: first that bidentate is the correct substrate
binding mode, which would in turn require that oxygen bind at a different location and
second that carboxylate activation is necessary to generate the Mn(III) species that is
observed under turnover. If oxygen binding occurs at the C-terminus metal center in
OxDC, structural elements exist that could facilitate long range electron transfer (LRET).
21
CHAPTER 1 INTRODUCTION
Oxalic Acid
Oxalic acid (Figure 1-1) is the simplest dicarboxylic acid and a metabolic product
for many species of plants and fungi.1–6 This acid is pervasive in nature and problematic
for human health and industry. Oxalic acid readily forms coordination polymers with
divalent cations, such as calcium, forming calcium oxalate, an insoluble material with a
Ksp of 10-31. Additionally, oxalic acid is a relatively strong organic acid with pKa's of 1.2
and 4.2. In plants, functions of soluble oxalate and calcium oxalate precipitants include
ion regulation, detoxification of heavy metals, and protection against insects and
foraging animals. It is involved in transferring atmospheric CO2 into the soil as CaCO3,
and is suggested to augment incident UV-radiation in light-limited environments.7–10 In
terms of human impact, oxalic acid forms roughly 60% of kidney stones and urinary
tract stones.11 In addition, oxalic acid poses a continuous problem for paper pulp and
sugar processing, where soluble calcium in processing water is exposed to free oxalate
in plant matter. This results in oxalate scales forming, reducing the diameter of piping
and increasing the energetic cost of these processes.12,13
Naturally Occurring Pathways for Oxalate Degradation
Three naturally occurring pathways exist for oxalate degradation, namely an
oxidase pathway, a decarboxylase pathway, and a chaperoned oxalyl-CoA
decarboxylase pathway (Figure 1-2). The oxidase pathway is favored in plants, while
the decarboxylase pathway is observed in fungi and soil bacteria. Oxalyl-CoA
decarboxylase is utilized solely by bacteria.
22
Oxalate Oxidase
Oxalate Oxidase (OxOx) catalyzes the decomposition of oxalic acid using an
oxidase pathway, resulting in the conversion of oxalate into two equivalents of carbon
dioxide and one equivalent of hydrogen peroxide, at the cost of oxygen and an
accessible proton source.14 X-ray structures of OxOx reveal that it crystallizes as a
single-layered hexamer in the form of a trimer of dimers (Figure 1-3).15,16 Each
monomer of OxOx contains a metal binding domain consisting of 3 Histidine residues
and 1 Glutamate, which is conserved in all isoforms and lies within the β-barrel fold that
is a defining characteristic of the cupin superfamily of enzymes.17–19 manganese is the
catalytic metal in this system, with a +2 resting oxidation state, as determined by
Electron Paramagnetic Resonance (EPR) spectroscopy.20 The oxidation state of the
enzyme is not maintained during catalysis, instead shifting through a Mn(III)
intermediate upon introduction of oxalate.20
The current literature mechanism for Oxalate Oxidase is presented as Figure 1-
4.21–23 In this mechanism, as observed crystallographically, the manganese ion begins
in a hexacoordinate form, with water occupying the two non-protein valences.
Spectroscopic data shows a Mn(II) resting state for the protein.20 Water is easily
displaced, resulting in binding of monoprotonated oxalate in a monodentate fashion.
The order of binding is unknown. It has been hypothesized based on theoretical work
that oxalate may bind bidentate and then reorient to monodentate to allow oxygen
coordination, as the energetic difference between the two conformers is small, however
glycolate, an oxalate analogue, has been crystallized coordinated to the manganese
center in a monodentate fashion, with water occupying the sixth valence.20,21 Oxygen
coordinates and abstracts an electron from manganese, generating the experimentally
23
observed Mn (III) intermediate.20,23 Oxalate becomes deprotonated though exposure to
a transient base and the carbon-carbon bond weakens, allowing CO2 to form as a
product. This step produces a carbon dioxide radical anion coordinated to the metal in
close proximity to the superoxide that retains coordination. Radical termination occurs
resulting in a peroxycarbonate, a highly labile species, which then decomposes into
carbon dioxide and a hydroperoxide anion, which can accept a proton from the acidic
environment to form hydrogen peroxide, the final product of the reaction.
Oxalate Decarboxylase
Oxalate Decarboxylase (OxDC), similar to OxOx, exists in crystallographic form
as a hexamer, however the OxDC hexamer is composed of two face-to-face stacked
trimers (Figure 1-5). Each monomer of OxDC contains two manganese binding
domains with identical coordinating residues, maintaining a 3-Histidine 1-Glutamate
coordination motif.24,25 OxDC contains several interesting structural features that do not
exist in the smaller OxOx. The existence of a second layer is unique to OxDC. If only
one trimer was considered, the total number of manganese would be the same as the
OxOx biological unit, however due to the double occupancy of manganese ions the
biological unit of OxDC contains twice as much manganese. While the coordinating
residues are identical, with identical spacing between them in the protein sequence, the
magnetic parameters are different, as observed by high-field EPR.26 The N-terminus
manganese ion is suggested to be the active site, based on a crystal structure that
contains formate, the product of the reaction, coordinated to the N-terminus
manganese.24 The C-terminus manganese is suggested to play a merely structural role,
however, mutation studies that knock out the C-terminus manganese drastically impact
the activity of the enzyme.27 As this C-terminus knockout mutant has not been
24
crystallized, it is difficult to say whether this is due to both sites being catalytically active
or whether the quaternary structure of the enzyme is impacted in a meaningful way.27
The N-terminus has several other features that suggest it is the main site for
catalysis. The decarboxylation reaction (Figure 1-2) requires a proton be shifted in the
oxalate molecule, which only has protons on carboxylate groups, to form the final
formate product, which has a carbon bound hydrogen atom. This requires the use of a
transient base to hold the proton, similar to OxOx. The N-terminus site has a
convenient glutamate residue that resides on a flexible loop, which has been observed
in two conformations in the literature, referred to as the 'open' and 'closed'
conformations.24,25 Both of these structures are crystallized under non-active, high pH,
conditions. Recent unpublished results, submitted to the PDB as identifier 5vg3, identify
the existence of a third intermediate conformation in the wild-type enzyme, which had
previously only been observed in a T165V mutant that altered the flexible lid guarding
the active site.28 The 'open' conformation points this glutamate residue, E162, away
from the active site. It is stabilized by a hydrogen bonding network that forms between
the two trimers of the hexamer unit. The 'closed' conformation brings E162 into the
active site cavity, closing off the solvent channel leading to the metal ion and bringing
E162 in proximity to the manganese.
In addition to the presence of a convenient proton acceptor, mutagenesis studies
have also identified Arginine 92 to be catalytically relevant. When R92 is removed, the
activity of the enzyme plummets.29 R92 is proposed to serve as an electrostatic
stabilizer, allowing for the enzyme to guide the oxalate molecule in place and add a
25
polarizing effect to the oxalate, using its positive charge to stabilize the nearby
carboxylate group of the oxalate when the enzyme is substrate-loaded.
The current literature mechanism, presented as Figure 1-6, for OxDC is very
similar to that of OxOx, although it only includes the N-terminus. The resting state of
the manganese ion is predominately in the +2 oxidation state, with water molecules
occupying the N-terminus open valencies. Roughly 16% of the manganese in OxDC at
the active pH exists in a +3 oxidation state.30 Oxygen and oxalate bind to the
manganese ion, requiring that monoprotonated oxalate bind monodentate to
accommodate the oxygen molecule. Electron transfer occurs upon oxygen binding,
resulting in the generation of a Mn(III) species and a bound superoxide radical, however
the order of binding is not currently known.30,31 The enzyme undergoes proton coupled
electron transfer (PCET), shifting the proton from oxalate to a transient base, which is
suggested to be E162 based on proximity, and resulting in a negative charge on the
oxalate. This charge is stabilized by the presence of the positively charged R92
residue. Introducing a negative charge on the oxalate weakens the carbon-carbon
backbone of the oxalate molecule, resulting in elongation of the bond.32 As the bond
weakens, the electron pair on the recently deprotonated oxygen pushes down, causing
a cascade, pushing electrons from the carbon-carbon bond towards the manganese-
bound part of the oxalate and liberating the first product of the reaction, CO2. The
resulting bound species is a carbon dioxide radical anion, which has been identified
experimentally using spin trapping techniques.33 This species is not proposed to react
with the nearby superoxide radical, and instead is to undergo PCET again, trading the
radical electron for the proton held by E162. This generates the final product of the
26
reaction, formate. Formate is then released and the catalytic cycle can be restarted. As
the bound superoxide still exists, only oxalate would need to be reintroduced.
OxDC requires the presence of oxygen for activity, although oxygen is not
consumed in the reaction. Under Nitrogen atmosphere conditions, burst kinetics are
observed, presumably due to trapped oxygen within hydrophobic pockets in the
enzyme, however, activity declines quickly until oxygen is reintroduced.34 This is a
curious result, as even a small amount of oxygen should be able to catalyze the
reaction according to the proposed mechanism.
Due to the close proximity of the highly reactive radical intermediates in this
reaction, and the comparatively slow speed of the rate-limiting PCET steps, these two
species could be expected to react, resulting in radical termination of the reaction and
formation of the peroxycarbonate species that is presumably generated in the oxidase
mechanism. This termination process would change the chemistry to that of OxOx. In
fact, in 0.2% of all turnovers this result is observed in the form of production of hydrogen
peroxide, however it is more likely due to loss of the radical anion intermediate than
any more complicated process. Additionally, superoxide escaping the cavity could
result in formation of hydrogen peroxide, which would account for this seemingly
bifurcated mechanism.23
Superoxide has been identified experimentally using spin trapping techniques
with 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO).35 The BMPO-
superoxide adduct can be identified in conjunction with the previously observed carbon
dioxide radical anion, allowing the relationship between the two to be explored.33 These
studies were the first experimental determination of BMPO bound superoxide as
27
identified via combined EPR, mass spectrometry and fluorescence measurements.
Wild type (WT) OxDC was considered in addition to a T165V mutant which impacts the
flexibility of the lid residues that are proposed to shield the entrance to the active site
cavity, stabilizing these residues in the 'open' configuration. If superoxide and the
carbon dioxide radical anion are truly generated in the same cavity, their relative ratios
should be similar if the lid based gating mechanism is altered, allowing small molecule
diffusion from the cavity. It was found during that work that when T165 is mutated and
the lid is disturbed, the relative amount of carbon dioxide radical anion increases
drastically, however, the superoxide radical only shows a small decrease proportional to
the relative catalytic efficiency. The relative impact between the two species is not
similar. These results suggest that perhaps the superoxide radical and the carbon
dioxide radical are generated at different sites.
Radical generation at different sites would make intuitive sense for this
chemistry. As radicals are highly reactive species, two being generated in the same site
should lead to radical termination based on proximity. However, manganese (III) is
known to be required for this reaction and the resting state of the enzyme is in the
manganese (II) oxidation state. If oxygen does not bind at the N-terminus site another
home must be found for it, in addition to an explanation for the open valences on
manganese. It could be that the manganese then takes on a bidentate orientation, or
that water occupies the other valence.
OxDC, unlike OxOx, does have a second metal binding domain. Although only
one is proposed to be catalytically active, after all, if both were showing catalytic activity
or cooperative behavior the role of the second site would have to be identified, knockout
28
mutations that remove the metal from the second site show marked impacts on the
ability of the enzyme to turn over substrate. Although these two sites are quite distant
within the same monomer, measuring 26 Å, if the interface between two monomers in
the trimer structure is considered as a route, the distance drops to 21.3 Å. This
distance remains quite large for a cooperative mechanism, but looking closely at this
interface a pair of tryptophan residues can be observed in a π-stacking orientation, with
one residue belonging to each monomer at the interface. W96 and W274 maintain a
distance comfortably within the range required for interaction in every crystallized form
of the enzyme (Figure 1-7). Preliminary unpublished kinetics data suggests that even
conservative mutations of these residues also have an impact on the catalytic ability of
OxDC. Consideration of this tryptophan dimer allows for the possibility of a long range
electron transfer (LRET) pathway, which would provide a role for the hitherto ignored C-
terminus manganese site. These residues shorten the total distance required for an
electron hop from 21.5 Å to 8.4 Å. Electron hopping has been observed in many
systems, such as Ribonucleotide Reductase, up to extreme distances (>40 Å) by taking
advantage of electron orbital interactions between aromatic residues.36–39
A recent crystal structure involving an E162 deletion mutant and metal
substitution to cobalt instead of manganese has resulted in a monodentate oxalate
coordination being observed crystallographically, however, this system is catalytically
inactive, thus bringing into question whether its behavior is truly representative of the
active enzyme.40
Electron Paramagnetic Resonance Spectroscopy
Radicals and transition metals serve as important vehicles for redox reactions in
many aspects of chemistry. From biological systems taking advantage of accessible
29
metal oxidation states and their proclivity to take on positive charges, to radical
polymerization and antioxidant activities in polymers and small molecules, radicals exist
in a variety of locations and for a great many purposes. However, radical species, due
to their reactivity, only exist in small concentrations or with very limited lifetimes.
Electron Paramagnetic Resonance Spectroscopy (EPR or ESR) serves as a highly
sensitive tool for the observation and characterization of these species and, by
extension, the chemistry they perform.
Spin Hamiltonian
When spin bearing particles are introduced to a magnetic field, the degeneracy of
the two spin states is lost, resulting in two general orientations: spin aligning parallel to
the magnetic field and spin aligning anti-parallel. Given the negative sign of the electron
charge, as contrasted with the positive charge carried by nuclei, the preferential
orientation for electrons is anti-parallel to the external field. The energy of a magnetic
moment in a magnetic field is shown in Equation 1-1.41
(1-1)
Where μ is the magnetic dipole and B0 is the applied magnetic field
(1-2).
The magnetic dipole, as defined in Equation 1-2, is a product of γ, the gyromagnetic
ratio and S, the z component of the spin angular momentum. Because the
gyromagnetic ratio is defined as shown in Equation 1-3, the spin orientation becomes
logical.
(1-3)
30
Here, q is the fundamental charge, 1.602∙10-19 C, m is the mass of the species, ge is the
g factor of the free electron, a dimensionless quantity, μB is the Bohr magneton, and ħ is
the Planck constant, divided by 2π.
As the strength of the magnetic field increases, the orientation polarization of the
electron spin with respect to the magnetic field increases in addition to the separation
between the newly split energy levels. This phenomenon is referred to as the Zeeman
effect, and leads to the most basic resonance phenomenon. The Zeeman Splitting is
defined in Equation 1-4:
(1-4)
In this equation g can deviate from the free electron value and provides information
about the environment surrounding the electron, μe is the Bohn magneton for the
electron, B0 is the applied magnetic field, and S is the z component of the spin angular
momentum. When the resonance condition is fulfilled, absorption will be observed, for
photons with commensurate energy as given in the relationship in Equation 1-5
(1-5)
where h is the Planck constant and ν represents the frequency of irradiation.
Given the high cost and limited availability of tunable microwave sources, the
relatively low field strengths needed to perform the EPR experiment, and the need for
narrow-band resonators, the frequency is held constant and the magnetic field is swept.
This also allows for the use of microwave resonators. EPR instrumentation exists in a
multitude of field and frequency ranges, with the most commonly used frequency in the
10 GHz range, referred to as the X-Band.
31
The experimentally determined g factor is, in fact, a second-order tensor, as
anisotropy is expected. For a rapidly tumbling molecule in solution, the tensor will
average out to an isotropic value, however, in frozen solution or single crystal form the
different axes of the tensor can be resolved. Systems are observed in all symmetry
types, from isotropic doped species to axially symmetric π conjugated systems to fully
rhombic species.
Hyperfine Interaction
Beyond the simplest example of an S=1/2 system that only demonstrates
Zeeman Splitting, further interactions are accessible via EPR. When electrons are in
either proximity to or orbitals pertaining to a spin bearing nuclei, such as Hydrogen,
additional spin couplings are observed. The coupling between electron and nuclear
spin is called the hyperfine interaction. This interaction has an isotropic component,
called the Fermi Contact Interaction, which represents the probability of an electron
being inside the nucleus, and an anisotropic dipolar component.
(1-6)
Hyperfine interactions introduce additional splitting into the EPR spectrum,
following the expected 2I+1 pattern, where an S=1/2 system with I=1/2 would result in
2∙(1/2)+1 = 2 lines, as allowed transitions will still be Δms = + 1. Therefore, even though
4 energy states are now present (ms=-1/2, mI =+1/2; ms=-1/2, mI =-1/2; etc.) only two
valid transitions exist, with the other two EPR transitions being double quantum
transitions and therefore spin forbidden, and the remaining two transitions being NMR
transitions, for which the energy is too high. An example of hyperfine splitting is shown
32
in Figure 1-8.The g value for this spectrum lies in the center of the two visible lines, with
the hyperfine value equal to half of the spacing between them.
Like the g factor, hyperfine coupling also takes on a tensor form, lending to the
existence of isotropic, axially anisotropic and rhombic forms. In addition, hyperfine
interaction need not be constrained to the nucleus bearing the electron. As hyperfine
has a dipolar component, it can be observed on neighboring nuclei if either the coupling
strength or electron delocalization is sufficient. This can lead to observation of
superhyperfine interactions, which are smaller hyperfine interaction with more distance
nuclei that generate additional splittings. An example of this can be observed in spin
trapped radical adducts, where the localization of the electron is predominately on the
nitroxide bond, but proton coupling can be seen inducing a further splitting on the
nitroxide EPR signal. As this term contains a dipolar component, the magnitude of
hyperfine interactions falls off as r-3, where r is the distance between the electron and
the coupled nuclei.41,42
Higher Spin Species and Zero-Field Splitting
Many catalytically interesting systems go beyond a simple radical spectrum.
From Photosystem II to Hemoglobin and Superoxide Dismutase, transition metals are
critical for many biological systems.43–47 However, transition metals often have S>1/2,
resulting in more spin manifolds that undergo Zeeman splitting. Additional splittings
arise, as the selection rule is unchanged at ΔS = +1, meaning that for an S=3/2 system,
3 transitions become available, with <-3/2|-1/2>, <-1/2|1/2>, and <1/2|3/2>. In the
absence of additional effects, these transitions will all be equal in intensity and spectral
position, therefore a one line spectrum should be observed. The higher spin manifold
experiences a stronger response to the applied field, as the Zeeman term (equation 1-4)
33
has a dependence on the magnitude of the spin angular momentum. This term only
applies when the electrons can interact, therefore a biradical system would instead
behave like two independent S=1/2 systems, while two interacting electrons form a
triplet state.
When metal ions become ligated, often the coordinating molecules will provide
an electronic impact. If bonding takes on an axial or rhombic form, the distortion will be
visible in the EPR spectrometer in the form of Zero Field Splitting (ZFS). ZFS, as its
name implies, comes about when the higher spin manifolds no longer retain degeneracy
at zero magnetic field. This effect can be small, appearing merely as a line broadening,
or very large, in some cases pushing into the THz regime — nearly an infrared
transition. The ZFS parameter in the spin Hamiltonian takes on the form in Equation 1-
7.
(1-7)
Where S is the z component of the spin angular momentum and D is the zero-field
splitting tensor.
ZFS has a drastic impact on the observed spectrum for higher-spin species. For
half-integer spin species, the ms=1/2 manifold is not affected by ZFS to first order,
meaning that half-integer spin species can be observed at any frequency range using
appropriate experimental parameters. For integer spin species, only the S=0 energy
level is unaffected, therefore if the magnitude of the ZFS is greater than that of the
microwave quantum being used (10 GHz or 0.3 cm-1 for X-Band) no transition will be
observed. These species are termed 'EPR silent', due to their lack of signal on
standard commercially available equipment. These species are still observable if the
34
microwave quantum and B0 can be increased to be greater than the ZFS, or under
special conditions using parallel mode EPR. An example of an S=3/2, I=0 species, as
in Figure 1-9 with the introduction of a small 2 GHz purely axial ZFS value is shown in
Figure 1-10.
As observed in Figure 1-10, introduction of ZFS causes a major change in the
spectrum for the simple S=3/2, I=0 system. Where previously the 3 transitions were in
exactly the same place, now they are spread out. Zero-field splitting is evidenced in the
energy separation observed in Figure 1-10 (middle) as the shift in the ms 3/2 manifold.
ZFS values can take on negative or positive values, with negative values generally
associated with an elongation of the axial bonds and positive values corresponding to
compression.48 Like the g and hyperfine tensors, the ZFS, often referred to as D, is a
tensor and can take on axial or rhombic forms. When a rhombic component is
introduced, it is generally referred to as E. The ratio of E/D is often used as a gauge of
the rhombicity of a system, with an upper limit of 1/3. With the introduction of E, the
spin Hamiltonian component pertaining to ZFS takes on the form of Equation 1-8.
(1-8)
Sx, Sy, and Sz are the respective components of the spin matrix. D is the axial
component of the ZFS parameter and E is the rhombic component of the ZFS
parameter.
Additional contributions can arise from nuclear quadrupolar interactions, which
will generate asymmetry in the hyperfine and only apply for nuclei with I > 1/2. Nuclear
Zeeman effects are not observable based on the field/frequency combinations used.
35
Pulsed EPR
EPR need not only operate in a CW mode. Pulsed EPR techniques are less
commonly used than the more ubiquitous CW, but they provide a wealth of additional
information that is not accessible via CW EPR. Pulsed EPR allows for observation of
spin-coupled nuclei at increased distances, resolution of very small hyperfine splittings
that might be washed out in a broad CW peak, and measurements of relaxation
parameters. Instead of holding the frequency constant and scanning the field range, as
in CW EPR, pulsed techniques rely on fixed magnetic fields and high power microwave
pulses to act as magnetic torque. This is particularly useful for short-lived species, as
the microwave pulses are on the order of 10 ns, resulting in simple experiments taking
<0.5 μs per shot. Longer relaxation times allow for more complex experiments. The two
most common pulsed EPR techniques used in metalloproteins are Electron Spin Echo
Envelope Modulation (ESEEM), which explores nuclei between 5 and 12 Å away by
observing modulation of the spin echo decay, and Electron Nuclear Double Resonance
(ENDOR), which allows for detection of strongly coupled nuclei, such as those that bind
to the transition metal itself but do not have inherent spin. This technique is appropriate
for distances up to 8 Å. ENDOR is of particular interest to our program, as the natural
substrate of OxDC, oxalate, contains two carbon atoms. ENDOR allows for 13C labeling
experiments and subsequent detection of the two carbon atoms, circumventing the
absolute need to use oxygen-isotopically enriched substrate.
Density Functional Theory
Density Functional Theory (DFT) is a computational method for calculating
molecular properties. This method was formalized by Hohenberg and Kohn in 1964
based on early work by Thomas, Fermi, Dirac, and Wigner and posits that all properties
36
of all states are formally determined by the ground state electron density; if the total
electronic energy is a function of the electron density, there must be an equation that
phrases energy in terms of density alone, which is a function of the spatial coordinates
of the system.49,50 DFT would offer an exact solution to the Schrödinger equation if the
explicit form of the functional were known, however non-classical contributions
stemming from electron-electron interactions, including self-interaction, exchange
interaction, and Coulomb correlation do not have explicit functional forms, therefore
much of DFT focuses on expressions for approximating those functional terms. This
technique is considered truly valid only for ground state calculations, as in the ground
state the density can be predicted and all positions and charges of nuclei are known.51
DFT scales similarly to Hartree-Fock methodologies, at N4, where N is the total number
of electrons in the system. This allows for larger basis sets or larger system sizes than
the gold-standard coupled cluster methodologies, which scale at N7.
Functionals
Many functionals are available, each with a correspondingly different focus.
These range from basic functionals, using only local density approximations (LDA) to
model the exchange term and electron correlation, such as S-VWN; to gradient
corrected models which depend on the local density and its gradient, such as PW91 or
B88; all the way to the more ubiquitous hybrid functionals, which include Hartree-Fock
exchange, such as B3LYP or M05-2X.52–56 Extensions of these models include
introduction of additional long-range correction terms for charge transfer excitations,
such as the CAM-B3LYP functional.57
37
Basis Sets
Basis sets, like functionals, come in a wide variety of forms. The most basic
basis sets are either combinations of Gaussian functions to represent orbitals or Slater
type orbitals, defined by Equation 1-9.
(1-9)
For a Gaussian-type orbital, with N being a normalization factor that ensures that <η|η>
= 1; x,y, and z corresponding to cartesian coordinates; and α is an orbital exponent that
determines how compact or diffuse the function is. In the case of Slater-type orbitals,
the basis function becomes (Equation 1-10):
(1-10)
Where is the orbital exponent term and Ylm is the spherical harmonic used to describe
the angular part of the function.
While Slater-type orbitals (STO) have better physical meaning, there is no
analytical technique for solving the overlap integrals, unlike Gaussian type orbitals
(GTO). Therefore nearly all modern basis sets use contracted GTOs; summing up
many primitive Gaussian functions in a linear combination to generate one Contracted
Gaussian Function (CGF), which takes on the form of Equation 1-11.51
(1-11)
The simplest expansion of a molecular orbital would utilize one basis function of
the form shown in equation 1-10 for each atomic orbital up to and including the valence
orbitals. This is called a minimal set. Minimal sets do not provide any useful
information with the current state of computing hardware, as modern hardware allows
for more precise calculations in reasonable time-frames. Preliminary optimizations are
38
often done using what are referred to as split-valence basis sets. In these basis sets
the valence and core orbitals are treated differently, as core electrons are known to be
mostly chemically inert. This format allows for minimal basis sets to be used for
nonreactive electrons and still take advantage of a more complete description used in
the valence. Examples of this format would be the ever-popular X-YZG Gaussian basis
set developed by Pople, which assigns X primitive Gaussian functions to each core
atomic orbital and Y and Z as the valence basis functions, with each valence being the
combination of Y and Z, which each are linear combinations of their value of primitive
Gaussians.58 For 6-31G, one of the most common Pople basis sets, each core atomic
orbital (AO) contains 6 primitive Gaussian functions, and each valence AO becomes a
combination of 3 primitive Gaussians and 1 additional primitive Gaussian. This allows
for better descriptions of the true STO style function, as part of the basis set can fit the
low interaction distance region, where interatomic Lennard-Jones style interactions
dominate and the other part can deal with the anharmonicity in the potential well at
higher distances.
Polarization functions can be added on top of a basis set, which either allow for
orbital diffusion by inclusion of p-functions for Hydrogen or d-functions for first-row
elements. Production quality data can be obtained by first performing a preliminary
optimization using a smaller basis set, such as 6-31G, followed by a more rigorous
optimization with more functions and tighter convergence criteria. It should be noted
that it is impractical to merely begin with a high-level optimization, since the starting
geometries may be too far from the local or global minima resulting in an unreasonable
time for the calculation. If the chosen basis set is too detailed the calculation may never
39
finish, instead resulting in convergence errors. Each first-row atom using 6-31G
contains 9 CGF, while moving to the Dunning cc-pVDZ set increases this number to 14
CGF for the same atom, including more polarization functions.59 Diffuse functions can
be added and are critical for describing anions.60,61
Specialized basis sets exist for a variety of purposes, including those with
enhanced polarization functions and basis sets focused on calculation of magnetic
parameters. Most specialized basis sets are limited to first and second row atoms and
are not parameterized for transition metals.60,62
Research Objectives and Overview
The goals of this research stem from the divergence of OxOx and OxDC and
focus on finding an explanation for how the two enzymes perform different chemistry
using a very similar active site, in addition to proposing a non-structural role for the C-
terminus manganese center. The introduction will serve to familiarize the reader with
the two enzymes in question, their reported chemistry, and the techniques that will be
used. Chapter 2 describes a series of computational studies investigating the
introduction of substrate into the active site of the protein and the effects of carboxylate
coordination on the redox potential of the metal center, in addition to 13C ENDOR
calculations which provide a theoretical basis for interpretation of previous experimental
work. Chapter 3 focuses on model complex studies, including 3 series of model
compounds demonstrating the effects of ligand flexibility on the magnetic properties of
the system, the effects of oxalate coordination and an EPR investigation into a
catalytically active OxOx model complex. Chapter 4 describes the construction and
operation of a rapid freeze quench (RFQ) apparatus that will allow observation of
intermediate species in the reaction and potentially allow for observation of the
40
substrate binding mode experimentally in an active protein sample. Chapter 5 provides
discussion of the current and future directions of the project and a summary of the
results from each chapter, along with discussion of their implications.
41
Figure 1-1. Oxalic acid
Figure 1-2. Enzymatic pathways identified for breakdown of oxalic acid. (A) Oxalate
Decarboxylase, (B) Oxalate Oxidase and (C) Oxalyl-CoA Decarboxylase
A
B
C
42
Figure 1-3. OxOx Quaternary structure. Coordinated manganese ions are represented
as purple spheres. PDB 2ETE.
43
Figure 1-4. Current literature mechanism for Oxalate Oxidase.24
44
Figure 1-5. OxDC quaternary structure, from PDB 1UW8, monomer is highlighted in
blue. OxDC exists crystallographically as a stacked dimer of trimers.
45
Figure 1-6. Currently accepted literature mechanism for Oxalate Decarboxylase.30
46
Figure 1-7. W96 and W274 π-stacking orientation at the interface of two monomers. Distances are in Angstrom and determined from the center of each aromatic residue for H and W. PDB 1UW8.
47
Figure 1-8. Hyperfine Interaction for an S=1/2, I=1/2 system with isotropic g and
isotropic hyperfine. (A) full field range for 9.5 GHz X-band. Hyperfine interaction is observed as the smaller additional splitting between the two Zeeman manifolds. (B) zoomed in region showing transitions. (C) representation of the EPR signal that would be recorded for this species.
A
B
C
48
Figure 1-9. Simulation of an S=3/2, I=0 system in the absence of additional effects. Resonance positions are highlighted in red. (A) full field range for 9.5 GHz X-band. Hyperfine interaction is observed as the smaller additional splitting between the two Zeeman manifolds. (B) zoomed in region showing transitions. (C) representation of the EPR signal that would be recorded for this species.
A
B
C
49
Figure 1-10. Simulation of an S=3/2, I=0 system with the addition of a 2 GHz ZFS value. Resonance positions are highlighted in red. (A) full field range for 9.5 GHz X-band. Hyperfine interaction is observed as the smaller additional splitting between the two Zeeman manifolds. (B) zoomed in region showing transitions. (C) representation of the EPR signal that would be recorded for this species.
A
B
C
50
CHAPTER 2 OXALATE BINDING MODES, CAVITY SIZE, REDOX AND HYPERFINE SPLITTING
CALCULATIONS
Background
Recent improvements in computing hardware have made DFT a very accessible
starting point for biological studies.63 Calculations that were not possible even ten years
ago are now readily accessible even without supercomputing resources. As the ability
to run calculations on larger and larger systems has grown, computational investigation
of metalloproteins has exploded.64,65 These important systems were long hindered by
the presence of the metal itself, as the number of electrons it has results in a drastic
increase in the number of functions needed to appropriately describe it.58 Due to the
poor scaling of ab initio methodologies, ranging from N4 for Hartree-Fock to N7 for
coupled cluster, large systems quickly become untenable. DFT methods scale similar
to, or in some cases better than, Hartree-Fock, making them one of the more accessible
methods, in addition to providing better predictions of molecular properties.
DFT is particularly applicable to OxOx and OxDC, as many crystal structures
exist, allowing for reasonable starting geometries. For OxDC, as the protein has been
crystallized in both the 'open' and 'closed' conformations, as discussed in Chapter 1,
both forms can be accessed in order to examine the binding cavity itself.24,25 As this
cavity is buried and accessed through a solvent channel with a proposed gating
mechanism, the channel itself must also be characterized. A large amount of
computational work has already been done for OxOx, suggesting that both
monodentate and bidentate binding is possible and energetically not locked into one
specific orientation.21 Previous OxDC computational studies using coupled cluster
theory and monodentate oxalate have shown that formation of a neutral substrate
51
based radical causes a large decrease in the free energy barrier for the first reaction
product, CO2.32 However, these coupled cluster calculations were done in the absence
of the transition metal and only looked at reaction barriers for the oxalate itself, thus
losing any information about the potential polarizing or stabilizing effects of the metal or
any second shell residues.
Additionally, OxDC has a wealth of available spectroscopic data showing
interactions with different small molecules. OxDC has been shown to interact with
several small carboxylates, as evidenced by the shift observed in the magnitude of the
D tensor as observed by parallel mode EPR.30 Experimental results are presented in
Table 2-1. Each of these preparations begin with OxDC in citrate buffer at pH 4.5.
Small carboxylates were then added to this solution and the spectrum was recorded.
Although citrate is also a carboxylate, it is too large to fit inside the active site cavity,
resulting in no effect on the observed signal.30,31
As shown in Table 2-1, each of the small molecules that were investigated
showed an impact on the magnetic parameters, with the product of the decarboxylation
reaction having the strongest effect, whether added as formate itself or added as
oxalate and converted. Acetate shows a much smaller effect than that of the native
substrate.
Computational Protocol
In general, calculations were performed using the CAM-B3LYP57 functional and
the cc-pvdz59 basis set for production quality data using the Gaussian09 computational
package.66 Initial geometries were taken from crystal structures as indicated in each
section and protonated. All optimizations were performed in the gas phase. Protein
backbone atoms were removed and only R-groups were considered. Alpha and beta
52
carbon atoms were frozen in place in order to restrict large scale deviations from the
crystal structure and better simulate the local environment of the metal. High-spin
multiplicities were used in all cases, as is most commonly observed for manganese and
as fits with experimentally observed states. In the case of the redox potential
calculations, SMD solvation was used, as recommended in the Gaussian white pages
for thermochemistry.67 Frequency calculations were done in all cases to verify energetic
minima were reached using "VeryTight" convergence criteria and "UltraFine" integration
grids. "VeryTight" criteria adds additional restrictions for the cutoffs on forces and step
size that determine convergence. Use of "UltraFine" grids increases the number of
points in the integration grid and is recommended for large systems with 'soft modes'
such as methyl rotations. Thermochemistry calculation output was used for obtaining
free energies for both the solvated and unsolvated cases and the same size grids were
used in all cases to ensure comparability.68
For hyperfine splitting constant calculations, calculations were performed using
ORCA.69 The CAM-B3LYP functional was employed. For manganese, the CP(PPP)
basis set was employed, which has flexibility in the inner s- and p-shells as Gaussian
basis functions have difficulty approaching the basis set limit because they do not
faithfully reproduce behavior close to the nucleus.70,71 This is of particular importance
when dealing with nuclear-electron interactions such as the hyperfine splitting. For all
other atoms the EPR-II double-zeta basis set was used, which is optimized for
calculation of hyperfine constants by DFT, in particular with B3LYP and its associated
functionals, but can only be employed for first- and second-row atoms.72 This
53
combination of basis sets is the only accepted methodology for determining EPR
properties via DFT.73
Cavity Size, Accessibility, and Potential for Bidentate Oxalate Binding Mode
While monodentate oxalate binding is proposed in the literature mechanism, this
binding mode is neither intuitive for this system nor what is most commonly observed in
inorganic oxalate complexes.74–78 Oxalate is highly symmetric, often resulting in side-on
bidentate binding with magnesium and manganese in protein structures as well.79–89
Manganese and magnesium are often physically and functionally interchangeable,
although redox processes that depend on manganese oxidation state are inactive with
magnesium.90,91 A crystal structure of a similar manganese metalloprotein with high
sequence homology to OxDC and identical coordinating residues shows bidentate
binding.92 RMSD overlays between Thermotoga maritima TM1287, a protein
crystallized with bidentate oxalate, and OxDC show that bidentate oxalate binding could
be accommodated by the manganese ion and that none of the coordinating residues in
the binding pocket would be impacted significantly (Figure 2-1). Monodentate binding
has been observed in a cobalt-substituted E162 deletion mutant of OxDC, however,
monodentate coordination is commonly observed for cobalt-oxalate complexes, and the
mutant is catalytically inactive.40,93
Computational Optimization of Oxalate Binding Modes and Localization in Crystal Structure
As a starting point, the static solvent channel for the OxDC active site was
identified and characterized using a CAVER,94 which allows analysis and visualization
of static solvent channels in the crystal structure and interfaces directly with PyMOL95.
This program inserts spheres into the structure at a user-defined starting point and
54
searches for channels by moving the centroids of these spheres while varying their
diameter. It stops at dead-ends based on a minimum cutoff value of the sphere
diameter and provides information about bottlenecks and relative sizes of static solvent
channels. Dynamic channels that only appear during motion cannot be identified by this
methodology, and the diameter of static channels can vary wildly in solution or
simulation. Cavity sizes generally do not change very much, as most internal residues
do not shift because they are restrained by the secondary structure of the protein.96
The N-terminus manganese ion was chosen as a starting point and a minimum
sphere radius of 0.7 Å was chosen in order to size the static channel in the low pH
crystal structure using the 'open' configuration, where E162 does not occlude the
accessibility of the active site. Using a sphere radius greater than 1 Å removes all
channels and using radii of ≤ 0.5 Å identifies spurious channels that travel the length of
the enzyme. Upon identification of the access channel and internal cavity boundaries,
DFT optimizations of monoprotonated oxalate, the native substrate, were performed in
both monodentate and bidentate binding modes. Both modes were demonstrated to fit
inside the available space (Figure 2-2), suggesting that monodentate binding is not the
only possible orientation.
Electrostatic Effects of R92
Based on previous experimental work demonstrating the importance of R92,24,29
simulations including this residue were performed focused on stabilization of the
oxalate. Optimizations were performed in the gas phase using the CAM-B3LYP57
functional and cc-pvdz59 basis set in Gaussian 09.66 Residues were extracted from
PDB 1UW8 and protonated. Only the R-group of each amino acid was considered.
Alpha carbons were methyl-terminated at the interface with the backbone and the alpha
55
and beta carbons of the R-group were frozen in place for the optimization to simulate
the effect of the carbon backbone. Unrestrained calculations often take on high-
symmetry octahedral configurations that are not representative of the native protein.97
Oxalate was added manually and initial optimizations were done using the 6-31G basis
set. Frequency calculations were also performed at the cc-pVDZ level to ensure a true
minimum was reached and not a transition state. Both monodentate oxalate and
bidentate oxalate were considered. In the case of monodentate oxalate, the sixth
coordination site was occupied by a water molecule.
Inclusion of R92 shows a decrease in oxalate-manganese bond distances for
coordinating bonds but an increase in distance for the non-coordinating carboxylate
group of oxalate in the monodentate case as depicted in Figure 2-4, using overlays of
geometry optimizations with and without R92. Relevant distances and angles for
monodentate oxalate coordination with and without R92 are presented in Tables 2-2
and 2-3, respectively, with corresponding figures 2-5 and 2-6 showing the atomic
indices for the tables. This results in the monodentate orientation being more head-on
than observed without R92. When this conformation is assumed, R92 is within
hydrogen bonding distance with both of the oxalate oxygen atoms that face toward it.
However, the strain placed on R92 by this interaction results in an increase in the angle
defined by the hydrogen atoms involved in the bonds and the central carbon of the
arginine functional group. The native, relaxed angle as so defined measures 62.6⁰. For
the monodentate coordination this angle is stretched to 73.1⁰: an increase of over 10⁰.
This strain is large enough that the arginine functional group deviates from planarity,
increasing the torsional angle, defined by the N-C-N-H atoms of the Arginine R-group,
56
for the hydrogen atom that coordinates with the end of the oxalate molecule. The
oxalate molecule shows a torsional angle of 25.7⁰ as defined across the carbon-carbon
backbone of the molecule, as compared to 2.3⁰ in the absence of R92.
When bidentate oxalate coordination is considered with the inclusion of R92, the
oxalate molecule takes on a much less planar configuration compared to the
monodentate system, adopting a torsional angle of 49.52⁰. Without R92, the torsional
angle is calculated to be 57.8⁰. Oxalate retains hydrogen bonding distances with E101
and R92. The energetic minima for the calculation, as verified by the lack of imaginary
frequencies, actually pushes the proton from the monoprotonated oxalate to the
neighboring glutamate residue that is in the first coordination sphere for the manganese
ion. Whether this is an artifact of the simulation, results from lack of inclusion of a
formal second sphere or water molecules in the solvent channel or is first-evidence that
E101 serves as the transient base in the reaction scheme is open to debate. R92
protons trap the non-protonated oxalate carboxylate group in a pincer stabilization
mode, resulting in a highly symmetric bond of 1.72/1.75 Å for the bridging distances.
The binding mode of the oxalate retains high symmetry as well, with bond distances for
coordinating carboxylates of 2.2 + 0.07 Å. Relevant distances and angles for bidentate
oxalate coordination with and without R92 are presented in Tables 2-4 and 2-5,
respectively, with corresponding figures 2-7 and 2-8 showing the atomic indices for the
tables.
Predicted ENDOR Spectra for Oxalate Binding Orientations
DFT calculations can serve well for prediction of EPR properties.98–103 Therefore,
using the optimized geometries generated for the R92 structures, monodentate and
bidentate oxalate calculations were performed in order to predict the hyperfine coupling
57
constant for 13C labeled oxalate. Hyperfine constants can be predicted with good
accuracy for first and second row atoms and specialized basis sets have been
developed for this purpose.60,104–106 Transition metals, such as manganese, are more
computationally intensive due to their high spin values, abundance of electrons, and
possibility for zero-field splitting. ZFS cannot reliably be predicted with high accuracy,
although the order of magnitude and sign are often attainable.73,98,100,107–109
Predicted spectra for oxalate binding were generated using the output
parameters from ORCA and simulated in EasySpin, using the Saffron function, which is
intended for simulation of pulsed EPR experiments, including MIMS ENDOR. Saffron
does not currently have the ability to take into account the polarizing effects of
temperature on the ENDOR spectrum, however this merely leads to enhanced spectral
intensity on the high-frequency side of the spectrum and does not lead to appearance of
new or distinct features.
When simulating the spectra, previously obtained experimental parameters were
used from high field measurements, as no robust or reliable procedure exists for
predicting the zero-field splitting parameter computationally. Experimental parameters
were deemed more realistic. Table 2-6 and Table 2-7, respectively, show the magnetic
parameters used for simulation of the E280Q ENDOR spectrum assuming bidentate
and monodentate coordination. Figure 2-10 and Figure 2-11 show the result of the
simulation overlayed upon the experimental E280Q ENDOR spectrum collected by our
group. Experimental parameters for the E280Q ENDOR spectrum at X-band are as
follows: X-band data collection parameters 9.735602 GHz, 345 mT, 360 ns , 601 points,
5 K cryostat temperature. Experimental spectra were processed by taking the log of the
58
recorded spectra and subtracting the mean of the first 20 data points as a baseline
estimate, following which the antilog was taken, 1 was subtracted from the spectrum
and the result was multiplied by -1. This produces the absolute ENDOR effect and
places all spectra on similar scales, and is a standard methodology for processing
ENDOR data.110 ENDOR spectra appear centered about the nuclear Larmor frequency
for a given field strength and the coupling strength is measured as the distance from the
center. Strong coupling appears as a large splitting and weaker couplings appear as
smaller distances from the center. The largest intensity peaks with the smallest
coupling are contributions from weakly coupled matrix nuclei with very small hyperfine
couplings.
In a highly symmetric side-on bidentate binding motif for oxalate, bond distances
for coordinating oxygens are similar. As such, their respective carbon distances are also
similar. As the dipolar component of the hyperfine interaction is dependent on distance,
the contributions to the total spectrum from each of the carbons should be very similar.
Figure 2-11 shows the calculated spectrum simulated in EasySpin using the parameters
shown in Table 2-6. Contributions from each individual carbon are shown, along with
the total calculated spectrum, confirming that the two similar distances yield similar
interaction strengths. The carbon atoms are labeled on the DFT structure provided as
an inset on the plot. As expected, the two carbon signals are very similar, with peaks
overlapping directly in some cases. This predicted spectrum faithfully reproduces many
of the features of the experimental spectrum, however, some features are shifted
slightly, resulting in a sub-optimal fit. Intensity discrepancies are also present in the
spectrum, which is common for simulated spectra. Least-squares rescaling is used for
59
intensity scaling and only explicit consideration of matrix nuclei will generate appropriate
intensities.
For the monodentate binding geometry, two distinct sets of hyperfine splittings
are expected, with the closer carbon atom providing the stronger ENDOR effect and the
farther carbon contributing the weaker couplings in the center of the spectrum. The
calculated parameters yield a spectrum—shown in Figure 2-11 with parameters given
in Table 2-7—that, again, shows moderate prediction of several spectral features,
however the small magnitude couplings are underestimated significantly and the sum of
the two distinct carbon lineshapes leaves much to be desired. Significant spectral
intensity is left in regions where the experimental spectrum displays no coupling,
indicating that this predicted parameter set is not appropriate.
As the initial system is not a crystal, where all atomic coordinates are well
defined, a combination of the two species should be considered. Simulations were
performed using both sets of calculated parameters in order to test whether a weighted
combination of both predicted spectra yields a better fit than either spectrum by itself
(Figure 2-12). Combinations explored ranged from 0% to 60% monodentate spectral
weight. No convincing simulation was generated by combination of the two spectra, as
the central transition would either become deformed via the combination of the two, or
significant intensity would be introduced in regions that do not correspond to the
experiment by the monodentate parameter set. Using 10% spectral weight for
monodentate oxalate added some curvature inside the low-magnitude central splitting,
however the edges continue to overestimate the coupling of the experimental
spectrum—a facet of the bidentate spectrum itself.
60
Using the bidentate set as a starting point, manual simulation was performed on
the spectrum in an attempt to refine the predicted values and obtain a more accurate fit.
The computational model included only the first coordination shell and R92, and would
therefore neglect any further stabilizing interactions from residues or hydrogen bonding.
Only the bidentate starting parameters were used, as the bidentate spectrum better
represented the experimental data. Magnetic parameters, including the two spin-
system weighting derived from high field simulation of E280Q was maintained in all
cases. The hyperfine coupling was changed in both spin systems simultaneously in
order to prevent both over-complication of the resulting spectrum and generation of
parameters that could not exist in reality. Figure 2-13 shows the contribution of each
carbon atom to the simulation. When comparing this to the two previously calculated
spectra, it is immediately apparent that this new fit retains the bidentate style coupling
pattern, with small differences in the medium and weaker coupling ranges.
Using a simple point-dipole model, the distances between Mn(II) and two 13C
nuclei can be estimated and compared. Using the basic assumption that the spins are
lined up along the z axis of the laboratory coordinate system, aligned with the magnetic
field, the relation between the magnitude of the hyperfine splitting and distance takes
the form of Equation 2-1.
(2-1)
Where μ0 is the magnetic constant, ge is the g factor for the system, μB is the Bohr
Magneton, γn is the gyromagnetic ratio for the nuclei of interest and z is the distance
between the two dipoles. Calculated distances and DFT optimized distances for
bidentate and monodentate binding, along with calculated distances for the manually
61
simulated spectra at X- and Q-Band are shown in Table 2-9. Crystallographic distances
for mono and bidentate binding for oxalate and divalent cations in protein systems is
presented in Table 2-10 for comparison. Experimental distances for oxalate
coordination in a bidentate mode range between 2.6 and 3.2 Å and are generally
symmetric to within 0.2 Å.
Adjusting the parameters for the bidentate fit results in a smaller isotropic
hyperfine splitting for carbon 1 and a slightly larger isotropic hyperfine splitting for
carbon 2, however, the predicted distances still fall within what would be expected for
bidentate binding, as the two predicted distances differ by ~0.15 Å and still fall easily
within the range observed for bidentate binding in protein systems.
Q-band ENDOR data also exists for this system, both for WT and E280Q,
allowing for the validity of the fit to be investigated by imposing the simulation
parameters obtained at X-band on the more-resolved Q-band spectrum. As the
computational model does not distinguish between the two and the EPR parameters do
not differ strongly, the calculated parameters should be transferable. Figure 2-14 shows
the X-band optimized parameters for E280Q plotted over Q-band WT high pH 13C
ENDOR collected by Troy Stich at UC Davis. Experimental parameters for Q-band data
acquisition are as follows: 33.4816 GHz Microwave Frequency, 1.218 T magnetic field,
400 ns , 281 points, 5 K cryostat temperature. Previous work shows that E280Q at high
and low pH and WT ENDOR at high pH presents with the same features and splitting.
When the X-band parameters are re-simulated at Q-band, it is apparent that the
majority of the features are still well represented, but slight changes could result in a
better description. The simulated fit was adjusted based on the Q-band spectra,
62
following which the new Q-band parameters were re-simulated at X-band and overlaid
on the X-band E280Q spectrum to ensure that the fit remained reasonable. Distances
were calculated in both cases based on the axial hyperfine after subtracting the
isotropic interaction. Figure 2-15 shows the X-band optimized simulation re-simulated at
Q-band and overlaid on high pH WT ENDOR data. Figure 2-20 displays the re-
optimized simulation at Q-band. Figure 2-16 shows the adjusted fit re-simulated at X-
band. Each simulation is labeled with the estimated distances for carbon 1 and carbon 2
and contains the individual contributions from each carbon atom of oxalate. Intensity
discrepancies near the center of the ENDOR spectrum, in the weakest coupling range
corresponding to matrix carbon, can resolved by adding natural abundance 13C
splittings. The end result of this process is a fit that accurately reproduces the X-band
spectrum with the exception of a small 4.2 MHz shoulder. The difference between the
simulated Q-band ENDOR spectra and those at X-band is an elongation of the distance
between the oxalate carbon atoms and the manganese center by ~0.1 Å. It should be
noted that the Q-band spectra were acquired with the sample poised at high pH, as
opposed to the X-band spectra which were taken at low-pH, and a slight change in
distance due to hydrogen bonding interactions would not be unreasonable. Therefore,
either of these two fits could be considered to accurately describe the system and the
difference between their simulation parameters is small.
The combination of DFT calculations and multifrequency ENDOR proves a
bidentate binding mode is not just possible, but observed experimentally. The model
does not differentiate between E280Q and WT and fits both sets of data. As the
spectral features are nearly identical between WT and E280Q, their binding modes are
63
characterized as similar. However, low active-pH experimental data of WT is still critical
in order to prove that rearrangement of the substrate is not observed.
Calculation of Redox Potentials
Redox potentials are particularly interesting for metalloenzymes as they generally
drive the chemistry taking place. Protein modulation of redox potentials via electron
withdrawing and electron donating groups and introduction of strain, resulting in bond
angle and distance distortions, contribute strongly to this effect.30,111,112 Non-protein
small molecules are also observed to interact with protein active sites, resulting in
modulation of potentials.30 Transition metals are also observed to alter redox potentials
for common molecules, such as dioxygen, and the rate of electron transfer for any redox
process is related to the potential of the acceptor group.113–115 Redox calculations are
difficult, as solvated geometry optimizations are often not feasible, resulting in the need
to use thermodynamic cycles. This begins with first optimizing the geometry in the gas
phase, followed by a solvated single point energy calculation. Using this information a
ΔGsolvation can be determined. These potentials require normalization to a standard
electrode and reference compounds are necessary in order to determined the solvation
energy of the electron.111,112,116 The cycle used is shown in Figure 2-17.
Using this thermodynamic cycle and the relationship between free energy and
potential, the following equations can be derived:
(2-2)
Equation 2-2 shows the general scheme for reduction of a Mn(II) ion in solution.
(2-3)
64
Equation 2-3 correlates free energy of reduction by taking the difference between the
free energy of the solvated oxidized and reduced forms of each complex.
(2-4)
Equation 2-4 is the free energy of reduction as related to the difference between the gas
phase free energy difference and the energy of solvation.
(2-5)
Equation 2-5 represents the change in total energy upon solvation. This value is
defined as the difference in solvation free energy between the oxidized and reduced
forms of the complex.
(2-6)
Equation 2-6 defines the free energy difference between the two species in the gas
phase based on their geometry optimized free energies.
(2-7)
Where F is the Faraday constant and ΔG⁰SHE is -4.28eV.117 Care must be taken
when choosing functionals, basis sets and convergence criteria when performing these
calculations as the energy differences are small on the absolute Hartree scale. In all
cases, for final structure optimizations and frequency calculations, an "UltraFine"
integration grid was used along with "VeryTight" convergence criteria.
The general structural model for the redox calculations is shown in Figure 2-18.
Protein residues are taken from PDB 1UW8 and were partially restrained as explained
in the experimental section. X and Y represent coordinating molecules used in the
calculations. X is water in all cases except bidentate-mode oxalate.
65
The first set of coordinating molecules used was X = water, Y = water in order to
estimate the redox potential of the protein itself. This value is not known experimentally.
The redox potential of the Mn(II)/Mn(III) couple in aqueous solution is 1.5415V.118
Proteins are well known to lower the redox potential of transition metal ions, as
controlling this potential is critical to their ability to perform catalysis. Structurally, there
is little deviation between the optimized Mn(II) di aquo complex and the Mn(III) form.
Bond distances for the coordinating residues from the protein and water are reduced, as
would be predicted for the higher charge to size ratio of the oxidized metal ion.
Structural overlay between the two forms as shown in Figure 2-19 (top right) shows very
little structural change for the complex. Water in site Y is within hydrogen boding
distance of the coordinating glutamate residue in both cases and the only real shift is an
angular twist of the histidine residues to accommodate the tighter binding of the water
molecules in the Mn(III) form. The calculated potential for the water/water form is 800
mV, which is well within the range seen for other manganese containing proteins.47,119
As the protein itself exists in a predominately Mn(II) resting state, a redox potential
lower than that of the oxygen/superoxide couple would be unreasonable. Likewise,
chemical oxidation experiments have been performed on this complex using potassium
hexachloroiridate(IV), which has an oxidation potential of 870 mV, providing an upper
limit to what would be a reasonable reduction potential.120
Following water optimization, the smallest existing carboxylate—formate—was
introduced in order to verify that carboxylates would modulate the redox potential of the
reaction. Optimized structures with formate are shown in Figure 2-19 (middle row). As
observed with the water optimizations, bond distances decrease when the Mn(III) form
66
is generated. The water molecule that remains coordinated shows a substantial change
during optimization, shifting into a more octahedral coordinating form. The protein
residues are largely unaffected by the change in oxidation state. The calculated
potential for formate, as shown in Table 2-11, is 328 mV. This represents a drastic
change in the redox potential induced by the carboxylate coordination. The formate
anion stabilizes the Mn(III) state and drops the potential almost into the range where
dioxygen could push the redox reaction by itself. The standard reduction potential for
the dioxygen/superoxide couple has been observed to be -330 mV at 105 Pascal
oxygen pressure.121,122 Formate has been shown to coordinate strongly to OxDC,
having been detected bound to the N-terminal active site via X-ray crystallography and
also shown in solution via EPR to modulate the zero-field splitting parameter of the
Mn(III) form.30,123
Acetate has been observed to coordinate to the Mn(III) form of the protein, as
demonstrated by modulation of the zero-field splitting parameter.30 As such, and as the
second smallest carboxylate, it was optimized in the same manner. The ZFS impact of
acetate is less than that of formate, spectroscopically.30 Acetate displays the same
behavior as formate and water, with minor conformational changes in the protein and
slight shifts of the coordinated water but almost no impact on the localization of the
acetate molecule. The calculated potential is 407 mV, which falls in line with previous
spectroscopic observations that acetate does not coordinate as strongly as formate.
When oxalate is considered, several possible binding orientations may be
possible, Oxalate can bind in a head-on monodentate mode, where only one
carboxylate is involved in binding to the metal center, or a bidentate orientation can be
67
seen where both carboxylate groups coordinate to the metal. A head-on bidentate
binding mode where manganese coordinates to both oxygen atoms of the same
carboxylate group is also possible. The bidentate orientation is facilitated by the single
carbon-carbon bond separating the groups in the oxalate molecule giving an
appropriate spacing between the carboxylate groups as to not strongly perturb the
system or make it energetically impossible. Additionally, a head-on bidentate mode
could be considered, where both coordinating oxygen molecules come from the same
carboxylate group. In inorganic small molecule complexes, the side-on bidentate
orientation is much more common, but the sister protein of OxDC, OxOx, is purported to
have a monodentate binding orientation.20 Therefore, both orientations must be
considered when identifying redox impacts on binding. Direct energy comparisons of
the two based on heat of formation calculations is not feasible due to differing number of
atoms between the monodentate and bidentate orientations, as monodentate requires a
water molecule to maintain the experimentally observed 6-coordinate geometry.
However, an additional optimization was performed by adding a water molecule behind
the oxalate in the bidentate orientation to give a directly comparable result. In terms of
gas phase energetics, bidentate binding with a hydrogen-bonded water behind the
oxalate is 28.5 kcal/mol more stable than the monodentate orientation with coordinated
water. Thermal energy at room temperature is 0.593 kcal/mol, meaning that based on
these calculations bidentate binding is strongly favored.
For monodentate coordinated oxalate with water occupying the sixth site, the
oxalate molecule takes on a head-on monodentate mode, with hydrogen bonding
between the non-coordinating carboxylate and the water ligand (Figure 2-20). Upon
68
oxidation, all coordinating bond distances are reduced, as expected, and the oxalate
anion shifts further into the horizontal plane as defined by the axially bound coordinating
residues. A reduction in the torsional angle of oxalate as defined across the carbon--
carbon bond and with the coordinating oxygen atoms is observed, bringing the oxalate
near planarity. Surprisingly, the calculated redox potential for the head-on binding
orientation is 543 mV. The conjugated pi network between the carboxylates in the
planar form does not appear to have a stabilizing effect on the monodentate system, as
would be expected. Monodentate oxalate therefore modulates the redox potential less
than formate. This suggests that electron density is retained in the oxalate anion and
not used to stabilize the Mn(II) form of the complex.
When oxalate is considered in a bidentate binding mode, two possible
orientations need to be considered. A head-on bidentate binding mode is possible,
where both the single bonded oxygen and double bonded oxygen of the carboxylate
coordinate. This orientation is not observed in proteins. Side on bidentate oxalate is
by far the most observed bidentate mode.80,81,84,87–89
Optimized bidentate orientations for this system show larger structural changes
than those observed with the other carboxylates. In most cases, the ligand binding is
simply pulled tighter between manganese (II) and manganese (III) forms. With
bidentate oxalate, a torsional angle shift becomes possible between the two binding
sites, allowing for a decrease in orbital interaction for the oxalate π system with respect
to the torsional angle. The increased torsional angle offers another avenue for the
complex to distribute molecular strain. An energetic minima is observed when oxalate
takes on a 'staggered' conformation, where the torsional angle of the oxalate backbone
69
is 90⁰.124,125 This conformational change allows for separation of the two sides of the
oxalate molecule when coordinated to Mn(II) and allows for orbital interactions to
stabilize the Mn(III) species in a planar conformation. As such, bidentate oxalate could
be expected to show the largest redox potential drop by stabilizing the Mn(III) redox
state in addition to potentially having a compounded redox modulating effect as it
coordinates two carboxylates. We find that based on the calculated potential, this is
indeed the case, with bidentate oxalate showing a 278 mV calculated redox potential.
Bidentate oxalate coordination shows strong geometric differences between the
Mn(II) and Mn(III) form and nearly all of the distortion is absorbed by the substrate
ligand (Figure 2-20). Oxalate goes from a nonplanar orientation in the Mn(II) form, with
a torsional angle of 71.85⁰ to a nearly fully planar system for Mn(III), with a torsional
angle of only 6⁰. Planar oxalate is observed in coordination complexes;74–76,78 however,
from a molecular orbital perspective, oxalate should remain non-planar to minimize the
orbital overlap from the two pi systems, as they interact in an anti-bonding fashion.
Manganese (III) has a much higher charge-to-size ratio, resulting in more electron
density being shifted from the oxalate towards the metal ion, which mitigates the
energetic penalty of interacting orbitals.
Although this result suggests that bidentate binding gives the strongest effect, it
also clashes with the currently accepted mechanism, as the literature proposed
mechanism involves monodentate binding and oxygen binding at the same manganese
site. Manganese does not commonly take on coordination structures in excess of six
bonds, therefore oxygen would necessarily coordinate at a secondary site. The C-
terminus manganese has been proposed to serve as a potential oxygen binding site,
70
therefore its redox properties in the presence of dioxygen should also be considered.
Knowing the redox potentials of both sites affords the ability to calculate a transition
probability for potential long-range electron transfer. Oxygen binding to metals can take
on many different forms, from bound dioxygen, to bound superoxide, to more exotic
forms. 114,121,126,127 Each of these forms could exist with or without a sixth ligand in the
form of water.
While the Gibb's Free Energy of the free electron in the gas phase is zero by
thermodynamic definition, the energy of a solvated electron in aqueous medium is not.
In order to account for this parameter, a correction energy is employed, which is
determined by comparison versus known potentials. Therefore, two model complexes
were employed in order to calculate the difference in the predicted reduction potential
and the experimental potential. The geometries for these two complexes,
Mn(acetylacetonato)3 and Mn(bipyridine)3, referred to as Mn(acac)3 and Mn(bipy)3,
respectively, are presented as Figure 2-21. As observed for the protein system, the
manganese(III) forms for each complex have shorter bonding distances to counter the
higher charge of the metal ion, but the angles remain similar and no geometric
distortions are observed. Octahedral coordination is retained and no imaginary
frequencies were obtained for either geometry optimization. The calculated potentials
obtained for the two model complexes return a reasonable result. Mn(III)(acac)3
calculations predict a reduction potential of -95 mV versus the literature values of
-174128 (recorded in acetonitrile as -373 mV vs Ag/AgCl and converted to SHE) to -110
mV129 vs SHE. Calculations for the reduction of Mn(III)(bipy)3 predict a 914 mV
potential. The literature value for oxidation of Mn(II)(bipy)3 is 1039 mV in aqueous
71
solution vs SHE, however the Mn(III) form is unstable.130 Reduction potentials tend to
decrease in aprotic solvents. In Mn(III)(acac)3, this difference is up to 200 mV; a
difference from methanol to acetonitrile.131
Conclusions
The redox impact of a series of small molecules was investigated on the active
site of Bacillus subtilis Oxalate Decarboxylase. All carboxylates were predicted to drop
the redox potential as compared to the water-only coordination model. Oxalate was
explored in two forms, head-on monodentate and side-on bidentate binding orientations.
In the head-on case, oxalate was predicted to have one of the weakest impacts on the
reduction potential. When bidentate binding is considered, oxalate has the strongest
impact on the reduction potential, as would be expected for a dicarboxylate and the
native substrate of the system. The bidentate binding orientation for oxalate is strongly
preferred. Previously collected 13C ENDOR spectra were also successfully simulated
using DFT parameters as a starting point, indicating that a bidentate binding mode is
experimentally observed in both WT OxDC at high pH and E280Q OxDC at both high
and low pH. Calculated reduction potentials predict bidentate oxalate to have the
lowest potential, indicating that this geometry is best poised toward catalysis.
72
Table 2-1. Simulated Magnetic Parameters for OxDC Mn(III) Signal in Parallel Mode Under Various Conditions.
Buffer D [cm-1] |E/D| A [MHz]
Succinate -2.38 0.13 140 Acetate -2.44 0.13 145 Formate/ Oxalate -4.0 0.11 150
Table 2-2. Bond distances, angles and dihedral for monodentate oxalate coordination
in the presence of R92. Labels correspond to Figure 2-5.
Distances (Å)
Angles (degree) Mn-O55 2.2
H77-C5-H80 73.14
Mn-O59 2.19
H77-N4-C5 120.58
Mn-C58 3.13
H80-N7-C5 121.38
Mn-C61 4.42
O59-Mn-O55 84.49
H56-O60 1.93 H57-E101 O 1.6
Dihedral (degree) H77-O60 1.78
O62-C61-C58-O59 25.72
H80-O63 1.83 Mn-O62 4.8 Mn-O60 3.6
Table 2-3. Bond distances, angles and dihedral for monodentate oxalate coordination
in the absence of R92. Labels correspond to Figure 2-6.
Distances (Å)
Angles (degree) Mn-O60 2.28
O64-Mn-O60 73.49
Mn-O64 2.15 Mn-O35 2.08
Dihedral Mn-O66 4.4
O36-H62 1.6
O69-C65-C63-O64 2.28
Mn-C63 3.3
Mn-C65 3.83 Mn-O69 3.54
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Table 2-4. Bond distances, angles and dihedral for bidentate oxalate coordination in the presence of R92. Labels correspond to Figure 2-7.
Distances (Å)
Angles (degree) Mn-O59 2.22
H82-C5-H81 62.26
Mn-O55 2.14
O55-Mn-O59 78.25
Mn-O36 2.19
H81-N14-C5 113.28
Mn-C56 2.88
H82-N7-C5 114.5
Mn-C54 2.75
O58-H82 1.69 O58-H81 1.72
Dihedral O57-H60 1.52
O55-C54-C56-O59 49.52
O37-H60 1.03 C54-C56 1.54
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Table 2-5. Bond distances, angles and dihedral for bidentate oxalate coordination in the absence of R92. Labels correspond to Figure 2-8.
Distances (Å)
Angles (degree) Mn-O59 2.31
O62-Mn-O59 76.36
Mn-O62 2.14 Mn-O57 2.07 Mn-C63 2.77
Dihedral Mn-C64 2.91
O59-C63-C64-O62 57.81
O60-H65 1.02 O58-H65 1.57 C63-C64 1.53
Table 2-6. E280Q Magnetic Parameters used for Bidentate Simulation. Hyperfine Splitting from DFT calculations. Other Parameters Derived From High-Field Work
System 1 Parameter
Value System 2 Parameter
Value
g 2.00087 g 2.00087 A [MHz] [-0.8140 -0.9758
1.3976; -0.41 -0.9346 2.0028]
A [MHz] [-0.8140 -0.9758 1.3976; -0.41 -0.9346 2.0028]
D [MHz] 1100
D [MHz] -950
E [MHz] 165 E [MHz] 190 Linewidth [MHz] 0.08 Linewidth [MHz] 0.08 Weight 0.56 Weight 0.44
75
Table 2-7. E280Q Magnetic Parameters used for Monodentate Simulation. Hyperfine
Splitting from DFT calculations. Other Parameters Derived From High-Field Work
System 1 Parameter
Value System 2 Parameter
Value
g 2.00087 g 2.00087 A [MHz] [0.4253 0.4862
1.2174; 0.4382 0.7807 2.4126]
A [MHz] [0.4253 0.4862 1.2174; 0.4382 0.7807 2.4126]
D [MHz] 1100 D [MHz] -950 E [MHz] 165 E [MHz] 190 Linewidth [MHz] 0.08 Linewidth [MHz] 0.08 Weight 0.56 Weight 0.44
76
Table 2-8. E280Q Adjusted Magnetic Parameters used for Bidentate Simulation Hyperfine Splitting Adjusted Manually Using Table 4-2 as a Starting Point. Other Parameters Derived From High-Field Work
System 1 Parameter
Value System 2 Parameter
Value
g 2.00087 g 2.00087 A [MHz] (X-band) [-0.684 -0.8758
1.2976; -0.41 -0.6346 2.0028]
A [MHz] [-0.684 -0.8758 1.2976; -0.41 -0.6346 2.0028]
A [MHz] (Q-band) [-0.7340 -0.8258 1.1276; -0.46 -0.7146 1.85028]
[-0.7340 -0.8258 1.1276; -0.46 -0.7146 1.85028]
D [MHz] 1100 D [MHz] -950 E [MHz] 165 E [MHz] 190 Linewidth [MHz] 0.08 Linewidth [MHz] 0.08 Weight 0.56 Weight 0.44
Table 2-9. Comparison of Calculated Distances from Dipolar Hyperfine Splitting with
DFT Optimized Distances.
System Z Component of Hyperfine Splitting [MHz]
Hyperfine Dipolar Calculated Distance [Å]
DFT/ORCA Calculated Distance [Å]
C1 Adipz C2 Adipz C1 C2 C1 C2
Bidentate (calculated)
1.5284
1.7801
2.962
2.815
2.88 2.75
Monodentate (Calculated)
0.5078
1.2021
4.277
3.209
4.42 3.13
Adjusted Simulation (X-band)
1.385
1.6834
3.061
2.868
- -
Adjusted Simulation (Q-band, WT pH8)
1.2716 1.6249 3.150 2.902 - -
77
Table 2-10. PDB Crystallographic Distances and Identifiers for Oxalate Coordination to Divalent Cations.
PDB ID Organism/Species Bound Ion
X- C1 [Å]
X- C2 [Å]
Binding Mode
Data Collection Temperature [K]
1DO8 Homo sapiens malic enzyme
Mn 3.1 3.0 bidentate 100
2RK7 Rattus norvegicus phosphoenolpyruvate carboxykinase, cytosolic [GTP]
Mn 3.0 3.0 bidentate 100
1NVM Pseudomonas sp. acetaldehyde dehydrogenase
Mn 2.9 3.0 bidentate 100
4WIU Mycobacterium tuberculosis Phosphoenolpyruvate carboxykinase [GTP]
Mn 3.3 3.3 bidentate 120
1GZ3 Homo sapiens mitochondrial NAD(P)+-dependent malic enzyme
Mn 3.1 3.1 bidentate 100
4L80 Chloroflexus aurantiacus malyl-CoA lyase
Mg 2.7 2.9 bidentate 100
2EB5 Escherichia coli 2-oxo-hept-3-ene-1,7-dioate hydratase
Mg 2.8 2.8 bidentate 100
1PYM Mytilus edulis phosphoenolpyruvate mutase
Mg 2.9 3.0 bidentate 120
3M0K Cryphonectria parasitica Oxaloacetate acetylhydrolase
Mg 3.0 3.1 bidentate 100
2DUA Variovorax sp. (strain Pal2) Phosphonopyruvate hydrolase
Mg 2.8 2.9 bidentate 100
5HI0 Bacillus subtilis Oxalate Decarboxylase
Co 2.8 4.1 monodentate 100
4NNC Burkholderia glumae OBCA, Oxalate Biosynthetic Component A
Co 4.0 4.8 monodentate 100
78
Table 2-11. Calculated values for redox cycling. Values are presented in eV unless otherwise labeled. Values labeled Ha are presented in Hartree.
X ΔGo (Ha) ΔGR (Ha) ΔGsolv(O) ΔGsolv(R) ΔGII ΔGsolv ΔΔGO
|R ΔGcorr
ΔG vs. SHE
water -2524.257 -2524.606 -6.486 -2.328 9.497 -4.158 5.338 5.080 0.801
formate -2637.254 -2637.468 -2.618 -1.681 5.802 -0.937 4.865 4.607 0.328
acetate -2676.545 -2676.752 -2.296 -1.630 5.610 -0.665 4.944 4.686 0.407
oxalate- mono -2825.783 -2826.002 -2.559 -1.687 5.953 -0.872 5.080 4.822 0.543
oxalate-bi -2749.380 -2749.588 -2.463 -1.636 5.642 -0.827 4.815 4.557 0.278
Mn(bipy)3 -2186.151 -2186.238 -0.481 -2.533 2.390 2.052 4.442 4.184 -0.095
Mn(acac)3 -2635.368 -2635.828
-
12.203 -5.1411 12.514 -7.061 5.452 5.194 0.914
79
Figure 2-1. 1.95 Å resolution crystal structure of Thermatoga maritima T1287. (A) with coordinated oxalate. (B) RMSD overlay with OxDC active site, (RMSD = 0606A., global RMSD 1.2 Å). Blue - T1287, Green - OxDC
Figure 2-2. DFT Optimization of Binding Modes for Oxalate Placed in CAVER Identified Binding Pocket. (A) Oxalate Monodentate with Water, (B) Oxalate Bidentate.
A B
A B
80
Figure 2-3. (A) mono- and (B) bidentate oxalate geometry optimization with the inclusion of residue R92.
Figure 2-4. (A) mono- and (B) bidentate oxalate geometry optimization overlays in the presence and absence of R92. For monodentate coordination, green represents the inclusion of R92 and cyan shows oxalate without. For bidentate binding, purple shows the inclusion of R92 and yellow is without.
A B
A B
81
Figure 2-5. DFT optimized monodentate oxalate coordination in the presence of R92.
Figure 2-6. DFT optimized monodentate oxalate coordination in the absence of R92.
82
Figure 2-7. DFT optimized monodentate oxalate coordination in the presence of R92.
Figure 2-8. DFT optimized monodentate oxalate coordination in the absence of R92.
83
Figure 2-9. Arginine bite angles for oxalate stabilization. (A) Monodentate oxalate with water coordination. (B) Bidentate oxalate. Native arginine has a 62.6⁰ bite angle, with monodentate increasing to 73.1⁰ and bidentate remaining similar to the native amino acid at 64.1⁰.
A B
84
Figure 2-10. Bidentate oxalate simulation at X-band using output from ORCA hyperfine calculations and previously determined EPR parameters for E280Q. Contributions from each carbon atom are shown in addition to the total simulated spectra. No additional fitting was performed. X-band data
collection parameters 9.735602 GHz, 345 mT, 360ns , 601 points, 5K. Data collected by Prof. Alexander Angerhofer.
85
Figure 2-11. Monodentate oxalate simulation at X-band using output from ORCA hyperfine calculations and previously determined EPR parameters for E280Q. Contributions from each carbon atom are shown in addition to the total simulated spectra. No additional fitting was performed. X-band data collection
parameters 9.735602 GHz, 345 mT, 360ns , 601 points, 5K. Data collected by Prof. Alexander Angerhofer.
86
Figure 2-12. Summed simulated spectra at X-band combining various weights of the
calculated monodentate spectrum combined with the calculated bidentate spectrum. X-band data collection parameters 9.735602 GHz, 345 mT, 360ns
, 601 points, 5K. Data collected by Prof. Alexander Angerhofer.
87
Figure 2-13. Bidentate oxalate simulation at X-band beginning with DFT calculated
parameters and adjusted for better fit. Contributions from each carbon atom are shown in addition to the total simulated spectra. Simulation parameters are given in Table 2-8. X-band data collection parameters 9.735602 GHz,
345 mT, 360ns , 601 points, 5K. Data collected by Prof. Alexander Angerhofer.
88
Figure 2-14. Bidentate oxalate simulation overlaid on Q-band WT pH 8.0 ENDOR data.
Experimental parameters 33.4816 GHz Microwave Frequency, 1.218 T
magnetic field, 400 ns , 281 points, 5K. Experimental data collected by Troy Stich at UC Davis.
89
Figure 2-15. Bidentate oxalate simulation re-optimized at Q-band overlaid on WT pH
8.0 ENDOR data. Experimental parameters 33.4816 GHz Microwave
Frequency, 1.218 T magnetic field, 400 ns , 281 points. Experimental data collected by Troy Stich at UC Davis.
90
Figure 2-16. Bidentate oxalate simulation re-optimized at Q-band overlaid on X-band
E280Q ENDOR data. X-band data collection parameters 9.735602 GHz, 345
mT, 360ns , 601 points, 5K.
91
Figure 2-17. Thermodynamic cycle for calculation of redox potentials. (g) notation indicates gas phase, (aq) indicates solvated. Roman numeral subtext indicates oxidation state of the metal in each calculation.
Figure 2-18. General coordination model for redox calculations. Protein residues are
used from PDB file 1UW8. X and Y represent coordinating molecules used in the calculation.
92
Figure 2-19. DFT optimized structures for different coordination models. Green represents Mn(II) structure, Cyan represents Mn(III) structure. An overlay of the two is presented on the right hand side to highlight any strong geometrical distortions or differences.
93
Figure 2-20. DFT optimized structures for different coordination models. Green represents Mn(II) structure, Cyan represents Mn(III) structure. An overlay of the two is presented on the right hand side to highlight any strong geometrical distortions or differences.
94
Figure 2-21. DFT optimized structures for Mn(bipy)3 and Mn(acac)3. Green represents Mn(II) structure, Cyan represents Mn(III) structure. An overlay of the two is presented on the right hand side to highlight any strong geometrical distortions or differences
95
CHAPTER 3 MODEL COMPLEXES - IMPACTS OF OXALATE, RIGIDITY AND COORDINATION
ENVIRONMENT
Introduction
Model complexes have long been viewed as an essential source of information in
the study of metalloproteins.119,121,132–135 The environment surrounding the metal center
is critical in modulating its properties, including optical spectra, redox potentials, and
free coordination sites, all of which contribute to the enzyme's ability to perform
catalysis. Multi-histidine coordination with readily exchangeable water-filled
coordination sites is a common motif for manganese containing proteins due to the
strong binding affinity of histidine for transition metals.134,136 Often, mixed coordination is
observed involving 1-3 histidines along with one or two negatively charged residues,
such as glutamate or aspartate.16,20,137 However, mixed coordination complexes are
difficult to prepare synthetically, especially when several of the groups representing the
coordinating residues share the same functional group.
As inorganic complexes can be synthesized, crystallized and identified
structurally with atomic precision, they are invaluable in testing hypotheses in protein
systems. As proteins are difficult to crystallize and often diffract quite poorly, small
structural details may be inaccessible. Additionally, the most interesting crystal
structures would contain the native substrate, which is a particularly challenging
endeavor as one needs to then deactivate the protein to prevent catalysis while
simultaneously retaining the active structure.
Three sets of structural models were used in order to measure relative effects of
different coordination environments on the manganese center. First, the effects of
96
planar oxalate coordination were investigated using a manganese oxalate metal-organic
framework. Following this study, a series of manganese macrocycles was investigated
in order to compare strain, rigidity and relative bonding geometry with zero-field splitting.
Finally, a catalytically active oxalate oxidase model complex was investigated in an
attempt to identify the divergence of the OxOx and OxDC mechanisms.
Experimental
Mn(pyim)(oxalate)
16 mg Mn(NO3)2 was combined with 19 mg 2(1-H-pyridyl)imidazole (pyim) in
50:50 water:methanol. 5 mg oxalic acid dihydrate was dissolved in 1 mL deionized
water. Solutions were carefully layered into 4 dram vials starting with 1 mL of the oxalic
acid solution, followed by a 6 mL water buffer layer and finally 6 mL of the metal-ligand
mixture. Vials were sealed and left to crystallize, resulting in x-ray quality crystals within
6 days. Elemental Analysis: Calculated for C10H11MnN3O5 (1): C, 38.94; H, 3.60; N,
13.64. Found: C,34.2; H, 2.79; N, 18.59%.
Cd(pyim)(oxalate)
This complex was prepared in the same manner as the Mn species using 15.4
mg Cd(NO3)2 with 19 mg pyim and layered in the same way. Elemental Analysis:
Calculated for C10H9CdN3O5 (1): C, 33.03; H, 2.50; N, 11.56. Found: C,34.06; H, 2.96;
N, 17.23%.
X-Ray experimental
X-Ray Intensity data were collected at 100 K on a Bruker DUO diffractometer using
MoK radiation ( = 0.71073 Å) and an APEXII CCD area detector.
Raw data frames were read by the program SAINT1 and integrated using 3D
profiling algorithms. The resulting data were reduced to produce hkl reflections and
97
their intensities and estimated standard deviations. The data were corrected for Lorentz
and polarization effects and numerical absorption corrections were applied based on
indexed and measured faces.
X-ray of Mn(pyim)oxalate
The structure was solved and refined in SHELXTL2013, using full-matrix least-
squares refinement. The non-H atoms were refined with anisotropic thermal
parameters and all of the H atoms were calculated in idealized positions and refined
riding on their parent atoms. The structure is a one dimensional chain with a repeat unit
of a Mn center, pyim ligand and an oxalate dianion. There are also two lattice water
solvent molecules. The oxygens of the water molecules are both disordered and
refined as O5/O5’ and O6/O6’ with their site occupation factors dependently refined.
Only one set of two protons on each water molecule was refined after locating them
from a difference Fourier map. In the final cycle of refinement, 2732 reflections (of
which 1875 are observed with I > 2(I)) were used to refine 205 parameters and the
resulting R1, wR2 and S (goodness of fit) were 7.30%, 15.23% and 1.272, respectively.
The refinement was carried out by minimizing the wR2 function using F2 rather than F
values. R1 is calculated to provide a reference to the conventional R value but its
function is not minimized.
X-ray of Cd(pyim)oxalate
The structure was solved and refined in SHELXTL2013, using full-matrix least-
squares refinement. The non-H atoms were refined with anisotropic thermal
parameters and all of the H atoms were calculated in idealized positions and refined
riding on their parent atoms. In addition to the one dimensional chains there are water
molecules (one per asymmetric unit) linking them together. The water protons and the
98
amine H3 were obtained from a difference Fourier map and refined freely. The Cd
center is disordered in two places where only traces of Cd (0.0174(1)% compared to
0.983(1)) seems to have asymmetric bonds to the linking oxalate ions. In the final cycle
of refinement, 2726 reflections (of which 2671 are observed with I > 2(I)) were used to
refine 189 parameters and the resulting R1, wR2 and S (goodness of fit) were 1.64%,
4.11%, and 1.166, respectively. The refinement was carried out by minimizing the wR2
function using F2 rather than F values. R1 is calculated to provide a reference to the
conventional R value but its function is not minimized.
EPR
EPR experiments at X-band were performed on a Bruker ELEXSYS E500
spectrometer fitted with a Super High Q resonator (SHQE). An Oxford Instruments
ESR900 continuous flow helium cryostat was employed to maintain sample
temperature. Parameters for X-band powder experiments are 3550 + 3500 Gauss
sweep width, 10 G modulation amplitude, 100 kHz modulation frequency, 80 ms
conversion time 25 dB attenuation (0.6325 mW), and 2048 pts.
High resolution single crystal rotation parameters for the first axis are 9.42 GHz
microwave frequency, 3550 + 3500 Gauss field sweep width, 10 G modulation
amplitude, 100 kHz modulation frequency, 40 ms conversion time, 35 dB attenuation
(0.06325 mW), 3501 pts, 2 degrees per increment, 180 increments, 2 scans per
increment (for a total scan angle of 360 degrees). Single crystals were mounted on a
custom MiTeGen 300 μm loop affixed to a Kel-F pin for two of the three axis collected.
For collection on the third axis crystals were mounted to a 5 mm solid quartz rod with a
flattened bottom and held in place using microscope immersion oil. Data collection
99
parameters for the second and third axis are as follows: 9.44 GHz microwave
frequency, 3550 + 3500 Gauss field sweep width, 10 G modulation amplitude, 100 kHz
modulation frequency, 40 ms conversion time, 28 dB attenuation, 5 scans, 10 degree
increment, 18 increments (for a total scan angle of 180 degrees). A Bruker model ER
218PG1 programmable goniometer with a resolution of 0.125⁰ and reproducibility of 0.5⁰
was used to control sample angle.
High Field EPR was performed at the National High Magnetic Field Laboratory in
Tallahassee, FL, using the 17 T transmission spectrometer fitted with a homebuilt
probe.138 Due to hysteresis in the magnetic field, the g factor for the pyim complex was
corrected to 2.000. Resolution of the manganese fine structure lines along with the
breadth of the spectrum precluded use of a high field standard for accurate g factor
calibration.
Manganese (II) Macrocycles
Mn(tacn)2, Mn(tacd)2, and Mn(tacud)2 were provided by Professor Ferman
Chavez at Oakland University as powder in vacuum sealed ampoules. Once received,
EPR samples were prepared by dissolving 10 mg of each macrocyclic complex in 1 mL
of dry methanol under argon and transferring the solution into a 5 mm O.D., 4 mm I.D.
quartz tube. Samples were freeze-pump-thaw cycled twice and flame sealed under
vacuum. High field EPR was performed on the 17 T transmission spectrometer at the
National High Magnetic Field Laboratory in Tallahassee, FL.
PCA was performed using MatLab R2017a. Bond angles and distances were
taken from crystallographic data. Principal Components 1 and 2 account for 100% of all
variance in both cases. PC 1 for bonds accounts for 91% alone and 62% for angles.
100
MnLCl/MnL(Otf)2
MnLCl and MnL(Otf)2 were also provided by Professor Chavez at Oakland
University.
3 mM samples were prepared in a glove box under nitrogen atmosphere at <1
ppm oxygen in 50:50 acetonitrile:toluene. Tubes were freeze-pump-thawed and flame-
sealed upon removal from the glove box to prevent premature oxygen exposure
Samples were analyzed as the ligand complex, the ligand complex with the addition of
20 equivalents bis(tetrabutyl ammonium) oxalate (TBAO), and finally the oxidized form.
The oxidized form was generated by cracking the sample tubes of samples containing
TBAO and allowing air exposure for 2 h. The sample turned dark pink as previously
observed, indicating that the species of interest was generated.
EPR spectra were collected using a Bruker ELEXSYS-II E500 EPR spectrometer
equipped with a Bruker DM4116 dual mode resonator fitted to an Oxford Instruments
ESR900 continuous flow helium cryostat. Sample temperature was maintained at 5 K.
EPR experimental parameters were as follows - perpendicular mode: 9.64 GHz
microwave frequency, 3550 + 3500 Gauss sweep width, 100 kHz modulation frequency,
10 Gauss modulation amplitude, 0.6325 mW power, 40 ms conversion time, 3501
points, 2 scans; parallel mode: 9.37 GHz microwave frequency, 3550 + 3500 Gauss
sweep width, 100k Hz modulation frequency, 10 Gauss modulation amplitude, 0.6325
mW power, 40 ms conversion time, 3501 points, 2 scans. Samples were pre-frozen in
liquid nitrogen before insertion into the cryostat.
High Field EPR spectra were collected at the National High Magnetic Field
Laboratory using the 17 T transmission spectrometer.139
EPR parameters were simulated using EasySpin.140
101
Discussion
Manganese(II)pyim(oxalate)2
The polymeric manganese(II)pyim(oxalate)2 exists as a series of manganese
centers bridged by bidentate oxalate with each manganese atom having two
coordination sites occupied by the pyim ligand oriented in an alternating eclipsing form
and four sites occupied by oxygen atoms from two different oxalate molecules, resulting
in an octahedral coordination environment. This complex crystallizes as an
orthorhombic system in the Pbca(61) space group, indicating D2h symmetry. The
asymmetric unit consists of one manganese atom, one pyim ligand, one full oxalate
molecule and two disordered water atoms in uncoordinated locations. The symmetry
axis encompasses the plane of the ligand.
Manganese-oxalate bond distances are highly symmetric, measured at
2.1801(49) and 2.1512(51) Å. The oxalate bite angle on the manganese atom is
76.344(172) degrees. Angles between the oxalate oxygen atoms and the imidazole ring
of the pyim ligand are 102.678(152) degrees for the oxygen opposing the pyridine ring
(O3-Mn-N2) and 96.691(165) degrees for the oxygen closer to the pyridine ring (O4-Mn-
N2).
Inter-manganese distances inside a single polymer chain are 5.6499(3) Å.
Between neighboring chains two manganese-manganese distances are observed, with
15.1374(8) Å being the two manganese aligned along the crystallographic c axis and
14.0844(8) Å to the closest manganese, which lies at a 70.868(0) degree angle as
defined by the two c-aligned manganese atoms.
102
Oxalate takes on a very planar conformation in this complex, with a torsional
angle of 176.946(5) degrees as defined by the carbon backbone and opposing oxygen
atoms across the carbon-carbon bond (O1-C9-C10-O4). The angle between oxalate
planes bonded to the same metal center is 85.7 degrees, resulting in the honeycombed
packing structure observed for the complex (Figure 3-2).
Mn(pyim)oxalate exhibits pi stacking stabilization along the crystallographic c
axis with pyim-pyim distances of 3.96 A, which is well within the accepted distance
range.141 The pyim molecule stacks antiparallel with imidazole in van der Waals contact
to the pyridine portion (N2-C6-N3-C8-C7) of the coordinated ligand and vice versa. This
interaction is not seen along the crystallographic b axis, where an 87 degree angle
exists between pyim planes.
Cadmium (II) pyim (oxalate)2
The cadmium form of the pyim polymer, (Figure 3-1, right) crystallizes in a
monoclinic system in the P21/c space group, indicating C2h symmetry. Loss of a
rotational axis of symmetry between the Mn and Cd forms of the coordination polymer
due to a change in oxalate-oxalate torsional angle accounts for the change in point
group. A 75.413 degree angle is observed between oxalate planes, which is a 10
degree reduction versus the manganese form of the complex. Cadmium in this
complex is inherently disordered, showing total displacement of 0.6203 Å between
locations and a relative occupancy of 98.26% for site 1 and 1.74% for site two.
In the cadmium form, as contrasted with the manganese form, the pyim ligands
all exist with the same relative orientation. Pyim still exhibits π-stacking behavior with
an even shorter distance of 3.70 Å center-to-center but the ligands no longer stack 1:1.
103
Instead, it is observed that the imidazole portion of the ligand stacks with the pyridine of
the neighboring chain, whose imidazole moiety continues this to the next chain. The
pyim ligands therefore stack in a zippered fashion with an angle of 60-62 degrees as
defined by the plane of the pyim ligand with the imidazole point nitrogen as the vertex
and imidazole point nitrogen of the pyim molecule above as the final point defining the
angle.
Chains of Cd(pyim)oxalate are aligned antiparallel, resulting in several possible
Cd-Cd distances with respect to the neighboring chain. Distances between neighboring
cadmium atoms in the same chain are found to be 5.8966(1) Å with 117.397(0) degree
angle for the chain as defined by three neighboring cadmium atoms. When the
neighboring chains are considered, cadmium distances are 6.07, 7.70 and 9.63 Å to the
nearest 3 cadmium atoms, respectively. The very short 6.07 Å distance is a sixty-
percent reduction versus complex Mn(pyim)oxalate. This distance is a result of the
antiparallel chain stacking orientation, as alternating cadmium atoms eclipse along the
crystallographic a axis with pyim ligand molecules pointed in opposite directions (Figure
3-3)
Although the coordination environment between the two complexes is identical,
the overall packing structure of the complex displays a large geometry change. While
the manganese form of the complex packs in a high symmetry configuration, the
cadmium form is flattened out along the oxalate-oxalate bonding axis, resulting in a
higher density structure. The torsional angle defined by C9-O2-Cd-O1 of the next
oxalate of 93.448(2) degrees causes a kinked packing structure of the cadmium form.
104
Oxalate in the cadmium form is more planar than the corresponding manganese
complex, with a torsional angle of 180.000(2) degrees as defined by the carbon
backbone and opposing oxygen atoms across the carbon-carbon bond. Bond distances
from cadmium to oxalic acid ligating oxygen atoms are nonsymmetrical, even when
accounting for the inherent disorder in the cadmium location. The cadmium-oxygen
bonds closer to the pyridine side of the pyim ligand have distances of 2.1470(1) Å to
the closer of the disordered cadmium pair and 2.2966(1) Å to the farther cadmium
location. Conversely, the bond distances closer to the imidazole side of the pyim ligand
are longer on both counts, at 2.3092(1) Å to the closer disordered cadmium and
2.7684(1) Å to the more distant. The carbon-oxygen bonds for the oxalate remain
relatively equal between the two sides, differing by less than 0.02 Å at 1.2454(0) Å and
1.2613(0) Å.
The X-band EPR signal (Figure 3-4) is dominated by the characteristic
manganese sextet with other spin manifolds obscured by overlapping peaks. High Field
EPR at 208 GHz allowed complete separation of the spectrum, resolving each of the six
possible transitions expected for the S=5/2 M(II) species (Figure 3-5). Additionally
splitting arising from the manganese hyperfine interaction are observed, resulting in
each ΔS transition appearing as a sextet.
The magnitude of the hyperfine interaction corresponds to what is classically
observed for high spin manganese (II) in an octahedral environment.142,143 Anisotropy in
the hyperfine splitting is not observed. Only manganese hyperfine splitting needs to be
considered for the powder spectrum simulation of MnCdpyim(oxalate)2 despite the high
relative natural abundance of spin-bearing isotopes the magnitude of any cadmium
105
hyperfine would be very small compared to that of manganese, as no unpaired
electrons reside on the cadmium and only delocalization across the oxalate π network
would facilitate this coupling.
The spectrum is best described using a single component, despite the inherent
disorder in the cadmium location. Due to the very small occupancy factor for the
second site it is likely that any signal resulting from manganese in that configuration is
too weak in relation to the rest of the signal to be observed.
Variation of temperature at high field reveals that the zero field splitting
parameter carries a negative sign, as decreasing temperature increases signal intensity
in the low field region of the spectrum (Figure 3-6).144 With negative D the low-field
parallel features decrease and the low-field perpendicular features increase as
temperature is lowered. Negative zero field splitting values for six-coordinate complexes
are indicative of elongation of the electron distribution along one axis.145
Mn(tacn, tacd, tacud)2
Macrocyclic ligands provide an interesting opportunity to investigate the effects of
strain on the EPR parameters of a system. As macrocycles can be generated with any
number of carbons between each successive coordinating residue, the amount of orbital
strain added to the manganese ion can be monitored. Any impact to the d orbitals of
the metal ion, such as elongation or compression about an axis or in more extreme
cases deviations farther and farther from the octahedral symmetry preferred by the
transition metal, will be visible in the EPR spectrum as an impact on the fine structure
parameters. The hyperfine interaction is not expected to change significantly, partially
because manganese(II) is an S-state ion with equal orbital occupation under high spin,
106
resulting in a system that is only really affected by the enforced geometry of the ligand.
Changes in the g-factor are likewise neither expected nor significantly observed.
Mn(tacud)2
Mn(tacud)2 has the highest degree of flexibility, correspondingly, the smallest
zero field splitting value of the series investigated. The undecane ring comprised of
solely single bonds allows for a large amount of motion, with extra atoms to provide
more degrees of freedom. This complex also exhibits perfect 180⁰ bonds across the
metal. The complex crystallizes in the P1 space group in a triclinic unit cell. A mirror
plane is present in the complex, which leads to the high degree of symmetry. The g=2
region of the spectrum (Figure 3-8) shows no distortion from anisotropic components
within the resolution of the experiment. When simulating high field data, it is important
to verify that the g=2 region does not show distortion, as it is easy to fit the higher spin
manifolds in a moderately broadened system such as this one. A correct simulation will
correctly reproduce both the higher spin manifolds and the g=2 region. Due to the large
spectral width of high field data, compared to the narrow line-width of the manganese
central splitting, presenting only the full scan range to show the higher spin manifolds
can be misleading, as the scale of the g=2 region (total width ~500G) is negligible
compared to the entire scan range. As shown in Figure 3-9, the g=2 region is difficult
to interpret when the full spectrum is considered.
When the entire spectrum is considered (Figure 3-9), the impact of the zero field
splitting is readily observed. A small ZFS exists in the complex with a magnitude of -
930 MHz (-0.03 cm-1). The negative sign of this parameter indicates axial elongation on
the bonds aligned with the D tensor.107,108 The negative sign can be confirmed by
simulating both positive and negative D values, as they manifest as mirror images at
107
high field. A good fit with a positive D requires an anisotropic g factor with magnitudes
that are not supported by the geometry of the system, whereas for a negative D a better
fit can be obtained with an isotropic g and hyperfine, which is more reasonable for the
system. Additionally, the following complexes in the series only simulate with a
negative D value. It would not be reasonable to expect the sign to change with an
increase of strain, as increasing strain will increase the magnitude of the ZFS, and
decreasing strain will return it to zero.
Mn(tacd)2
In order of decreasing ring size, the second complex in the series is Mn(tacd)2.
The decane ring involves one less carbon atom, but the impact of this change is readily
visible in the EPR spectrum. The zero-field splitting of the complex increases drastically
to -2470 MHz (0.082 cm-1). The central g=2 sextet (Figure 3-11) begins to show the
effects of a moderate Mn(II) ZFS. Splitting of the peaks is observed, but this splitting is
not a result of any anisotropy in the system. The central g value of the complex differs
slightly from that of the undecane (tacud) complex, showing a decrease from 2.0013 to
2.0008. This difference is observable at high field, but not necessarily informative. The
hyperfine also decreases from 244 MHz for tacud to 233 MHz for tacd, which suggests
that electrons are less strongly coupled to the manganese ion, which would be expected
if the orbital is elongated. The space group of the complex shifts to P21/c, but the mirror
plane is retained, resulting in preservation of the 180⁰ angles across the metal ion.
When observing the entire field range it is readily apparent that this spectrum is
significantly different compared with that of the undecane macrocycle. While that
spectrum had strong overlap between the ZFS-influenced high spin manifolds, in this
complex the peaks are well separated. The simulation predicts two high field peaks,
108
which match well with the inflection points of the highly broadened high field peak
observed in the experimental spectrum. The bounds for the ZFS value are
predominately simulated based on the low-field peaks, which match very well. An
anisotropic broadening term could be incorporated to add a low-field Gaussian
broadening to account for the difference in peak height and specific broadening could
combine the high field peaks, however this would be hard to justify physically.
Mn(tacn)2
The final complex in this series, and the most geometrically strained, is the
cyclononane complex. It represents the minimum number of carbons between each
nitrogen that will still take on a tridentate chelating mode. This complex becomes
strained enough that the 180⁰ angles across the metal ion are distorted and the mirror
plane that was inherent in the other two complexes is lost, resulting in an orthorhombic
system in the Pbca space group. The increased complexity of the g=2 region is, again,
a result of the slightly larger ZFS, increasing from -2470 MHz for the cyclodecane to -
2650 MHz for the cyclononane ligand (Figure 3-12). The field range is higher on this
complex but this is not due to a large g factor shift. Instead, data collection gave better
lineshapes and less dispersion at 214.4 GHz than 208 GHz. The g factor decreases
slightly from the tacd complex, from 2.0008 to 2.0003. This is still within the generally
observed range for manganese (II) complexes. The hyperfine coupling, again, sees a
small decrease from the previous complex from 233 MHz to 223 MHz. This change is
on the order of 3.5 Gauss.
The high field spectrum, shown in Figure 3-13 shows the stronger effect of the
constrained geometry on the ZFS of the complex. The separation between the
observed high field peaks increase even further, resulting in a total field range of 800
109
mT. High spin manifolds are resolved in terms of location, but the hyperfine interaction
is still broadened out, as previously observed in the other two complexes in the series.
Simulation parameters for all complexes are presented in Table 3-2. When comparing
the parameters side-by-side, a clear trend emerges. All parameters decrease as the
strain in the system increases. The zero-field splitting increases in magnitude but
retains the same sign as the rest of the series, but curiously the g factor and hyperfine
interactions both decrease. A decrease in the hyperfine indicates weaker coupling
between the nuclei and the electrons, which makes logical sense with the greater
elongation of the orbitals aligned with the D tensor. The D strain parameter is relatively
consistent across all of the samples and represents a Gaussian distribution about the
principal ZFS value in order to simulate broadening in liquid samples, as simple peak-
to-peak Gaussian broadening is insufficient to describe higher spin species (S > 1/2).
The upper limit before spectral distortion is observed in the simulation is less than 100
MHz in the E parameter for both the undecane and cyclononane macrocycle, however
the cyclodecane warrants a higher E term to obtain a good fit. This indicates that the
undecane and cyclononane are truly axial, as introduction of a meaningful E results in a
worse fit.
Due to the fact that these complexes exist as a series with similar coordination
environments, the series can be analyzed as a whole to determine which elements
display the highest degree of change. While it is not generally possible to determine the
orientation of the D-tensor in anything except a single crystal of a complex, usually
doped into a diamagnetic matrix, knowing all of the bond distances and angles allows
principal component analysis to be performed, identifying the variables with maximum
110
variability. The D tensor is not generally aligned to the crystallographic axis. Analysis
of structural components and correlation with the ZFS tensor can yield information
about what changes most between each complex and which features seem to be
retained. Bonds or angles that show maximum variance in the complexes can be
assigned as the main contributors to increasing ZFS, as fixed components should
contribute the same in each complex. The bond distances and angles used are
presented in Table 3-4. Figure 3-14 shows PCA of the bonds in each complex.
Variables are defined as the distance between the labeled atom and the manganese
ion. In cases of mirror symmetry (tacud and tacd) N4, N5, and N6 are defined as being
opposite N1, N2 and N3, respectively, in order to maintain bonds in an order that allows
for PCA to be performed.
Once the data is transformed into the principal component axes and plotted,
several bonds become immediately apparent as strong contributors. N4-Mn shows the
strongest effect, encompassing nearly all variation in principal component 1. Other
bonds show variable effects in component 2, with the highest contributors being N3-Mn
and the symmetry related N6-Mn. Principal component 1 for bonds accounts for 91% of
the variance and the combination of the two components accounts for 100% of the
variance. Therefore, N4-Mn is expected to have the strongest impact on the zero-field
splitting.
However, bonds are not the only possible observable that can be used to provide
information about the ZFS tensor. Electronic orbitals are sensitive to both bond
distances and angles. Therefore, verifying that the N4-Mn bond is still contributing
strongly to the angular variation serves as additional confirmation that it would be
111
relevant to the ZFS tensor. When PCA is performed on all angles of the complexes
through the metal ion, with the same naming considerations in place, N4 continues to
be a strong contributor, providing strong contributions to variance in componant one
through the N3-Mn-N4 angle and nearly all of the component 2 contributions through
N2-Mn-N4. Angles involving N2 also show strong contributions in component 1,
including N2-Mn-N3 and the symmetry related N2-Mn-N6.
MnLCl
Recent work by Pawlak et al. has yielded an oxalate oxidase (OxOx) model
complex sharing the 3N-1O coordination environment native to both OxOx and oxalate
decarboxylase (OxDC).146 It is capable of performing oxidase chemistry, facilitating the
conversion of oxalic acid into hydrogen peroxide and carbon dioxide while consuming
one equivalent of dioxygen. Characterization of the reaction product and tuning of the
reaction parameters revealed that in order for the complex to produce the expected
equivalents of reaction product acidified conditions were required, as in OxOx.[10,11,14]
Before introduction of oxygen to the sample, it remained colorless but upon exposure it
quickly took on a dark pink color. This color change was correlated with the loss in
Mn(II) content as observed by perpendicular-mode EPR spectroscopy. The
identification of the product was not possible but Mn(III) and Mn(IV) were proposed as
possible species due to oxidation being the most likely result of oxygen exposure.
Due to characteristically high zero-field splitting values, Mn(III)-containing systems are
not generally accessible using traditional X-band EPR. Instead, high field/high
frequency spectrometers or specialized parallel mode resonators have to be used to
detect and characterize Mn(III).30,48
112
Parallel-mode EPR at X-band has been successfully used for the investigation of
integer spin species that are considered 'EPR silent'.44,48,147,148 At low fields, the mixing
of ms = ±2 states makes the observation of Δms = ± 4 transitions near g ≈ 9 partially
allowed.41,149
Mn(III) with the 3N-1O coordination motif has been proposed to play a key role in
oxalate degradation in both OxOx and OxDC. Therefore, further investigation into this
model complex was of interest in order to determine whether Mn(III) was indeed the
active species and what interaction this species has with the oxalate substrate during
the catalytic cycle.
The MnLCl (L=1-benzyl-4-acetato-1,4,7-triazacyclononane) complex exhibits the
3N-1O coordination motif observed in OxOx.16,20 In the protein the Mn is coordinated by
3 histidine ligands and one glutamate. The model complex instead uses a hindered
triazacyclononane ring with a carboxylate substitution to provide the required
coordination in a non-axial fashion (Figure 3-16).
This enforced geometry faithfully reproduces the relative coordination of the
protein active site. The previously proposed catalytic cycle for the MnLCl system
suggests that the ligand stabilizes the transition state, a μ-oxygen bridged dimer, upon
oxygen exposure resulting in an increase in the initial rate as compared to MnCl2 in
solution.146 As ligands are well known to modulate redox potentials, this impact should
be seen in the local environment of the electron cloud.
The catalytic cycle for OxOx requires both a source of dioxygen and of protons to
carry the reaction to completion and generate two equivalents of carbon dioxide and
one equivalent of hydrogen peroxide. In the absence of a proton source, only one
113
equivalent of carbon dioxide is produced. It was reported that before addition of HCl
only 0.7 eq. of CO2 per oxalate molecule was generated.146 However, upon acidification
of the solution additional CO2 was produced, up to two equivalents per oxalate.
Reaction with an excess of bis-(tetrabutylammonium) oxalate (TBAO) still generated
two molar equivalents of CO2 per molecule TBAO, confirming that the complex is
catalytically active.
Pawlak previously demonstrated that in the anaerobic form of the model complex
a Mn(II) signal is observed at g=2. This signal was recorded at X-band at 77 K. Upon
exposure to oxygen, a color change was observed in the complex and the clear,
colorless complex changed very quickly to a dark pink. This color change was
correlated with a loss in Mn(II) signal, and within 100 minutes the Mn(II) signal was
undetectable. This suggests either that the signal intensity was too weak for the
detection system or that an 'EPR silent' species was generated. Weak signals can be
observed more readily at liquid helium temperatures due to the enhanced spin
polarization.
Parallel-mode EPR was used to identify the loss of perpendicular-mode signal
previously observed for the MnLCl complex upon exposure to oxygen.146 Mn(III)
complexes often have very large zero-field splitting parameters due to the uneven
distribution of electrons in the d-manifold whereas Mn(II) in its high spin configuration
allows for maximum electron separation, as the d5 system can utilize each of the d
orbitals resulting in a much more symmetric electronic distribution and usually much
smaller fine structure. Upon oxidation, the high spin configuration is maintained,
resulting in a global spin of S=2. This S=2 state retains EPR activity, but the absence of
114
the so-called Kramer's doublets (for half-integer spin) may lead to ‘EPR silent’ species
when the ZFS is larger than the microwave photon energy. This can be overcome by
moving to higher microwave frequencies in combination with higher magnetic fields in
order to approach the high-field limit wherever possible. However, Mn(III) complexes
often show a large zero field splitting strain which broadens the expected spectra and
makes them difficult to observe even at high fields. Mn(III) in rhombic environment (E ≠
0) can often be observed in X-band using a parallel-mode cavity since under low field
conditions the spin states are mixed allowing for the detection of a ∆mS = ±4 transition
near g ≈ 9 when the parameter 3E2/D is smaller than the microwave photon energy.
Under anaerobic conditions the spectrum of the model complex MnLCl is
dominated by the characteristic Mn(II) sextet at g=2 near 3500 G (Figure 3-17).
Transitions between higher spin manifolds are not well resolved. The monotonously
decaying shoulders at low and high field suggest the presence of only a small zero field
splitting. The high field spectrum shows a simple sextet (Figure 3-19) with no additional
splittings and no peaks appear outside the central g=2 region, also suggestive of a
small zero field splitting parameter (ZFS). The absence of additional splittings in this
spectrum also demonstrates the absence of appreciable g-anisotropy (Figure 3-19).
The characteristic sextet hyperfine splitting exists in all paramagnetic manganese
complexes as a result of the electronic spin coupling to the nuclear spin of the only
stable isotope, 55Mn, which is present at 100% natural abundance and has a nuclear
spin of 5/2. Typical Mn(II) hyperfine splitting constants generally range between 200
and 275 MHz, with lower values corresponding to weaker coupling to the manganese
center.[22–26] Mn(II) in MnLCl exhibits hyperfine splitting on the order of 250 MHz, which
115
is on the higher end of the range and indicates that the electrons in the system are not
much delocalized into the surrounding ligands.
MnLCl is not soluble in acetonitrile:toluene without the addition of TBAO.
Therefore direct comparison cannot be made before and after oxalate addition in the
same solvent system to monitor ligand perturbation via oxalate. However, the signal
observed is consistent with an octahedrally coordinated Mn(II), indicating that
coordination is likely not affected by the solvent. MnLCl displays a singlet line in powder
form due to rapid spin exchange, as is commonly observed in magnetically
concentrated samples, such as the previous manganese metal-organic framework
shown in Figure 3-4.
Upon introduction of oxygen to the sample the Mn(II) signal in perpendicular-
mode EPR (Figure 3-17) decreases drastically and a Mn(III) signal (Figure 3-18) is
observed in the low-field region of the parallel mode spectrum at ~800 Gauss. This
signal is often observed for high spin Mn(III) in octahedral coordination environments.150
It displays the typical manganese sextet hyperfine splitting. Measuring 100 MHz the
magnitude of the hyperfine coupling constant is significantly smaller than for Mn(II) but
is larger than what is observed for manganese superoxide dismutase--one of the few
Mn-proteins with similar coordination environment to OxOx whose Mn(III) signals have
been characterized by parallel-mode.
Simulation of the low field Mn(III) (Figure 3-20) signal reveals that it is
spectroscopically similar to protein-based Mn(III) previously reported in the literature,
including Mn Superoxide Dismutase,150 OxOx,20 and OxDC.35 The ratio of E/D defines
the ratio of the rhombic portion of the zero field splitting parameter with respect to the
116
axial component. This term can be determined at X-band through simulation and
reveals information about the relative symmetry of a system. An axially symmetric
system is defined as Dx=Dy≠Dz. This is often observed in heme systems, where metal
coordination by the heme is planar, resulting in all four horizontal coordination sites
occupied by nitrogen and axial sites offering varied coordination. A rhombic system is
defined as having no two components of the D tensor being equal. This rationale
applies to other Hamiltonian tensors as well. The ratio of E/D has a limit, where 1/3 is
defined as the maximum degree of rhombicity or strongest distortion from octahedral
symmetry.
The E/D value obtained by simulating the spectrum suggests a moderately
distorted octahedral geometry. Carboxylate ligands are well known to lower the redox
potential of a metal system.100 Electron transfer from the manganese to bound oxygen
results in the S=2 Mn(III) signal along with a superoxide radical, which is left to diffuse
away in solution. Upon oxygen binding, electron abstraction is predicted if the standard
potential of the oxygen/superoxide couple is equal or higher than that of the
Mn(II)/Mn(III) couple. Mn(II) does not spontaneously oxidize to Mn(III) in solution, so
the ligand, along with oxalate itself, must lower its reduction potential to make this
transition possible. After gaining an extra electron, oxygen becomes the highly reactive
superoxide species, which will abstract protons from nearby molecules, whether it be
solvent or otherwise. Superoxide will further disproportionate to generate the hydrogen
peroxide end product of catalysis. This leaves the Mn(III) species as the driving oxidant
to break down oxalate in solution, resulting in the two carbon dioxide molecules that
complete the catalytic reaction.
117
When placed into an octahedral ligand field, the 5D ground state term of the
Mn(III) ion splits into the predicted 5T2g and 5Eg terms. With a high spin configuration,
equal population is predicted in the three T2g orbitals, precluding a Jahn-Teller
distortion. This is not true for the E2g orbital pair consisting of the dx2-y2 and dz
2 atomic
orbitals. The S=2 total spin introduces the expected Jahn-Teller distortion, which in
combination with spin-orbit coupling will break the degeneracy of the E2g state into a
5A1g and 5B1g state, as predicted by character tables for conversion from Oh symmetry to
D4h. This conversion is a result of elongation or contraction of the axial bonds along the
z-axis and can be observed by the corresponding rise in the magnitude of the zero-field
splitting parameter.48
ZFS parameters for Mn(III) complexes are overwhelmingly negative, with very
few examples in the literature carrying a positive sign (Table 3-7) and the majority
carrying a ZFS value between -2 and -4 cm-1. Mn(III) complexes are observed across
the entire range of E/D.
Magnetic parameters for OxDC (Table 3-5) show similar magnitudes to that of
the MnLCl complex, although the change upon oxidation is not nearly as drastic.30 The
semi-rigid protein backbone in OxDC would mitigate major changes in the local
environment and preserve the activity of the enzyme. This is corroborated by the higher
rate of reaction for the enzyme versus the MnLCl-oxalate complex. The magnitude of
the hyperfine interaction is almost identical both in the S = 5/2 Mn(II) state and the S=2
Mn(III) state, indicating that the manganese electrons are still well localized and the
same axial distortion is observed in the zero-field splitting parameter. The magnetic
118
similarity observed between Mn(III) species in OxDC and MnLCl indicates the
coordination environment between the two is similar.
This system is the first model complex for OxOx that effectively catalyzes oxalate
oxidation and it is therefore of great interest to compare its mechanism with that of
OxOx. However, even OxDC has a minor oxidase pathway and the present data may
allow meaningful insight into parts of the OxDC mechanism. OxDC shares the same 3-
histidine-1-glutamate coordination environment with OxOx. The proposed mechanisms
for the two proteins are identical for the first two steps, a one-electron oxidation of the
oxalate mono-anion followed by CO2 abstraction which generates a CO2⸱− radical anion
intermediate.151,152 This radical is further oxidized by superoxide which had been
generated before or directly by dioxygen to produce a second equivalent of CO2. This
last step can happen through inner or outer sphere electron transfer and is therefore not
dependent on the presence of the complex which has to be re-oxidized to Mn(III) by
dioxygen. In OxOx an inner sphere electron transfer is quite likely since both the
superoxide and the CO2− radical anion may be bound in the same active site. In the
case of OxDC the predominant reaction is a decarboxylation which requires the
reduction and protonation of the CO2− radical anion. A second Mn(II) center in the
OxDC monomer which is currently proposed to fill only a strictly structural role may
actually participate more actively in this process by storing the extra electron needed for
the final reduction reaction.123
Despite the identical coordination environment of OxOx and OxDC, oxidase
chemistry is favored for this mono-nuclear complex. There is only one place for
dioxygen to bind and abstract an electron. In the presence of substrate this generates
119
the two reactive intermediates—the carbon dioxide radical anion and superoxide
radical—in close enough proximity to react with each other and leading down the
oxidase pathway.
The slow rate of reaction of this complex compared with the native OxOx might
allow for the detection of an intermediate oxygen bound species and may be prepared
using rapid freeze-quench techniques. Comparison between the Mn(III) signals
observed for each of these systems could yield structural or spectroscopic information
explaining how OxDC can use the same active site geometry to yield such different
chemistry.
Conclusions
Mn and Cd(pyim)(oxalate)2
The pyim(oxalate)2 system enforces a bidentate binding orientation for oxalate.
When only the manganese form is considered, no spectral information can be
recovered due to Heisenberg spin exchange both within and across chains. When
manganese is doped into the cadmium analogue, the magnetic parameters are
recovered and spectral splitting is observed. As the geometry of the oxalate is well-
resolved and symmetric in a side-on bidentate mode, this complex has potential to
serve as a model for ENDOR experiments.
Mn(tacn)2, Mn(tacd)2, Mn(tacud)2
The series of Mn based triazamacrocylces studied show a strong correlation
between structural rigidity of the ligand ring and the magnetic ZFS parameter identified
via HFEPR. The smallest and most strained system showed an axial ZFS of -2650
MHz, which decreased with each carbon added to the ring. The least rigid system,
1,4,7-triazacycloundecane, reduced the magnitude of this parameter to only -930 MHz.
120
Given that the only difference in these systems is the number of carbons, PCA was
performed to identify the location of the D tensor, which is traditionally only accessible
via single crystal rotation.
MnLCl
Mn(III) was identified and characterized as being a reaction intermediate during
the breakdown of oxalic acid catalyzed by MnLCl. The species was characterized via
X-band parallel-mode and high field EPR. Spectral parameters obtained via X-band
simulation are as follows: g = 2, A = 150 MHz, D = 96 GHz, E = 10.7 GHz. These values
fall well within the range observed for Mn(III) in oxalate decarboxylase suggesting a
similar coordination environment. The MnLCl model system is to our knowledge the first
3N-1O model complex that shows oxalate oxidase activity. The reaction is driven by
Mn(III) which is generated by dissolved dioxygen in the presence of substrate. This
appears to follow a similar reaction pathway as found in OxOx and OxDC.
121
Table 3-1. Crystal Data and Structure Refinement for Mn(pyim)oxalate2 and Cd(pyim)oxalate2.
Identification code 1 2
Empirical formula C10H11MnN3O5 C10H9CdN3O5
Formula weight 308.16 363.6
Temperature 100(2) K 100(2) K
Wavelength 0.71073 Å 0.71073 Å
Crystal system Orthorhombic Monoclinic
Space group P b c a P 21/c
Unit cell dimensions a = 9.2321(7) Å, α= 90° a = 8.4698(3) Å, α= 90°
b = 15.1953(12) Å, β= 90°
b = 18.4304(6) Å, β= 102.0044(6)°
c = 19.2432(14) Å, γ = 90° c = 7.7892(3) Å, γ = 90°
Volume 2699.5(4) Å3 1189.32(7) Å
3
Z 8 4
Density (calculated) 1.516 Mg/m3 2.031 Mg/m
3
Absorption coefficient 0.997 mm-1
1.857 mm-1
F(000) 1256 712
Crystal size 0.386 x 0.144 x 0.036 mm3 0.299 x 0.212 x 0.085 mm
3
Theta range for data collection 2.117 to 24.998°. 2.210 to 27.494°.
Index ranges -10≤h≤10, -18≤k≤18, -22≤l≤22
-11≤h≤11, -23≤k≤23, -10≤l≤10
Reflections collected 28920 22265
Independent reflections 2372 [R(int) = 0.0486] 2726 [R(int) = 0.0155] Completeness to theta = 25.242° 97.30% 99.90%
Absorption correction Analytical Analytical Max. and min. transmission 0.2707 and 0.2302 0.9075 and 0.7190
Refinement method Full-matrix least-squares on F2
Full-matrix least-squares on F2
Data / restraints / parameters 2372 / 0 / 205 2726 / 0 / 189
Goodness-of-fit on F2 1.272 1.166 Final R indices [I>2sigma(I)]
R1 = 0.0730, wR2 = 0.1523 [1875]
R1 = 0.0164, wR2 = 0.0411 [2671]
R indices (all data) R1 = 0.0926, wR2 = 0.1632 R1 = 0.0167, wR2 = 0.0413
Extinction coefficient n/a n/a
Largest diff. peak and hole 0.567 and -0.973 e.Å-3
0.578 and -0.263 e.Å-3
122
Table 3-2. Simulated EPR parameters. All parameters were fit manually using EasySpin.
Parameter Simulated value
g 2 A 245 MHz
D -2300 MHz (0.077 cm-1)
E 150 MHz (0.005 cm-1)
E/D 0.065
Table 3-3. Simulation parameters for Mn macrocycles
Simulation parameter
Mn(tacud)2 Mn(tacd)2 Mn(tacn)2
g 2.0013 2.0008 2.0003
A [MHz] 244 233 223
D [MHz] -930 -2470 -2650
E [MHz] <100 300 <100
Dstrain [MHz] 600 600 500
123
Table 3-4. Crystallographic distances for coordinating residues in Mn(tacud)2, Mn(tacd)2, and Mn(tacn)2
Distances
Mn(tacud)2
Mn(1)N(1) 2.2894(10) Mn(1)N(3) 2.3011(13)
Mn(1)N(2) 2.2846(10)
Mn(tacd)2
Mn(1)N(1) 2.2626(12) Mn(1)N(3) 2.2336(12)
Mn(1)N(2) 2.2744(12)
Mn(tacn)2
Mn(1)N(1) 2.2771(11) Mn(1)N(4) 2.2556(11)
Mn(1)N(2) 2.2701(10) Mn(1)N(5) 2.2636(10)
Mn(1)N(3) 2.2485(11) Mn(1)N(6) 2.2662(11)
124
Table 3-5. Crystallographic angles for coordinating residues in Mn(tacud)2, Mn(tacd)2, and Mn(tacn)2
Angles
Mn(tacud)2
N(1)Mn(1)N(2) 85.93(4) N(1)Mn(1)N(3A) 101.52(5)
N(1)Mn(1)N(2A) 94.07(4) N(2)Mn(1)N(3) 92.95(4)
N(1)Mn(1)N(3) 78.48(5) N(2A)Mn(1)N(3) 87.05(4)
Mn(tacd)2
N(1)Mn(1)N(2) 83.74(4) N(1)Mn(1)N(3A) 99.96(4)
N(1)Mn(1)N(2A) 96.26(4) N(2)Mn(1)N(3) 79.03(4)
N(1)Mn(1)N(3) 80.04(4) N(2A)Mn(1)N(3) 100.97(4)
Mn(tacn)2
N(1)Mn(1)N(2) 77.64(4) N(2)Mn(1)N(6) 101.12(4)
N(1)Mn(1)N(3) 77.76(4) N(3)Mn(1)N(4) 101.18(4)
N(1)Mn(1)N(4) 173.63(4) N(3)Mn(1)N(5) 101.96(4)
N(1)Mn(1)N(5) 95.54(4) N(3)Mn(1)N(6) 178.41(4)
N(1)Mn(1)N(6) 103.88(4) N(4)Mn(1)N(5) 78.52(4)
N(2)Mn(1)N(3) 78.75(4) N(4)Mn(1)N(6) 77.34(4)
N(2)Mn(1)N(4) 108.38(4) N(5)Mn(1)N(6) 78.37(4)
N(2)Mn(1)N(5) 172.89(4)
125
Table 3-6. Simulated EPR parameters for MnLCl in Perpendicular and Parallel Mode
State S g A [MHz] |D| [GHz] |D| [cm-1] E/D
As prepared
5/2 2 240 <0.1 - -
Oxidized 2 2 150 96 3.21 0.111
OxDC 5/2 2 249 1.340 0.0447 0.17
OxDC 2 2 140 (-)71.3 (-)2.38 0.13
126
Table 3-7. Simulated EPR Parameters for Various Mn(III) Complexes Determined Using High Field EPR.
Compound gx gy gz D [cm-1
] E [cm-1
] E/D
[Mn(III)F3(H2O)(2,2'-bipyridyl]·2 H2O153
1.996 1.996 2.005 -4.253 0.493 0.1159
[Mn(III)F3(H2O)(1,10'-phenanthroline]153
1.965 1.965 1.984 -4.035 0.182 0.04511
[Mn(III)F3(H2O)(4,4-dimethyl-2,2'-dipyridyl]
153
1.986 1.986 2.005 -3.90 1.202 0.3082
[Mn(III)F3(H2O)(5,5-dimethyl-2,2'-dipyridyl]·0.5 MeOH
153
2.007 2.007 2.006 -4.046 0.223 0.0551
Tetramethylethylenediaminetrimethyl Mn(III)
154
2.0042 2.0042
2 -21784 0 0
Mn(5,10,15,20-tetraphenylporphyrinato)Cl
155
1.882 1.882 -2.271 0 0
Mn(2,3,7,8,12,13,17,18-octakis(dimethylamino) porphyrazinato)Cl
155
1.984 1.984 -2.331 0 0
Mn(2,3,7,8,12,13,17,18-octakis(dimethylamino)porphyrazinato) diethyldithiocarbamato
155
1.983 1.983 -2.621 0 0
Mn( 1,4,8,11-tetraazacyclotetradecane)2
156
2 2 1.99 +0.604 0.034 0.0563
(tetraphenylporphyrinato) manganese(III) chloride
157
1.982 1.982 2.005 -2.2905 0 0
(phthalocyanato)manganese(III) chloride
157
2.02 2.02 2.005 -2.28 0 0
Mn(1,3 diphenyl 1,3 propanedione)3157
1.97 1.97 1.99 -4.35 0.26 0.060
{Mn3+
}TiO2 157
1.991 1.991 2.002 -3.41 0.1161 0.0340
Tris(2,4-pentanedionato)manganese(III)
158
1.991 1.991 1.991 -4.522 0.252 0.0557
Mn(5,10,15-tris(pentafluorophenyl)corrolyl) (triphenylphosphine oxide)
159
1.9804 1.9804
1.9944 -2.692 0.0303 0.0113
Mn(N,N-bis(2-pyridylmethyl-ethylamine))F3
160
1.96 1.96 1.98 -3.672 0.702 0.1912
Mn(N,N-bis(2-pyridylmethyl-ethylamine))N3
160
2.02 2.02 1.98 +3.501 0.82 0.2342
Mn(2,2‘:6‘,2‘ ‘-terpyridine)F3 160
1.972 1.972 1.96 -3.822 0.752 0.1968
127
Figure 3-1. Manganese pyim oxalate (A) and cadmium pyim oxalate (B).
Figure 3-2. Manganese(pyim)oxalate2 crystal packing orientations. (A) Chains aligned along crystallographic c axis and (B) aligned along crystallographic b axis.
Chains aligned along C Chains aligned along B
A B
A B
128
Figure 3-3. Cadmium(pyim)oxalate2 crystal packing orientations. (A) Chains aligned along crystallographic b axis and (B) chains aligned along crystallographic a axis.
Chains aligned along B Chains aligned along A
A B
129
Figure 3-4. Manganese(pyim)oxalate2 X-band EPR signal at 5K (purple dash). 0.3% Mn doped cadmium(pyim)oxalate2 X-band EPR signal at 5K (black trace).
130
Figure 3-5. 208 GHz High Field EPR of 0.3% Mn doped cadmium(pyim)oxalate2 collected at 5 K.
Figure 3-6. 208 GHz EPR collected at variable sample temperature. Population
increase is observed at the low-field position with deceasing temperature. Scaling factors are included in the legend of the plot and are applied to keep the most intense transition of each spectrum at the same relative intensity for comparative purposes.
131
Figure 3-7. Ligand geometries for Mn(tacn)2 (A), Mn(tacd)2 (B) and Mn(tacud)2 (C).
Crystallographic representations are presented below each ligand.
A B C
132
Figure 3-8. Mn(tacud)2 spectrum collected at 5K and 208GHz, focusing on the g=2 region. Experimental spectrum is presented in black with simulation presented in blue.
133
Figure 3-9. Mn(tacud)2 spectrum collected at 5K and 208GHz, encompassing all observed features. Experimental spectrum is presented in black with simulation presented in blue.
134
Figure 3-10. Mn(tacd)2 spectrum collected at 5K and 208GHz, focusing on the g=2 region. Experimental spectrum is presented in black with simulation presented in blue.
135
Figure 3-11. Mn(tacd)2 spectrum collected at 5K and 208GHz, encompassing all observed features. Experimental spectrum is presented in black with simulation presented in blue.
136
Figure 3-12. Mn(tacn)2 spectrum collected at 5K and 214.4GHz, focusing on the g=2 region. Experimental spectrum is presented in black with simulation presented in blue.
137
Figure 3-13. Mn(tacn)2 spectrum collected at 5K and 208GHz, encompassing all observed features. Experimental spectrum is presented in black with simulation presented in blue.
138
Figure 3-14. Principal Component Analysis based on the bond distances in the complexes for coordinating atoms.
139
Figure 3-15. Principal Component Analysis based on the angles in the complexes for
coordinating atoms.
140
Figure 3-16. Coordination environment of MnLCl
Figure 3-17. Perpendicular mode EPR spectrum of MnLCl with 20 eq. TBAO. Black line indicates spectrum acquired before exposure to ambient oxygen. Blue line indicates spectrum acquired 2 hours after oxygen exposure.
141
Figure 3-18. Parallel mode EPR spectrum of MnLCl with 20 eq. TBAO. Black line indicates spectrum acquired before exposure to ambient oxygen. Blue line indicates spectrum acquired 2 hours after oxygen exposure.
142
Figure 3-19. High field EPR spectrum of pre oxidized MnLCl at 208 GHz. The oxidized sample did not show enough signal intensity to be reliable, which is common for manganese (III) in solution.
143
Figure 3-20. Simulated parallel mode EPR signal observed for the oxidized complex. Black line represents experimental data, red represents simulation.
144
CHAPTER 4 DESIGN AND IMPLEMENTATION OF A RAPID FREEZE QUENCH FOR SHORT
TIMESCALE KINETICS AND REACTIVE SPECIES
Rapid Quenching Techniques
Quickly quenching chemical reactions in order to study both products and
intermediates has a long history in chemistry. Many assays rely on rapid quenching in
order to establish time points for use in kinetics. Several formats of reaction quenching
exist, whether it be a drastic pH change that moves a process outside of its functional
range, adding an additional reagent that competes with the original process at a higher
rate to remove excess material, or quenching a reaction by stopping the movement of
the atoms entirely by freezing a solution.161 Each of these processes has its own
timescales, advantages and issues. Table 4-1 presents information on different
timescales available based on technique, along with some of the limitations of each.
Time scales presented are minimum time scales.
Chemical quenching is the most prevalent form of reaction quenching. This
process stalls a reaction by drastically changing the conditions, however, this technique
is diffusion limited. Therefore, dependent on the solvent viscosity, the quenching time
becomes variable. In addition, if the active species is not solvent accessible it becomes
even more difficult to estimate a time scale. Many chemical quenching reagents are
available, from strong acids and bases to irreversible inhibitors to oxidants and
reductants. Chemical quenches introduce more reagents into the reaction, which allows
for unwanted and complicating interactions. Additionally, if the reaction products or
intermediates are transient or highly reactive, employing this format of quenching could
destroy the objective of the study. Therefore, careful consideration must be given to the
reagent to be used to ensure it will not participate in unexpected chemistry.
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Often, the first thought for cryogenic quenching is to simply immerse a sample
tube in liquid nitrogen with the idea that the sample will freeze very quickly and therefore
prevent further chemistry. For low time resolution applications or non-reactive systems
this approach is valid. Nitrogen is a poor conductor of thermal energy, with a thermal
conductivity of 139.6 mW/(m∙K), and inherently has a comparatively specific heat of
2.04 kJ/(kg∙K).162 This can be compared to water, with corresponding thermal
conductivity of 591 mW/(m∙K) at room temperature and a specific heat of 4.184
kJ/(kg∙K).163 A higher coefficient of thermal conductivity allows for faster heat transfer
between the medium and a higher specific heat increases the total amount of energy
the reservoir can absorb without losing the ability to cool the sample. Nitrogen suffers
from the classic 'Nitrogen glove effect' or 'Leidenfrost effect' due to a disadvantageous
combination of these two properties, resulting in an insulating barrier of gasses forming
between the sample and the solution as the temperature difference between LN2 and
room temperature is extreme. The onset of this temperature range for N2 is 126 K,
therefore at the boiling point of 77 K, this effect is always observed.164
In order to overcome the poor thermal conductivity of LN2, an alternative cryogen
should be utilized. Isopentane is often used for this purpose, as its melting point is 112
K. It is used commonly in tissue preparations, as the flash cooling of isopentane
prevents ice crystal build-up.165–167 While this means that isopentane will freeze solid if
left in contact with liquid nitrogen for a long enough period of time, the thermal
conductivity at liquid nitrogen temperatures approaches 180 mW/(m∙K) and isopentane
does not demonstrate the Leidenfrost effect in that regime.168 Regardless of the solvent
used to quench, the material and diameter of the sample tube itself also contributes to
146
the quenching time. Isopentane complicates this, as introduction of a tube into
supercooled isopentane will generate an isopentane film on the outside of the tube,
increasing its diameter. Therefore, utilizing 5 mm sample tubes— common for
cryogenic EPR applications—results in isopentane residue left on the outside of the
tubes, causing them to freeze inside the cryostat. Alternatively, instead of using a
sample inside a tube, one can spray the solution directly into the isopentane. This
results in a much faster freezing time and allows for interfacing of a microfluidic mixer or
grid mixer, which also drops the time required considerably as compared to manual
pipetting followed by freezing. However, using an isopentane spray approach carries its
own share of problems, as the solution becomes dispersed along the top of the
isopentane solution, meaning that the sample must then be collected from the top of a
moving liquid. In addition, isopentane is very flammable and small particles are more
likely to build up a static charge, which also makes them more difficult to insert into a
sample tube. Therefore, while the timescale approaches the important range to see
reaction intermediates for many biological processes, this technique is not sufficient for
even medium-throughput applications.
Based on the reaction time for OxDC, with a turnover rate of 50 s-1, the
quenching time necessary for this experiment must be less than 20 ms in order to
investigate the pre-steady state reaction.23 Therefore a different technology must be
employed to successfully catch this reaction in the act of turnover.
Shorter timescales are accessible using specialized equipment that eschews the
need for an isopentane bath entirely. None of these systems are currently commercially
available and instead must be built. The two types of rapid freeze quench (RFQ)
147
devices that appear in the literature are a wheel based RFQ, first reported by Gary
Gerfen at Albert Einstein College and a plate based RFQ, which was developed by
Daniella Goldfarb at the Weisman Institute.169–171 Each will be discussed, along with the
potential complications of each device.
In the wheel based RFQ, shown in Figure 4-1, two beryllium-copper wheels are
arranged in parallel with a very small spacing between them. Beryllium-copper is used
because it is much harder than pure copper and retains the vast majority of copper's
excellent thermal properties. These wheels are pre-cooled in liquid nitrogen and driven
by DC motors to rotate against each other, generating a crushing surface. These
wheels are partially immersed in a liquid nitrogen bath and remain in contact throughout
the experiment. The sample is mixed using a high pressure syringe ram, pushing the
two reactants through a mixing grid using very small diameter tubing. The output of the
mixer is connected to a variable length of tubing to change the dwell time of the
reaction, thus determining the time scale. Longer times are accessible by lengthening
the tubing. The output of the dwell or aging tubing is attached to an emitter nozzle,
which is made from a 22 Gauge stainless steel needle, which has been bent to be
parallel to the wheel surface and to which 25 100μm holes are added in order to
generate a larger surface area for the spray. The output is sprayed directly onto the
turning wheels, which then grind the sample to a fine powder. The powder is deposited
into the nitrogen bath, which can then be drained slowly and the sample can be
shepherded to a small collection cup. The area for sample collection is too small to be
able to use X-Band tubes, and is instead designed for use at high field, where Teflon
cups are the standard.
148
Sample collection is the main issue that plagues the wheel based RFQ.
Additionally, two DC motors are necessary to provide the ability to grind the sample, as
one motor by itself driving through a contact interaction will cause the interface of the
wheels to cant, allowing sample to fall straight through. This system would be very
difficult to adapt to X-Band use or extend to be viable at multiple field ranges.
Additionally, beryllium-copper is a highly toxic material, requiring the use of respirators
to machine. This system must also be constantly purged with dry nitrogen in order to
prevent ice build-up.
The plate style rapid freeze quench uses a PDMS based microfluidic mixer and a
similar syringe ram setup for delivering pressure to the system.169 However, instead of
using crushing wheels this system sprays from the outlet nozzle directly onto a
precooled aluminum disc. This disc is kept in contact with a liquid nitrogen bath.
Aluminum was used due to ease of machining, even though it lacks the specific heat of
copper. Aluminum does still retain good heat transfer properties, and therefore
outperforms isopentane. This device was designed such that the aluminum disc would
rotate, allowing for duplicate measurements in addition to being able to move the
emitter nozzle further away from the center of the disc, which allows the aging hose to
be swapped, resulting in the ability to generate several time-points of data at the same
time using small sample volumes. The entire assembly is kept inside a nitrogen gas
box in order to prevent moisture accumulation on the plate, which would effectively
dilute the sample. However, this system does not lead to easy collection of sample and
transfer to a tube. It suffers from the same issue as the wheel based RFQ in that the
operators are left to their own devices for loading a sample tube, making this system,
149
again, more useful for higher field measurements. Also, because the plate cannot be
removed from the nitrogen bath without jeopardizing the sample and scraping the
sample from the plate with a blade means that the blade would also have to be
maintained at nitrogen temperatures, else the entire experiment is in vain.
Each of these approaches has issues in terms of sample collection, reaction
stability or ability to alter the system for use under multifrequency conditions. The ideal
system must allow for various sizes of sample tubes, with a straightforward
methodology for sample collection and would be portable, in case the products are
difficult to characterize, so the measurements could be repeated at the National High
Magnetic Field Laboratory in Tallahassee.
Design of a Portable Rapid Freeze Quench
The first part of designing this apparatus was determining which methodology to
follow. While the wheel based system affords a finer powder, neither is particularly easy
to collect a sample from, and the plate based design has less moving components in
contact with LN2, likely resulting in a longer operational lifetime. Additionally, the
material choice is very important. Beryllium-copper is ideal for wheels that must
counter-rotate due to its hardness. Making a wheel-based RFQ using aluminum wheels
would likely result in a nonfunctional system, as the wheels may deform. However,
copper shows better thermal properties than aluminum, therefore copper was deemed
the better material. However, copper oxidizes over time and copper oxide leaching into
the sample results in a paramagnetic impurity in a region that eclipses the entire
expected signal. Oxygen-Free Copper (C10100) has an internal limit of 0.5 parts per
thousand residual oxygen, meaning the only copper oxide impurities would be surface
oxidation based. This is easily remedied by polishing all copper pieces with ultra-fine
150
sandpaper before operation of the equipment. Once brought to temperature, it is
important to maintain a low oxygen environment, therefore a nitrogen purge gas must
be introduced. Cold nitrogen is ideal, as it will prevent surface warming of the sample in
addition to providing a buffer against ambient moisture.
Rotating Plate and LN2 Interface
In the end, a rotating plate RFQ approach was chosen. This was deemed a
more transportable solution, as the unit itself is more self-contained. The plate itself
was designed with a large conical section on the bottom, allowing for a large thermal
mass of copper to be used and a high surface area with the LN2 cooling space (Figure
4-2). This design allows for long time-frame operation to collect full data sets, as the
majority of the nitrogen will be used in pre-cooling the plate itself. Once the copper
plate reaches temperature it will require very little nitrogen to maintain cryogenic
temperatures. Additionally, this format allows for use of a single motor, whereas the
wheel based RFQ requires two.
The plate is surrounded by a custom build vacuum insulated liquid nitrogen
dewar. Many freeze-quench set-ups, like that depicted in Figure 4-1, utilize styrofoam
boxes as an insulating material due to their decent insulating properties combined with
how inexpensive they are. We decided to make a more permanent structure, as then
we could both control the volume, use it as a mounting piece for other parts in the
system, and retain better insulation by introducing a vacuum pump. Additionally,
styrofoam containers have a short lifetime with continuous nitrogen exposure and are
prone to absorbing liquid oxygen, making them hazardous as they are already
inherently flammable. The RFQ freeze plate is shown again in Figure 4-3 mounted
inside the custom dewar. The darker grey area represents the vacuum space, with the
151
outllet shown on the right hand side. In the completed RFQ this outlet goes to a three-
way valve with a vacuum gauge attached so the dewar pressure can be monitored.
In addition to maintaining the temperature of the freeze plate, the nitrogen dewar
provides an easy way to generate a cold nitrogen atmosphere. Stainless steel gas
tubing was fitted to the bottom of the nitrogen dewar, shown in Figure 4-3 (B) allowing
house nitrogen gas to be cooled using the existing LN2 reservoir. This addition allows
for cold nitrogen gas to be used as a purge gas and prevent moisture buildup on the
freeze plate. This also alleviates the concern that the purge gas could cause sample
melting.
Sample Tube Holder and Scraper
One of the major concerns with building this apparatus was that it should be
portable and adaptable to multifrequency conditions, in case further experiments
needed to be done in Tallahassee, and to expand the number of instruments it could be
interfaced to. For example, the sample holder needed to be removable, such that
multiple sizes could be made depending on the application. For X-band EPR, the most
relevant diameter is < 4mm, as this is the sample tube size that will fit into the ENDOR
resonator. Other resonators, such as the dual mode (Bruker 4116DM) or high-Q can
use 4 mm tubes without issue. The sample tube holder is made entirely of oxygen-free
copper, again, due to its superior heat transfer properties. The majority of the sample
holder remains in contact with liquid nitrogen at all times in order to ensure samples can
be preserved. Four holes are drilled, with scallops at the top to guide sample to the
tube. The bottom of each hole is slightly tapered in order to prevent sample tubes from
falling into the nitrogen reservoir when the sample holder is removed. The sample
152
holder is shown in Figure 4-4 in both solid model and wireframe, allowing the smooth-
bored holes to be visualized.
While the sample holder has 4 holes, only two sample tubes may be loaded at
one time. Due to the difficulty of sample collection using plate based RFQ
methodologies, a scraper was introduced into the design (Figure 4-5). The scraper
maintains contact with the freeze plate and serves to gather up the frozen powder
sample and funnel it towards the sample tubes. The freeze plate has a slight overlap
with the sample holder, mitigating the chance that the sample will merely fall onto the
vacuum shielded dewar and be lost. Once the sample is collected, it can be pushed
down into the tube using a pre-cooled plunger to achieve good packing. The scraper is
made of oxygen free copper and is mounted using 3 long copper pins. These pins are
in contact with liquid nitrogen so that the scraper does not have to rely solely on surface
contact with the freeze plate to maintain its temperature. Since the scraper is
removable, it can be pre-cooled in a large LN2 dewar before insertion into the RFQ
apparatus. The scraper also has an adjustment screw, shown in Figure 4-5 (A) as the
hold between the mounting pins. This allows for the scraper to be engaged and
disengaged, resulting in the ability to spray more sample if needed or interface
additional steps into the sample collection process. The scraper can be removed and
the sample holder rotated in order to collect duplicates of the same sample.
Figure 4-6 shows the interface of the scraper with the sample tube holder in
wireframe and the interface with the freeze plate. The scraper takes advantage of the
scalloped back of the sample tube holder to guide frozen sample into place without
coming into contact with anything warm and requiring minimal operator involvement.
153
Removable Crush Wheel
One of the principal advantages of using a wheel-based RFQ is that the powder
output from the system is well homogenized. Not wanting to lose this advantage, we
introduced a wheel component into this RFQ design. The wheel is removable, so that it
can be pre-cooled and not merely rely on contact with the freeze plate to maintain
temperature, and can be disengaged using a level. This allows the operator the choice
of using the crushing wheel or not. This wheel does not have its own motor and is
instead driven by the spinning of the freeze plate. Placement of the wheel near the
scraper affords a space on the other side of the scraper to spray sample. Having only
two points of contact with the holding bracket for the wheel minimizes heat loss, as the
surface area in contact with a cold source is much higher than that of the mounting pins.
Full Assembly
The entire assembly is surrounded by an aluminum frame, which allows for
mounting the motor that drives the wheel significantly above the interface (Figure 4-8).
This helps prevent the motor from freezing up when the plate cools, as the two are
connected through insulating spacers. This also allows for mounting of the crush wheel,
and power, and allows gas supply and vacuum ports on the back of the apparatus. .
The RFQ is operated by a single switch controlling the motor, as seen at the top of the
apparatus affixed to the motor housing. The mixer is also operated by a single switch.
The built RFQ apparatus is shown in Figure 4-9. The orange tubing shown was
added to diffuse the output of the cold nitrogen line so as to achieve even coverage
without blowing the test sample from the plate.
154
Mixing Station and Flow Control
A commercially available mixer was utilized, consisting of a Kollmorgen Motion
Technologies Servo Disc DC Motor coupled to an Update Instrument, Inc. Model 715
Syringe-Ram Controller. The DC motor is coupled to a threaded shaft, to which the
plungers are affixed. The plungers fit snugly into 1/4" thick quartz barrels with 6.5 mm
I.D. and 106.05 mm total length, which is where the solutions to be mixed are stored
before the experiment. The syringe-ram controller allows for the velocity of the plunger
to be varied from 0.8 to 8 cm/second and the displacement to be controlled, which
determines the amount of sample that will be pushed into the mixer system. Unlike the
microfluidic approaches to RFQ, this set-up as-is does not allow for sub 100-μL sample
sizes. However, other approaches were designed exclusively for W-band spectroscopy,
where the total sample volume cannot exceed 0.4 μL due to the inverse relationship
between resonator frequency and cavity size. X-band EPR allows for samples from
100-200 μL to comfortably sit inside the resonator cavity using either 4 or 5mm O.D.
sample tubes.
Tubing used for coupling the mixer to the syringe ram is 1/16" OD 0.03" ID
Polyetheretherketone (PEEK). The two hoses have matching lengths of 17" in order to
balance the pressure of the system, resulting in a dead volume pre-mixer of 63 μL due
to the volume of the connecting tubing. Aging hoses are made from 1/16" O.D., 0.015"
I.D. PTFE tubing, with lengths, calculated volumes, and aging times for a 4 cm/s ram
displacement velocity shown in Table 4-2.
The mixer used in this system is an Update Instruments, Inc. Wiskind grid-type
mixer, consisting of layered PEEK grids that introduce turbulence into the two mixing
streams.172 A microscope image of the mixer is shown in Figure 4-10. This mixer is
155
characterized by high efficiency and short mixing lengths and as such does not depend
on turbulent mixing inside the aging hose itself. Complete mixing is achieved inside the
mixer, and as such, a reliable start-time for the reaction can be established and the
Reynolds number in the reactor hose is unimportant.
Emitter Nozzle
For optimum performance, the output of the mixer must be coupled to an emitter,
which will reduce the average droplet size, increase surface area and result in faster
and more even freezing. The simplest emitter nozzle used is a solder-plugged 22-
gauge needle with several small diameter holes drilled into it, mounted radially above
the plate. 22 gauge is chosen because of its matching inner diameter with the peek
tubing used in the mixer output, resulting in dimensions of ID = 0.413 mm,
OD = 0.7176 mm.171 Use of the emitter nozzle increases the post-reactor path length,
and should therefore be avoided if absolutely minimal time points are necessary.
RFQ Mixer Testing
In order to ensure that the RFQ behaved as expected, a prototype reaction was
employed. A 500 μM 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) solution
was prepared in water and various amounts of an equimolar ascorbic acid solution was
added. These values ranged from 0% to 100% equivalence (0 to 500 μM) with the
remaining volume made up of water. This reaction requires thorough mixing. First, the
experiment was performed using manual pipetting and 30s mixing time in order to
establish a baseline for the experiment. EPR parameters were 9.40733 GHz
microwave frequency, 3348 + 250 G sweep width, 1024 points, 20 dB attenuation (2
mW power), 40 ms conversion time, 1 G modulation amplitude, 100 kHz modulation
frequency. These test experiments were performed on a Bruker ELEXSYS-II E500 X/Q
156
band EPR spectrometer fitted with an Oxford Instruments ESR900 liquid helium cryostat
and Bruker SHQE High-Q resonator. Initial cryostat temperature was maintained at 60
K in order to remain under the operating temperature of the RFQ (77 K). Reactions
were performed without a glassing agent, as addition of DMSO or glycerol would
change the viscosity of the system, increasing back-pressure in the syringe ram and
negatively impacting the mixing efficacy. The bimolecular rate of reaction for reduction
of TEMPOL by ascorbate in aqueous solution is 7 M-1s-1.173 This is of the same order of
magnitude as the unsubstituted reaction of TEMPO and ascorbate, which carries rate
constants of 2.4 + 0.3 M-1s-1 at neutral pH and 2.20 + 0.03 under acidic conditions.174,175
A plot of spectra acquired versus concentration of ascorbate in solution is shown as
Figure 4-11. The concentrations used are high enough to result in a one-line spectrum
centered at g=2, as the hyperfine interaction is lost in frozen solution. This is partially
due to the lack of a glassing agent, as inhomogeneous freezing will result in an
apparent concentration effect. These conditions at room temperature would still result
in a 3 line TEMPOL signal, which remains distinct up to ~5mM radical concentration. At
high reductant concentrations the beginning of the nitroxide hyperfine interaction is
observed, however with very low intensity.
Following this test reaction with manual pipetting, reactions were performed
using the RFQ mixer itself. 1 mM TEMPOL was loaded into the first syringe ram barrel
and 1 mM ascorbate was loaded on the opposing side. This reaction is particularly
suitable for mixer testing as the TEMPOL solution has a light yellow tint in this
concentration range and the ascorbate solution is clear. Therefore, the solution in each
ram can be checked after ejection to ensure that leaks were not present, as a leak on
157
one side of the ram will result in solution contamination as backpressure pushes the
contents of one barrel through the tubing to the second.
Before cooling to liquid nitrogen temperatures, the copper components of the
RFQ were polished using 600 grit sandpaper to remove the surface oxide layer.
Copper oxide is paramagnetic and would result in a strong g=2 signal that would eclipse
the predicted spectrum. Once polished, dry nitrogen gas flow was started in order to
mitigate the effects of ambient oxygen. The crush wheel was removed and cooled
separately in a shallow dewar. For the RFQ test experiments, the syringe ram velocity
was maintained at 4 cm/s and the length of tubing was varied from 265 mm to 1650
mm. This corresponds to a solution flow rate of 1.33 mL/s. As path length is the only
altered variable, it will be directly proportional to aging time and therefore total reaction
time. For a second-order kinetic process a sharp decay is expected. Once the system
was set up and the solutions were loaded, syringe ram displacement was set to 200 mm
and the output was collected as waste to clear the tubing lines of the system. This
corresponds to ~660 uL of solution produced, which is sufficient to clear all lengths of
tubing used, with the maximum dead volume of the system with 500 mm tubing ~60 μL.
Immediately following sample ejection the nozzle was aimed at the RFQ plate and
displacement was changed to 600 mm and the system was re-engaged. As the system
has a constant flow of cold nitrogen gas across the plate, haste is required as to not
freeze any residual solution on the nozzle output. This generates 2 mL of solution spray
— far more than enough to ensure packing of the sample tube. Minimal volumes were
not used for this test. Sample packing was achieved by allowing the crush wheel to
grind the frozen solution to a smaller particle size and the use of very fine tweezers to
158
push sample into the tube, followed by compacting with a pre-cooled stainless steel rod.
As the tube is not visible when inside the copper sleeve, packing could only be inferred
by mechanical resistance until the tube was removed. Sample tubes had the following
dimensions: 3.8 mm O.D., 2.39 mm I.D., 68.3 mm length and were machined from Kel-F
rods. Packed samples in these tubes were inserted in pre-cooled 5.0 mm O.D. 4.0 mm
I.D. quartz tubes and kept in a nitrogen dewar until insertion in the cryostat.
EPR parameters for this data set are as follows: 3348 + 250 Gauss field sweep,
1000 points, 5 K cryostat temperature, 1 scan, 1 G modulation amplitude, 100 kHz
modulation frequency, 9.40 GHz microwave frequency. 24 dB attenuation (0.7962 mW),
40 ms conversion time.
Figure 4-12 shows stacked plot spectra acquired with different aging times used
in this trial, with an inset showing the maximum intensity for each spectrum plotted
against the calculated aging time. It is clear that varying the aging time causes a
decrease in signal intensity, which is the expected result for a longer reaction time in
this system. As the solution concentrations remain constant over the course of the
experiment, signal decrease is a result of increased mixing time and not altered mixing
efficiency. Figure 4-13 takes the maximum intensity value for each peak in Figure 4-12
plotted against aging time. The trace shown is an exponential decay, with a fit value of
y = 1.869∙e^(-0.025576∙x)
In order to verify if the time-points are reproducible, an additional experiment was
performed using the same EPR acquisition parameters, but including variation of the
syringe ram speed. Data points were collected in triplicate. Accurate measurements
were performed for the mounting height of the emitter nozzle versus the RFQ plate to
159
determine absolute post-mixer quench times. Testing was done to determine the
minimum possible quench time for this system as-built without incorporating a different
mixer geometry. A threaded hose was used to connect the mixer output to the spray
nozzle, incorporating an unavoidable delay time based on hardware compatibility. The
calculated post-mixer time remains under 1 ms when using the highest ram
displacement speed, however a significant amount of variation appears when using this
speed at this shortest possible reaction time, suggesting that the system might not be
consistent at high ram displacement speeds. Figure 4-14 shows the RFQ reaction of
1 mM TEMPOL and 1 mM ascorbic acid with 90% confidence interval. Points were
generated by taking the peak-to-peak height of the EPR transition. The mean of each
set of data points is presented as a diamond marker and individual data points are
shown as triangles, with upward-pointing red triangles representing data collected using
8 cm/s displacement and downward-pointing blue triangles representing points collected
at 4 cm/s displacement. Analysis of this plot shows two anomalous points. First, at the
minimal mixing time one point is significantly higher in intensity. This could indicate a
leak on the ascorbate side of the mixer for this data point. The system does not
currently have a way to load sample without interacting with the seals at the top of the
syringe barrels. A weak-sealing o-ring combined with the high pressure derived from
the 8 cm/s ram speed used for that data point could result in solution leaking from the
bottom of the syringe barrel, as it relies on o-ring seals at both sides. This would
account for having a higher-than-predicted signal, as no TEMPOL would be reduced.
Indeed, this sample had a signal proportional to the pure TEMPOL stock solution. The
other anomalous point corresponds to the 18 ms time point. This data point was also
160
collected at 8 cm/s displacement speed. This suggests that either the system cannot
operate functionally at 8 cm/s, or that the drive speed calibration is incorrect. Further
testing is required to determine which of these two situations is the case. A different
mixer design, such as transitioning to a microfluidic assembly, might be a better
solution, as smaller sample volumes could be used and the even-smaller channel size
would not require the use of such fast displacement speeds.
Conclusions
A rapid freeze quench apparatus was designed, constructed and tested. This
system allows for very rapid cooling of the sample stream coming out of a syringe-ram
based mixer and is capable of low millisecond quenching times using fast syringe ram
displacement, however mixer constraints lead to variability in intensity at very short time
scales. The RFQ system is poised to perform ENDOR experiments using WT OxDC in
the active pH range, which will provide a definitive experimental answer as to the native
substrate binding orientation in the active enzyme.
161
Table 4-1. Timescales of Various Rapid Quench Techniques
Technique Time Scale Limitations
Chemical Quenching 5 ms 161 Stability of system, choice of quenching agent
LN2 ~s Cooling time
LN2 with isopentane bath ~500 ms Film of frozen isopentane increases tube diameter
LN2 with isopentane spray 5-30 ms 176,177 Sample volume, static
Rotating plate RFQ <5 ms 169 Sample collection and packing
Wheel based RFQ 50 μs 170 Poor interface for X-band sample tubes
Table 4-2. Reaction Time Added by Aging Hoses using a 4 cm/s ram displacement
velocity. Sample Calculations Provided in Appendix B.
Hose Label Length [in]
Length [mm]
Aging Hose Volume [uL] Aging Time [ms]
20 - 68.5 2.47 1.86
25 - 85.0 3.07 2.31
40 - 132.0 4.77 3.59
50 - 160.6 5.80 4.37
64 8.38 212.7 7.68 5.79
80 10.44 265.1 9.57 7.21
100 12.94 328.6 11.86 8.94
125 16.38 415.9 15.01 11.31
160 20.75 527.1 19.03 14.33
200 25.81 655.6 23.67 17.83
250 32.00 812.8 29.34 22.11
320 41.63 1057.3 38.17 28.76
400 51.81 1316.0 47.51 35.79
500 64.94 1649.4 59.54 44.86
162
Figure 4-1. Wheel Based RFQ. (A) Full assembly with motors. (B) Closer detail of wheel interface with base visible. Image provided by Professor Gary Gerfen.
Figure 4-2. RFQ Copper Freeze Plate (A) side and (B) top.
A B
A B
163
Figure 4-3. (A) RFQ freeze plate mounted in double-walled vacuum dewar. (B) RFQ plate with cold nitrogen line.
Figure 4-4. Sample holder, sized for 4 mm O.D. sample tubes. (A) solid assembly and
(B) wireframe, showing sample holes.
A B
A B
164
Figure 4-5. Sample collection scraper (A) from top and (B) from side, showing holding
pins that maintain nitrogen contact.
Figure 4-6. Scraper interface with sample tube holder (A) wireframe showing mounting
and nitrogen contact and (B) top view showing interface with freeze plate.
A B
A B
165
Figure 4-7. Removable crushing wheel with handle and mounting bracket.
166
Figure 4-8. (A) RFQ full assembly with insulating box and (B) RFQ full assembly wireframe.
A B
167
Figure 4-9. Photographs of completed RFQ. (A) Full assembly, (B) detail zoom of scraper and crush wheel, (C) detail of nitrogen line.
A B
C
168
Figure 4-10. Microscope image of the grid-type mixer used in the RFQ experiment.
Hole diameter shown in center is 1/16" (159 μm).
169
Figure 4-11. Prototype reaction for RFQ mixer testing. 500 μM TEMPOL is mixed with varying amounts of an equimolar ascorbic acid solution and diluted to a given final volume. Reduction of the radical species is observed, resulting in a decrease in the EPR signal.
170
Figure 4-12. RFQ reaction testing. 1 mM TEMPOL is loaded on one side of the syringe ram with 1mM ascorbate on the opposing side to give final concentrations at full mixing of 500 μM. The output distance from the grid mixer is changed by adding various lengths of tubing, increasing the dwell time of the reaction.
171
Figure 4-13. Exponential decay fit on the maximum intensity for each tubing length used in Figure 4-12. Fit equation 1.869∙e(-0.025576∙x).
172
Figure 4-14. RFQ reaction performed in triplicate using 1mM stock TEMPOL and Ascorbic acid solutions. Delay time was varied through the use of various lengths of aging hose in combination with variation of the ram speed. Error bars represent a 90% confidence interval. X-axis represents absolute time post-mixer accounting for air-distance. Blue triangles represent individual data points at 4cm/s displacement speed, red triangles represent data points collected at 8cm/s displacement speed and yellow diamond markers represent the mean of the collected data points.
173
Figure 4-15. Second Order kinetics plot of the RFQ test reaction. Slope of best fit line is
14.9 Conc-1s-1. Literature kinetics for TEMPOL reduction is 7 M-1s-1. Initial point for predicted kinetics from signal intensity of 200 μL of 500 μM TEMPOL and represents an upper limit.
174
CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS
Conclusions
Oxalate Decarboxylase catalyzes the heterolytic cleavage of the oxalic acid
carbon-carbon bond via a transition metal supported radical mechanism. DFT
calculations, shown in Chapter 2, suggest that electron transfer is more strongly
facilitated if oxalate, or any small carboxylate, binds to Mn(II) before oxidation. The
strongest effect was predicted for bidentate oxalate, which would preclude oxygen
coordination at the same metal center. Previous work has shed doubt on oxygen
coordination at the N-terminus by spin trapping studies.35 These calculations were
facilitated by a new low pH crystal structure of WT OxDC, allowing for the active site
geometry to be preserved. Two binding modes were identified and compared against
experimental ENDOR data, demonstrating that only the bidentate mode correctly
predicted the experimental data. Chapter 3 investigated the effects of geometry on the
magnetic parameters of Mn(II), suggesting that the environment of the buried active site
in OxDC is fairly rigid, as evidenced by its ZFS parameter, and that the coordination is
maintained as the Mn(III) form is generated. EPR studies on a catalytically active OxOx
model show very similar spectroscopic signals to those observed for OxDC, suggesting
that in either chemistry the active site does not change significantly, leaving the
mechanistic divergence of the two systems more likely related to the C-terminus metal
coordination site in OxDC. Chapter 4 detailed the design and construction of a RFQ
apparatus to investigate the binding mode experimentally in a catalytically active
system.
175
Future Directions
Building from this work, several options arise. First, the DFT calculated
potentials presented in Chapter 2 should be verified experimentally using protein film
voltammetry (PFV). While this technique is difficult, it has been applied successfully to
many systems, notably Cytochrome c and iron-sulfur cluster electron transfer
reactions.178–183 In general, a protein solution is coated onto a polished glassy carbon
electrode, either through dropping concentrated protein solution and drying or via
repeated dip-and-dry cycles. Not all proteins behave well with film formation, and no
guarantee can be made that the quaternary structure is preserved. Of particular interest
would be the difference in redox potential for the native enzyme and the enzyme with
the addition of substrate in order to determine if the strong potential drop predicted with
oxalate coordination is observed experimentally and the relative magnitude compared to
formate coordination.30
Performing molecular dynamics (MD) simulations would also be of interest for
this system. Many structural features have been identified that are proposed to have
catalytic importance or relevance, including the hydrogen bonding network around the
active site flexible lid, the π-stacked tryptophan dimer of W96 and W274, the
importance of R92, and even the wrapped interfaces between monomers for the trimer
structure and between trimers for the crystallographic hexamer. These simulations
could be performed with and without oxalate, resulting in another indicator of oxalate
binding orientation and a more real-time look at what impacts R92 has. Additionally,
given that three orientations of the flexible lid have been identified, seeing the transition
between these states could help to explain which states are most relevant and which
bonds control substrate access. MD simulations can also be performed replacing water
176
molecules at random with dioxygen, potentially showing where oxygen localized in the
protein.184 This includes oxygen trapped in hydrophobic pockets, which has been
suggested to be the source of the anaerobic burst kinetics that have been previously
observed, and oxygen coordination to metal centers.15 This could provide a more
concrete role for the C-terminus manganese, although in order to make a more
compelling argument oxygen simulations would need to be performed in the presence
and absence of oxalate in the modeled system. Due to the drastic increase in
performance and decrease in hardware cost, combined with the explosion of GPU
computing for these kinds of systems, this type of experiment has never been more
accessible. Using distributed computing resources or high enough powered cards,
microsecond MD timescales are achievable in reasonable time periods for even
moderately large systems, and the second timescale regime may be accessible within
the next 5-10 years.185–188 Oxygen diffusion MD simulations are not common, but are
certainly within the realm of possibility now.184
Spin trapping experiments have been successfully applied in this for identifying
radical intermediates, including the formate precursor radical and superoxide.33,35
Given that the reaction has been shown to be driven by Mn(III) and that C-terminus
knockout mutants show no activity, it may be possible to 'kick-start' the reaction using a
chemical oxidant on an inactive species. If electron transfer is the sole purpose of the
C-terminus and the true binding events at the n-terminus only require generation of Mn
(III) to drive catalysis, introducing substrate to a C-terminus knockout like E280Q may
be able to generate reaction products. If this can be identified, then the role of the C-
terminus becomes more clear.
177
Identification of the source of hydrogen peroxide production is also relevant to
the mechanism. This side-product of the decarboxylase mechanism has been referred
to as either a bifurcation in the mechanism or a misfire. Therefore, understanding
whether it is a necessary product, whether it merely comes about after oxidation of one
metal site or the other and then is a reaction product of superoxide, or whether it
represents an anomaly, where the carbon dioxide radical anion and the superoxide
radical react in an oxidase scheme, is important to the global understanding of the
protein itself. Work done in our group has found evidence of hydrogen peroxide
production at low pH in the absence of substrate. The source of this hydrogen peroxide
is still to be determined.
Additional DFT calculations might also be performed focusing on the stability and
redox potential of the C-terminus site. Oxygen-metal binding can take on many
oxidation states and coupling between a high-spin Mn(II) and a triplet ground state such
as oxygen can take on several total spin combinations. Simple calculations may be
performed comparing the total electronic energy of Mn(II) in the C-terminus using the
same atomic restriction scheme employed in Chapter 2, but changing the spin
multiplicity. Oxygen can bind with ferromagnetic or antiferromagnetic orientations,
resulting in a global spin of 3/2 or 7/2 for the combined system. Additionally, based on
the active pH range of the enzyme, any superoxide generated would likely protonate,
therefore these calculations should also be performed using hydroperoxyl as the bound
species. Changes to the reduction potential by known inhibitors may also be
calculated, such as nitric oxide.189
178
APPENDIX A DFT FINAL GEOMETRIES AND FREQUENCY TABLES
Table A-1. Mn(II) n-terminus water/water coordination - atomic coordinates
Atom x y z Atom x y z H 5.827246 -0.56219 0.492176 O -2.62587 -0.48937 -1.01098 C 5.704159 0.527578 0.413262 O -2.87743 -2.70814 -1.0527 H 6.675802 1.003656 0.603675 H -6.70111 -0.84695 1.683291 C 5.179819 0.928193 -0.99768 H 3.557261 -2.99132 3.414943 H 5.112687 2.025256 -1.06249 C 3.171789 -3.87029 2.874772 H 5.917052 0.622448 -1.75614 H 3.558545 -4.76657 3.379515 C 3.842719 0.339299 -1.35542 C 1.631151 -3.89175 2.849958 N 3.626728 -0.37344 -2.51605 H 1.27974 -4.79084 2.323866 C 2.307731 -0.64479 -2.61914 H 1.241374 -3.96722 3.878308 H 1.868736 -1.19923 -3.44403 C 1.014939 -2.69407 2.175184 N 1.653231 -0.1481 -1.58876 N 1.06996 -1.43627 2.741102 C 2.601013 0.462663 -0.79399 C 0.456996 -0.56693 1.911064 H 2.327582 0.966196 0.127564 H 0.359987 0.495479 2.114309 H 5.005504 0.852068 1.198648 N 0.011433 -1.18866 0.839008 H 1.725661 4.721515 2.205415 C 0.348055 -2.51715 0.99707 C 0.833636 5.364847 2.230994 H 0.069617 -3.26107 0.258797 H 1.161767 6.401243 2.392754 H 3.58069 -3.84444 1.854121 C 0.037227 5.252384 0.918785 Mn -0.53111 -0.27327 -1.11926 H -0.81847 5.946904 0.943534 O -1.3003 0.642098 -3.08797 H 0.665916 5.57866 0.077621 H -2.20776 0.493366 -2.75751 C -0.45047 3.873192 0.608601 H -1.22312 0.034 -3.83824 N -1.55796 3.292062 1.19142 O -0.65877 -2.2374 -2.15995 C -1.68962 2.035946 0.709952 H -1.55798 -2.55776 -1.75173 N -0.7209 1.761794 -0.14282 H -0.05248 -2.98791 -2.12719 C 0.052372 2.902803 -0.21674 H -2.4947 1.359515 0.9805 H 0.914389 2.963491 -0.87455 H 4.337329 -0.63519 -3.18853 H 0.226247 5.065982 3.09947 H 1.476893 -1.20982 3.639799 H -6.05207 0.73715 2.150113 H -2.19527 3.742549 1.836684 C -6.1342 0.023449 1.316649 H -6.7404 0.498941 0.528917 C -4.76685 -0.37105 0.797695 H -4.24209 0.535255 0.462159 H -4.16582 -0.79447 1.622207 C -4.74661 -1.34972 -0.37166 H -5.18622 -2.32252 -0.11284 H -5.31625 -0.93735 -1.22153 C -3.31669 -1.56235 -0.83716
179
Table A-2. Mn(II) n-terminus water/water coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
25.2792 0.179113 674.11 0.51705 1152.71 0.381568 1570.14 1.94193 48.2626 0.102202 680.393 3.87782 1175.93 2.79163 1575.16 1.31426 56.7518 0.274764 681.556 0.289608 1176.62 1.19144 1659.01 3.29094 71.0416 0.149556 684.89 0.053664 1180.39 0.980838 1691.03 6.19688 82.7968 0.043999 690.504 0.23793 1194.06 0.177095 2546.32 100 90.0393 0.048433 693.039 0.495512 1196.01 0.220009 2938.63 1.1513 102.935 0.196547 694.12 0.194314 1199.22 0.180088 2964.73 0.49743 106.083 0.315119 700.76 0.161092 1221.98 0.024819 2964.93 0.468711 118.279 0.131776 726.152 0.057187 1223.42 0.025648 2966.49 0.386956 139.332 0.09142 781.022 1.44507 1226.57 0.009031 2968.66 0.249174 146.677 0.176629 784.279 1.06202 1230.22 0.009776 2980.42 1.1412 160.831 0.476028 784.756 1.15196 1273.54 0.035271 2985.14 0.91505 168.892 0.908634 835.928 1.76429 1297.32 0.092741 2988.9 0.862398 182.096 0.320251 845.933 0.506875 1301.95 0.10498 2990.6 0.787269 193.763 0.812313 847.05 0.661054 1302.14 0.121245 2993.28 0.580956 210.275 0.56575 852.014 1.43518 1306.56 0.088359 3002.89 0.708685 219.473 0.000121 859.854 0.131949 1310.9 0.220182 3003.06 0.905175 220.676 0.04219 866.059 0.126581 1311.85 0.210873 3003.68 2.63202 223.835 0.488383 866.253 0.769541 1313.46 0.167173 3005.44 1.35344 225.099 0.697384 866.615 0.21092 1372.22 0.095965 3006.34 0.418821 230.233 0.000807 866.819 0.064614 1376.09 0.171314 3008.33 1.29561
253.27 1.55668 867.338 0.152176 1378.42 0.177226 3011.16 1.16869 266.415 1.77265 870.468 0.10151 1408.3 0.428135 3011.62 0.864646 283.317 1.49041 871.723 0.056065 1418.06 0.300972 3015.31 1.18023 326.611 2.71733 872.275 0.049057 1418.74 0.382878 3025.21 0.905012 379.697 8.95269 877.982 0.542718 1419.05 0.426945 3026.48 0.832934 411.794 0.642829 923.901 11.0806 1443.9 0.236961 3030.64 0.750404 470.905 6.04105 1000.79 0.190792 1450.54 0.442822 3283.27 0.090524 490.157 0.098863 1001.83 0.197265 1452 0.233868 3287.32 0.111585 490.827 0.092364 1004.52 0.184911 1452.12 0.502436 3290.74 0.16657 492.924 0.064557 1066.39 4.18637 1452.54 0.524073 3292.09 0.248435 494.949 0.065301 1069.65 2.08592 1454.56 0.193947 3299.86 2.07769 496.731 0.13111 1074.47 1.00746 1454.66 0.123211 3303.2 0.193035 498.647 0.195111 1089.78 2.75188 1455.19 0.091027 3651.98 8.43813 590.248 13.8686 1110.12 1.87317 1464.19 0.167571 3652.9 3.74712 609.379 7.87229 1126.39 1.95765 1483.54 1.71801 3657.51 4.80538 616.484 7.11188 1138.88 3.52798 1485.25 1.52339 3681.22 8.19295 628.104 3.86396 1147.39 0.872357 1489.34 1.74033 3841.51 5.45219 630.238 9.34477 1151.27 0.762255 1563.75 3.18026 3863.72 2.44494
180
Table A-3. Mn(III) n-terminus water/water coordination - atomic coordinates
Atom x y z Atom x y z H 5.80448 -0.77678 0.484117 H 3.376829 -4.9801 3.24949 C 5.719453 0.317439 0.429644 C 1.491222 -4.02865 2.730223 H 6.708471 0.752542 0.621764 H 1.113962 -4.9001 2.176855 C 5.218495 0.76863 -0.97449 H 1.085105 -4.11356 3.750424 H 5.169508 1.868467 -1.01846 C 0.91836 -2.7962 2.08048 H 5.954984 0.461481 -1.73377 N 0.773145 -1.5875 2.733386 C 3.866527 0.23081 -1.35578 C 0.244289 -0.68474 1.895608 N 3.41759 0.092441 -2.65748 H 0.024865 0.345633 2.154466 C 2.089758 -0.12133 -2.66753 N 0.040413 -1.25137 0.71191 H 1.512306 -0.25405 -3.57718 C 0.453663 -2.56942 0.816137 N 1.607979 -0.13613 -1.42711 H 0.374168 -3.26051 -0.01482 C 2.718072 0.072443 -0.60481 H 3.450973 -4.02373 1.749851 H 2.617599 0.12303 0.474046 Mn -0.51301 -0.33528 -0.97282 H 5.042559 0.660646 1.226251 O -0.906 0.592364 -2.78636 H 1.866199 4.597437 2.296586 H -1.39867 1.428936 -2.73184 C 0.989573 5.260788 2.331849 H -1.38569 0.013004 -3.40394 H 1.342937 6.284489 2.515359 O -0.70546 -2.05506 -2.24521 C 0.200123 5.205187 1.011976 H -1.66163 -2.40666 -2.03963 H -0.63794 5.91922 1.046376 H -0.13967 -2.78575 -2.52895 H 0.84387 5.536447 0.184246 H -2.59019 1.501225 0.834314 C -0.32851 3.850221 0.664715 H 3.996038 0.153581 -3.4901 N -1.54111 3.361093 1.106092 H 0.998826 -1.41487 3.708123 C -1.71121 2.108817 0.645222 H -2.21588 3.874406 1.663983 N -0.66085 1.732876 -0.07538 C 0.206018 2.818023 -0.06621 H 1.158058 2.790366 -0.58812 H 0.367737 4.961947 3.189409 H -6.0325 0.856631 2.088855 C -6.13542 0.165662 1.240152 H -6.72298 0.676644 0.461536 C -4.77735 -0.259 0.720881 H -4.2338 0.644932 0.410055 H -4.18894 -0.71401 1.535538 C -4.77883 -1.20926 -0.47174 H -5.22126 -2.1918 -0.25966 H -5.32765 -0.78435 -1.32908 C -3.353 -1.45584 -0.93262 O -2.42054 -0.53391 -0.65258 O -3.06506 -2.47135 -1.56643 H -6.72946 -0.69376 1.587078 H 3.440805 -3.20783 3.334974 C 3.031677 -4.05648 2.765781
181
Table A-4. Mn(III) n-terminus water/water coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
45.0752 0.425139 669.139 6.8335 1157.2 2.09707 1554.03 2.81202 51.1412 0.282144 680.893 0.84282 1169.32 2.79795 1564.52 3.88422 66.6269 0.319244 681.352 0.336572 1173 1.88995 1592.99 6.60957 78.6438 0.553157 683.709 0.432788 1184.21 3.5398 1616.5 16.4044 96.0777 0.231058 698.765 1.9659 1189.45 0.350038 2532.44 100 108.139 0.496568 700.883 2.43566 1197.48 0.190638 2954.19 1.24584 120.628 0.334664 707.289 0.861019 1202.28 0.148736 2966.42 0.104048
133.73 0.64722 712.894 2.07737 1222.23 0.015848 2974.49 0.097544 150.54 0.798292 722.505 0.134217 1227 0.109995 2974.94 0.201401
159.073 0.274922 769.681 0.779727 1227.75 0.033963 2979.05 0.153858 181.983 0.998762 778.329 4.77335 1232.72 0.030207 2990.28 1.06383 190.153 0.428055 785.404 23.538 1277.07 0.07036 2992.13 0.410322 210.719 0.480285 797.781 10.5683 1300.74 0.66203 2996.42 0.51524 218.684 0.001817 817.248 2.38441 1303.44 0.920784 2996.91 0.921043 218.763 0.101056 829.815 1.98649 1304.54 0.514042 2997.24 0.890638 222.637 0.576593 840.907 1.30875 1307.26 0.240807 3003.74 1.55219 223.149 0.142713 855.289 0.977166 1307.91 0.245769 3004.77 5.89123 229.571 0.00055 856.99 1.63939 1312.49 1.05094 3005.52 0.359443 237.383 0.414009 868.243 0.774872 1315.53 0.087086 3010.24 1.23748 238.714 0.158477 868.356 0.24754 1341.45 0.222012 3011.92 0.284747 268.581 1.30583 869.293 0.458957 1362.21 0.107857 3014.12 0.965235 279.765 2.60213 870.284 0.143606 1365.79 0.073773 3020.81 0.544271 317.949 1.33699 871.006 0.054955 1395.07 0.886195 3021.33 1.05112 336.223 2.45332 872.65 0.214524 1416.18 0.511721 3025.17 0.127369 355.376 8.76494 875.517 0.076712 1418.91 0.676274 3032.86 0.62264 391.371 4.51882 876.345 0.087429 1420.21 0.731001 3034.76 0.484797 447.654 0.892729 886.777 0.709764 1440.25 0.37368 3041.79 0.380688 484.672 0.070971 996.188 0.120881 1451.05 0.388009 3285.14 0.218341 494.473 0.128079 1001.64 0.488171 1453.38 1.01961 3299.45 0.233738 495.043 0.322557 1016.22 0.12643 1453.5 1.00512 3303.7 1.02418 497.365 0.180454 1059.46 2.43642 1453.97 0.76907 3305.45 4.88779 499.644 0.048291 1063.25 7.82132 1454.87 0.262586 3310.43 1.67536 511.294 2.92187 1065.13 2.20007 1455.95 0.455437 3316.59 0.82511 535.616 16.2014 1071.39 0.444323 1456.51 0.555066 3621.12 16.7291 601.105 18.4854 1108.37 0.664854 1463.15 0.527164 3625.59 11.1168 636.067 1.96071 1128.63 0.954288 1495.23 2.04833 3628.87 11.4485 656.773 4.18355 1140.26 5.96986 1496.19 2.60333 3730.82 10.3677 659.977 4.39725 1146.62 6.75848 1504.67 2.08832 3825.81 13.3042 666.009 0.749566 1150.4 3.65706 1549.85 7.04835 3849.45 9.69627
182
Table A-5. Mn(II) n-terminus water/formate coordination - atomic coordinates
Atom x y z Atom x y z H 5.831242 -0.06086 0.712327 C 3.291672 -3.04651 3.593977 C 5.66939 0.99796 0.463246 H 3.717893 -3.83938 4.226648 H 6.620243 1.537362 0.58252 C 1.752998 -3.13058 3.573807 C 5.138156 1.14907 -0.99325 H 1.443886 -4.1111 3.184822 H 5.034983 2.219973 -1.22997 H 1.360829 -3.06762 4.602939 H 5.894539 0.755558 -1.69129 C 1.095896 -2.08006 2.717008 C 3.826774 0.459454 -1.25217 N 0.868618 -0.78322 3.136471 N 3.621866 -0.43783 -2.28025 H 1.083811 -0.40556 4.04889 H 4.334954 -0.8211 -2.88606 C 0.264652 -0.11571 2.125365 C 2.302825 -0.73531 -2.33207 H -0.02891 0.927835 2.185516 H 1.843015 -1.42833 -3.02948 N 0.096858 -0.90378 1.08546 N 1.644857 -0.07575 -1.40424 C 0.609491 -2.13447 1.43994 C 2.581749 0.664645 -0.72265 H 0.600891 -2.95329 0.720068 H 2.299387 1.309356 0.104348 H 3.696593 -3.1578 2.577612 H 4.94863 1.408491 1.185886 Mn -0.56861 -0.46369 -1.02702 H 1.519981 5.26122 1.567714 O -1.48 0.30332 -3.13434 C 0.608482 5.872305 1.490192 H -2.31207 0.179839 -2.63827 H 0.902081 6.932413 1.489009 H -1.25131 -0.61636 -3.38363 C -0.17544 5.522083 0.21307 O -0.36456 -1.99322 -2.49243 H -1.05413 6.18317 0.126111 C -0.07869 -3.21938 -2.21117 H 0.448728 5.73187 -0.66796 H -0.49471 -3.96237 -2.93592 C -0.6089 4.093616 0.126067 O 0.610457 -3.60821 -1.27489 N -1.71981 3.562555 0.750953 H -2.57988 1.575852 0.779425 H -2.40205 4.075289 1.293657 C -1.78727 2.239856 0.447199 N -0.78064 1.88034 -0.31838 C -0.04677 3.026794 -0.5292 H 0.83782 3.020905 -1.16006 H 0.005937 5.69015 2.393962 H -6.10247 1.033872 2.147031 C -6.1463 0.19002 1.440102 H -6.76641 0.504989 0.584608 C -4.76212 -0.22901 0.989745 H -4.27253 0.628723 0.507703 H -4.14599 -0.48744 1.869612 C -4.6979 -1.37964 -0.00885 H -5.09531 -2.31439 0.411478 H -5.29639 -1.12585 -0.90109 C -3.25849 -1.60894 -0.43509 O -2.64308 -0.51474 -0.7999 O -2.74349 -2.71682 -0.37785 H -6.68225 -0.63205 1.942567 H 3.636155 -2.07715 3.988336
183
Table A-6. Mn(II) n-terminus water/formate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
24.0209 1.0282 676.1745 87.8062 1176.724 6.019 1559.872 66.9193 37.1558 4.9142 679.5747 0.9061 1178.222 77.2049 1564.551 12.2253 43.6832 5.9986 679.989 13.2558 1181.282 30.8229 1574.262 15.6201 57.0542 0.8319 688.0293 11.8464 1187.194 16.0222 1685.523 86.4107 71.5123 3.1203 690.7712 9.3742 1192.66 9.2063 1750.861 442.2696 76.3455 0.868 692.4889 3.9258 1194.167 8.6933 2931.821 240.5672 81.6184 3.5554 706.404 1.5665 1196.87 5.6231 2937.558 18.7423 94.2837 1.7646 726.1737 1.2909 1211.393 0.2988 2952.08 20.3075
107.3651 3.5587 760.0011 110.0589 1222.349 0.1852 2953.863 23.6626 118.8059 12.279 772.9453 28.5521 1224.133 0.2698 2955.223 19.5592 124.0108 1.3563 782.3375 12.5164 1228.326 0.1402 2956.747 10.1873
127.082 2.5185 783.3856 55.8753 1265.076 2.4838 2968.815 24.3577 139.2811 1.449 801.1671 315.6969 1292.472 1.5255 2971.222 43.7005 148.1392 0.5847 823.4257 7.4451 1295.508 1.2285 2982.302 21.7697 157.4557 4.5743 844.4672 51.6106 1297.351 1.7776 2983.117 20.146 168.0865 17.6902 862.9961 1.2139 1298.536 0.222 2986.35 14.1175 187.0529 5.7474 864.818 2.033 1302.197 7.115 2998.287 13.282 190.6308 16.6915 864.964 13.2118 1305.474 2.9341 2998.487 39.8583 200.2531 43.926 865.2513 3.6643 1316.424 1.9915 2999.453 36.9992 212.5855 8.5672 865.5856 0.5688 1345.192 265.5604 3000.233 38.5659 221.0408 0.7805 867.5879 1.7297 1372.366 5.257 3000.883 37.1845 224.9765 15.5053 869.1098 4.5436 1382.791 16.3754 3002.076 32.1871 225.1079 3.9648 870.1969 0.794 1385.394 3.9305 3003.766 24.4452 225.9194 46.1348 870.5469 5.9876 1404.13 65.1077 3007.451 15.2241 231.7606 0.0655 880.5212 10.214 1408.699 5.328 3012.376 45.4132 322.3519 18.3829 894.8198 9.911 1415.712 5.4265 3018.487 22.2701 328.4785 46.9569 910.5374 212.8447 1417.078 5.3894 3019.786 18.0187 335.0114 45.2757 942.226 37.1117 1418.246 5.0832 3024.511 18.5802 436.6942 59.32 990.2409 2.5578 1442.926 5.3181 3235.145 107.2586
481.593 4.2344 997.7637 5.2975 1448.489 8.9603 3276.726 1.653 483.5819 0.9243 999.7008 3.2142 1449.606 7.413 3280.815 3.775
487.69 1.3704 1064.55 46.9532 1449.762 6.2436 3290.443 4.4815 492.8807 1.5933 1068.101 4.2408 1452.544 2.0658 3297.206 43.177 493.4165 2.9634 1070.041 23.8677 1452.594 2.6801 3307.924 25.3448 498.7536 2.1908 1080.433 60.7593 1455.274 0.511 3613.979 215.3194
572.568 122.7513 1109.528 0.9429 1455.398 0.6287 3664.784 80.9821 575.2943 92.7342 1119.862 1.7306 1462.624 4.726 3668.971 89.2015 591.2007 73.8694 1129.637 12.1567 1468.776 29.9377 3671.758 64.1182 592.5834 70.5909 1134.403 10.5816 1477.408 26.5076 3693.802 216.695 673.6613 3.7687 1140.015 5.8732 1479.43 29.9634
184
Table A-7. Mn(III) n-terminus water/formate coordination - atomic coordinates
Atom x y z Atom x y z H 5.844358 -0.44453 0.634776 O -2.94155 -2.62398 -0.93453 C 5.724655 0.633669 0.458638 H -6.68343 -0.56892 1.867587
H 6.697912 1.122931 0.59885 H 3.567392 -2.58471 3.771312 C 5.199345 0.905567 -0.98236 C 3.179705 -3.50982 3.316499 H 5.131961 1.991642 -1.15243 H 3.564878 -4.35826 3.89954 H 5.938234 0.527895 -1.70698 C 1.638824 -3.52728 3.296107 C 3.859763 0.291143 -1.28283 H 1.286671 -4.4686 2.850899 N 3.485646 -0.28958 -2.48045 H 1.247066 -3.50811 4.326038 H 4.108438 -0.53364 -3.24158 C 1.026307 -2.39422 2.51478 C 2.151454 -0.49973 -2.48415 N 0.871795 -1.13103 3.054678 H 1.612409 -0.96348 -3.30364 H 1.110631 -0.85871 3.999893 N 1.60397 -0.08006 -1.35468 C 0.31031 -0.33136 2.12729 C 2.665346 0.404199 -0.59801 H 0.077119 0.715019 2.295735 H 2.501148 0.821696 0.390373 N 0.097498 -1.00469 1.011479 H 5.03261 1.032095 1.215327 C 0.538423 -2.29637 1.242779 H 1.767329 4.99352 1.862141 H 0.473673 -3.04708 0.460575 C 0.875563 5.636544 1.831388 H 3.583111 -3.58034 2.295975 H 1.203555 6.683409 1.895301 Mn -0.59766 -0.2638 -0.93832 C 0.076704 5.407191 0.536245 O -1.02449 1.03106 -2.86035 H -0.77888 6.100947 0.498075 H -1.84026 1.549847 -2.81168 H 0.704742 5.656226 -0.33157 H -1.25676 0.232652 -3.36778 C -0.41705 4.007523 0.354162 O -0.68178 -1.45989 -2.39877 N -1.55539 3.512252 0.965903 C -0.50624 -2.77368 -2.36377 H -2.20803 4.049093 1.52484 H -1.00553 -3.2724 -3.22 C -1.68838 2.203147 0.667133 O 0.155565 -3.37312 -1.55264 N -0.68955 1.799077 -0.09906 H -2.51255 1.576119 0.99381 C 0.106144 2.918303 -0.30244 H 5.844358 -0.44453 0.634776 H 0.999504 2.868201 -0.91733 H 0.269635 5.418628 2.724372 H -6.02887 1.048861 2.183754 C -6.11402 0.261173 1.420214 H -6.72093 0.662851 0.592929 C -4.7491 -0.18443 0.937623 H -4.22844 0.689061 0.520227 H -4.14276 -0.52771 1.793612 C -4.73441 -1.26634 -0.13688 H -5.14291 -2.22654 0.202206 H -5.31385 -0.94768 -1.01974 C -3.30631 -1.52679 -0.58276 O -2.46244 -0.45504 -0.55002
185
Table A-8. Mn(III) n-terminus water/formate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
36.6067 1.6007 640.8704 121.1436 1158.516 39.3089 1558.57 67.5333 49.7807 4.7797 680.004 4.1674 1163.865 62.8477 1563.414 26.3064 59.2442 2.5892 680.216 0.7214 1181.079 31.7725 1573.189 32.037 68.8233 0.2687 681.0496 0.7755 1192.712 7.7892 1621.814 91.4919 71.6987 3.9754 686.6966 1.4106 1195.169 2.1523 1798.955 356.7114
79.536 1.6957 687.416 3.0194 1200.51 4.3641 2948.454 14.9111 100.5569 4.2997 692.1356 1.0718 1208.921 4.8138 2963.233 4.9304
104.797 9.6973 694.8844 2.2006 1222.196 2.8823 2966.944 8.9215 118.2574 0.9739 724.8864 0.7948 1223.391 12.6727 2966.972 10.407 138.2794 3.6409 753.0201 78.7648 1225.851 0.0748 2969.062 6.0802 141.7886 10.984 767.0508 186.2977 1231.235 2.7322 2980.733 22.9368 146.9543 11.6437 770.8131 121.9972 1235.766 417.3009 2985.303 16.1895
171.496 8.7034 774.3974 13.4605 1274.814 2.5139 2988.569 17.256 179.0773 1.9859 785.9321 18.4714 1298.01 2.8766 2989.641 13.5552 188.8008 12.6312 826.7854 6.158 1301.076 1.6117 2993.024 12.2242 208.2311 26.6137 831.7291 33.4254 1303.469 2.4657 3003.396 10.8116 212.9352 0.7846 856.0918 12.1759 1304.603 5.9182 3004.24 22.4769 218.2664 0.0386 866.5781 6.797 1307.173 3.2892 3005.384 61.9979 222.6864 0.303 867.1397 2.8762 1309.43 0.9382 3007.62 11.2541 223.3782 0.1954 867.637 4.7646 1312.629 5.9118 3008.067 20.9666 230.4381 10.4929 868.4245 6.5213 1349.598 3.3189 3009.824 18.7393 230.8718 0.0497 868.7813 0.384 1357.126 2.5244 3011.426 21.9666 242.1181 7.3687 870.9348 3.0471 1372.019 8.0072 3016.134 13.4579 245.7105 47.2326 873.1071 0.9311 1384.808 23.1859 3025.609 16.5869 267.7121 70.2494 873.5009 0.9573 1403.203 6.8279 3027.451 16.0558 303.7757 51.3635 883.3204 11.855 1418.058 7.6459 3029.513 7.744 335.5254 54.2095 888.3766 11.0553 1418.937 0.9243 3033.928 13.1686 364.1776 12.3052 925.9621 25.7424 1419.287 11.1899 3043.827 55.2792
456.117 31.452 994.2559 3.0051 1442.936 4.4982 3286.72 73.0902 472.6836 121.0257 998.9628 2.8086 1450.483 8.6662 3289.218 5.2139 485.9339 7.8623 1003.426 3.8622 1451.739 8.1799 3291.094 2.9629 491.9338 2.5511 1046.397 4.6258 1452.123 10.6655 3298.714 9.7021 493.3717 63.4518 1058.691 36.3899 1453.775 6.5596 3299.944 50.5707
496.676 4.7747 1063.468 30.6022 1455.015 4.0593 3310.713 16.0007 500.8605 15.8035 1081.269 56.5005 1455.391 1.5838 3647.455 231.8397 502.1449 10.6574 1109.29 0.8831 1455.702 2.1536 3647.813 33.8563 580.6275 173.6298 1129.187 0.7006 1463.359 4.4442 3656.865 99.0069 604.3596 111.8123 1148.274 15.6575 1484.753 28.4468 3724.202 62.1091 620.3918 90.2204 1149.934 26.3083 1488.564 33.8174 3861.141 91.2445 637.4393 87.2494 1152.879 20.8403 1492.207 27.4332
186
Table A-9. Mn(II) n-terminus water/acetate coordination - atomic coordinates
Atom x y z Atom x y z H 5.796129 -0.78796 0.821787 H -6.59619 1.011259 2.113971 C 5.805126 0.220278 0.382857 H 3.391301 -1.77198 4.404574 H 6.83524 0.604486 0.402987 C 2.876828 -2.72437 4.199525 C 5.264011 0.192369 -1.07753 H 3.182922 -3.44504 4.972289 H 5.337113 1.203692 -1.50898 C 1.345698 -2.54742 4.19276 H 5.92471 -0.44569 -1.68683 H 0.863663 -3.51611 3.997537 C 3.848461 -0.29899 -1.20757 H 1.001994 -2.22802 5.191115 N 3.441489 -1.22899 -2.14231 C 0.849389 -1.57381 3.155597 H 4.046888 -1.78303 -2.73304 N 0.982246 -0.20632 3.312797 C 2.087492 -1.27823 -2.13492 H 1.378829 0.263568 4.114944 H 1.486331 -1.94227 -2.75509 C 0.451512 0.394249 2.221849 N 1.604626 -0.42597 -1.25819 H 0.423134 1.470798 2.080029 C 2.685581 0.184189 -0.67418 N -0.00619 -0.50219 1.378826 H 2.560517 0.947619 0.088242 C 0.230638 -1.73426 1.947661 H 5.185136 0.870904 1.017573 H -0.06382 -2.6352 1.416257 H 2.463659 5.255907 0.67588 H 3.228546 -3.08992 3.223739 C 1.665201 5.98871 0.486696 Mn -0.63095 -0.39211 -0.79509 H 2.131806 6.965677 0.291873 O -0.98072 -0.32036 -3.00673 C 0.795986 5.551211 -0.70549 H -0.87939 -1.32034 -3.17385 H 0.037869 6.32503 -0.91334 H -1.93812 -0.17094 -3.01117 H 1.42019 5.489408 -1.60906 O -0.37071 -2.51204 -0.92425 C 0.127329 4.225153 -0.52927 C -0.76747 -3.23799 -1.88702 N -1.04147 4.017336 0.176663 O -0.74961 -2.88165 -3.09454 H -1.61117 4.730685 0.611846 C -1.31564 -4.59802 -1.52491 C -1.34145 2.693318 0.121919 H -0.62802 -5.11693 -0.84174 N -0.43156 2.033097 -0.55913 H -2.25304 -4.42015 -0.97526 C 0.482784 2.976215 -0.97252 H -1.50459 -5.20555 -2.418 H 1.33846 2.699607 -1.58252 H -2.22534 2.245521 0.566451 H 1.067864 6.077748 1.407761 H -5.74087 2.563938 2.009969 C -5.94481 1.625261 1.470166 H -6.52844 1.879989 0.570002 C -4.66444 0.902117 1.107202 H -4.05193 1.561571 0.476669 H -4.07428 0.707188 2.020636 C -4.82331 -0.40457 0.337308 H -5.35864 -1.16757 0.91973 H -5.39527 -0.22 -0.58877 C -3.4559 -0.94863 -0.03704 O -2.65969 -0.06713 -0.56415 O -3.18196 -2.12566 0.17464
187
Table A-10. Mn(II) n-terminus water/acetate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
27.6214 0.079625 634.214 5.50209 1147.7 1.76465 1501.21 32.5869 30.1703 0.018632 671.254 5.00682 1168.23 5.3079 1560.34 4.10316 51.7082 0.117478 677.628 0.489562 1177.66 2.94415 1566.94 0.874216 59.2658 0.156769 679.039 0.123397 1185.79 1.48973 1575.96 2.78062 60.4454 0.305752 681.288 0.184289 1188.81 1.76215 1646.43 41.8253 67.5754 0.493472 688.557 0.55565 1191.49 0.765751 1679.87 5.93346 74.1923 0.23911 690.891 0.619425 1194.03 1.60019 2876.57 100
80.426 0.186332 694.609 0.235755 1196.76 0.564537 2936.79 1.53965 87.3718 0.182145 704.57 0.194807 1209.9 0.022517 2951.35 1.73708 91.4703 0.4714 725.039 0.072318 1221.48 0.047548 2952.18 1.02316
123.13 0.18296 772.741 2.39151 1221.67 0.007576 2953.42 1.89445 133.254 0.147814 780.411 1.19028 1228.2 0.010779 2954.8 1.57869 138.923 0.425063 787.157 2.07346 1263.73 0.176856 2967.17 2.17085 146.068 0.159451 830.915 0.7384 1291.8 0.135497 2969.78 3.86793 151.422 0.758688 843.778 3.21912 1296.13 0.117116 2979.82 1.99049 165.344 0.123927 862.538 0.105623 1296.96 0.159754 2983.07 1.70521
171.45 0.379079 864.581 0.159325 1297.95 0.023215 2985.95 1.18574 184.328 0.301439 864.725 1.00547 1304.36 0.069258 2997.33 3.29174 196.889 3.86336 864.97 0.361389 1314.01 0.124179 2997.82 1.74696 202.763 1.44531 865.312 0.124188 1324.58 0.648038 2999.55 2.57652
220.9 0.0038 867.09 0.176318 1350.63 0.54783 2999.76 3.86103 223.481 0.014983 868.106 0.661945 1368.39 1.22855 3000.13 2.28232 224.957 1.13732 869.537 0.052601 1373.11 0.442989 3002.69 2.48949 226.509 0.042823 870.262 0.345136 1390.08 0.4114 3003.53 2.40429 231.993 0.002178 882.413 0.818773 1407.96 0.364054 3005.19 1.60601 290.635 3.33222 904.167 1.4953 1416.44 0.52623 3013.25 3.83124 304.402 4.25073 919.649 17.275 1416.93 0.477723 3018.07 1.86012 317.517 2.24786 933.564 1.65059 1418.99 0.310755 3019.67 1.60571 343.288 2.98369 946.268 3.91877 1442.4 0.431151 3024.23 1.67992
480.61 0.307114 990.111 0.239648 1443.96 2.90874 3066.37 0.90567 481.265 0.451204 995.662 0.586675 1448.35 0.685244 3147.3 0.487746 483.974 0.079633 998.188 0.362717 1449.74 0.674237 3178.48 1.83628 486.721 0.228045 1030.39 1.66047 1449.76 0.494178 3253.94 3.94141 490.373 0.210816 1063.79 6.00778 1452.44 0.266133 3275.71 0.148992 492.558 0.472308 1064.43 4.31931 1452.78 0.188484 3277.97 0.620299
499.24 0.413141 1067.03 1.20935 1453.1 1.24224 3284.25 0.699899 510.773 8.23066 1073.47 2.25658 1455.22 0.041225 3287.42 2.2146 572.445 5.10632 1106.26 0.132125 1455.34 0.034129 3297.81 3.87578
576.02 12.0514 1111.49 11.4846 1462.21 0.351408 3665.01 6.85893 590.063 5.32095 1117.18 0.340553 1468.82 2.79025 3668.15 6.93504 591.664 7.96171 1128.2 0.908647 1476.25 3.49742 3671.04 5.40025 599.778 6.07199 1139.18 0.373782 1478.88 2.11574 3820.87 5.06535
188
Table A-11. Mn(III) n-terminus water/acetate coordination - atomic coordinates
Atom x y z Atom x y z H 5.822512 -1.15736 0.565855 H 3.471698 -2.4373 4.084875 C 5.851684 -0.11164 0.228867 C 2.924687 -3.34995 3.80083 H 6.893605 0.234665 0.249881 H 3.220027 -4.14837 4.495973 C 5.278527 0.02223 -1.21337 C 1.399437 -3.13373 3.848967 H 5.355473 1.069474 -1.54683 H 0.886224 -4.06776 3.578639 H 5.909794 -0.56564 -1.89928 H 1.083291 -2.89832 4.878025 C 3.84696 -0.4164 -1.3574 C 0.908442 -2.04752 2.92775 N 3.295953 -0.91324 -2.52333 N 1.042289 -0.71779 3.28394 H 3.812544 -1.25763 -3.32397 H 1.418054 -0.3785 4.160566 C 1.949386 -0.85897 -2.43881 C 0.549453 0.047035 2.290166 H 1.281871 -1.20696 -3.22174 H 0.529106 1.132481 2.316725 N 1.573547 -0.34231 -1.28132 N 0.110016 -0.71089 1.306463 C 2.74856 -0.07427 -0.59879 C 0.324136 -2.02032 1.692553 H 2.733345 0.373189 0.389837 H 0.031364 -2.84003 1.045294 H 5.278915 0.498365 0.943286 H 3.246987 -3.63547 2.788986 H 2.669415 4.962217 1.10486 Mn -0.59873 -0.17094 -0.72505 C 1.879886 5.722387 1.012948 O -0.91751 0.441246 -2.81347 H 2.359448 6.704608 0.89973 H -1.27617 -0.45691 -3.13425 C 0.973425 5.43015 -0.19568 H -1.65201 1.063766 -2.91177 H 0.225344 6.23196 -0.30489 O -0.62952 -1.97386 -1.19454 H 1.572357 5.449176 -1.11777 C -1.26691 -2.55755 -2.18416 C 0.2709 4.111136 -0.13729 O -1.63159 -1.96092 -3.19893 N -0.90097 3.895176 0.569235 C -1.48515 -4.02717 -2.00057 H -1.43399 4.602757 1.061211 C -1.25939 2.600255 0.461956 N -0.37835 1.936258 -0.26889 C 0.578355 2.871202 -0.64906 H 1.418597 2.588285 -1.27517 H 1.308437 5.733475 1.953892 H -5.58027 2.351373 2.365034 C -5.82738 1.481003 1.738598 H -6.42281 1.842709 0.884986 C -4.57605 0.765315 1.273452 H -3.96518 1.476803 0.700219 H -3.96968 0.464796 2.145682 C -4.7887 -0.45237 0.38046 H -5.3128 -1.27511 0.882646 H -5.3711 -0.17936 -0.51568 C -3.44558 -0.99109 -0.08002 O -2.48213 -0.06987 -0.30125 O -3.27594 -2.1809 -0.23573 H -6.48013 0.823216 2.334217
189
Table A-12. Mn(III) n-terminus water/acetate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
33.2913 0.090592 646.427 7.92312 1159.51 1.47243 1493.04 2.84189 44.4179 0.141973 679.665 0.110696 1162.56 2.41546 1562.16 8.49098 45.9008 0.301707 681.742 0.243289 1165.77 8.23115 1565.14 1.48863 55.4857 0.403781 683.482 4.28999 1178.2 3.34415 1577.89 4.02838 69.8402 0.390843 686.805 0.122686 1190.62 0.744679 1656.32 22.2632 86.2741 0.10938 688.83 1.41813 1194.72 0.265775 1722.6 17.2593 100.334 0.083505 689.804 1.01319 1200.21 0.393863 2874.51 100 107.478 0.185768 691.622 0.236043 1203.95 5.64395 2945.67 1.75941 115.647 0.183446 693.276 0.632568 1220.6 0.020542 2958.04 0.578047 124.849 0.307926 724.687 0.102831 1220.94 0.109848 2967.76 0.933377 133.228 0.027928 770.804 7.11309 1224.48 0.031227 2968.61 0.506194
148.69 0.51938 775.633 1.71052 1231.05 0.016785 2968.71 0.758383 175.328 0.65924 780.111 8.18948 1271.97 0.233402 2980.57 2.06244 179.848 0.302983 787.225 21.4739 1297.29 0.305126 2984.25 1.99202 182.642 0.155717 832.714 0.329406 1301.68 0.190005 2985.81 1.5664 197.511 0.521772 833.98 3.21419 1303.76 0.274456 2989.49 1.61893 208.615 0.290494 853.171 1.23921 1306.68 0.325419 2993.21 1.27479 218.224 0.012489 866.654 0.680639 1309.09 0.351174 3002.59 2.2886 222.148 0.005053 867.012 0.266194 1314.21 0.409014 3004.37 0.821236 222.821 0.01211 867.733 0.410947 1315.64 0.558601 3006.24 0.636236 230.536 0.149478 868.029 0.636804 1335.35 22.7878 3007.14 6.41809
231.24 0.007764 868.864 0.018529 1353.17 0.2582 3008.21 2.1945 246.411 0.707969 870.185 0.421403 1359.27 0.420974 3008.89 2.40243
272 1.05864 872.67 0.144096 1363.82 0.28857 3011.21 2.25942 299.023 5.07899 873.736 0.107028 1403.78 2.75263 3015.82 1.3078 309.168 1.40686 873.951 0.91127 1404.68 35.7537 3026.26 1.59071 338.003 7.95075 877.024 2.39504 1418.96 0.721066 3027.59 1.61784 364.285 0.204577 893.383 1.11831 1419.19 0.729967 3028.54 1.04342 388.303 5.29191 952.58 5.32905 1420.27 0.460663 3034.37 1.31748 451.383 9.92885 996.864 0.389896 1442.66 0.417516 3085.16 0.444975 480.519 0.23072 1001.14 0.27651 1445.05 1.64676 3172.5 0.277148 490.161 0.866846 1003.34 0.44045 1450.39 0.786929 3200.77 0.454823 490.682 0.033789 1030.51 4.21567 1451.72 0.931603 3289.89 0.229584 495.712 0.083814 1060.59 4.43773 1452.32 1.03684 3290.79 0.206869 497.405 0.647319 1064.76 2.60172 1454.26 0.34319 3294.7 0.670792 500.285 0.43143 1064.98 2.26151 1454.82 1.43455 3297.16 1.43849 557.563 0.912596 1065.75 6.66592 1455.12 1.82684 3300.35 6.36371 573.755 11.315 1080.31 14.1697 1455.41 0.326505 3306.29 1.29618 589.983 7.25138 1108.17 0.045849 1456.4 0.252499 3646.26 13.9781 619.181 8.1862 1127.65 0.207068 1463.38 0.374268 3648.7 12.4087 634.584 7.19536 1148.13 1.95642 1485.71 3.03566 3655.77 9.78807
638.03 11.4343 1153.79 1.73981 1491.31 3.27842 3840.96 7.77119
190
Table A-13. Mn(II) n-terminus water/oxalate coordination - atomic coordinates
Atom x y z Atom x y z H 4.997458 -3.43174 0.317302 O -2.07667 1.354843 -0.2364 C 5.458201 -2.54955 -0.15171 O -3.5347 -0.19513 0.477636 H 6.540791 -2.72505 -0.23606 H -4.74842 4.171025 2.694839 C 4.838398 -2.2768 -1.55445 H 2.768527 -3.19445 4.130553 H 5.347526 -1.41282 -2.00809 C 1.836739 -3.7672 3.997376 H 5.045523 -3.13299 -2.21619 H 1.820102 -4.5628 4.756872 C 3.358262 -2.00988 -1.53991 C 0.594857 -2.86384 4.127123 N 2.410831 -2.89338 -2.0111 H -0.31551 -3.46627 3.998171 H 2.58698 -3.82325 -2.36692 H 0.545789 -2.44342 5.146024 C 1.196624 -2.30396 -1.91853 C 0.543286 -1.74905 3.115157 H 0.25488 -2.77731 -2.17518 N 1.368134 -0.64222 3.174287 N 1.307923 -1.09161 -1.42002 H 2.037834 -0.43687 3.902819 C 2.647348 -0.89931 -1.17978 C 1.101705 0.134911 2.098047 H 3.022424 0.028555 -0.75943 H 1.612455 1.071321 1.893549 H 5.296655 -1.6902 0.515562 N 0.16365 -0.40652 1.354458 H 5.050389 3.46957 0.316721 C -0.19729 -1.5832 1.977466 C 4.699451 4.50324 0.179609 H -0.96075 -2.23044 1.549512 H 5.563972 5.129397 -0.08533 H 1.871584 -4.23671 3.003419 C 3.620003 4.573146 -0.91494 Mn -0.55549 0.053111 -0.76243 H 3.324726 5.624409 -1.07284 O -1.27625 0.417537 -2.85922 H 4.041664 4.229458 -1.8706 H -1.71008 1.277212 -2.74921 C 2.408312 3.740739 -0.64221 H -2.05015 -0.21475 -2.85108 N 1.412162 4.031421 0.26909 O -1.37239 -1.894 -0.91225 H 1.346414 4.864213 0.839371 C -2.53871 -2.36429 -0.76025 C 0.48561 3.037031 0.218947 O -2.91484 -3.23398 0.031418 N 0.82945 2.114992 -0.65363 C -3.63952 -1.84987 -1.71751 C 2.01805 2.54886 -1.19921 O -4.80165 -2.39796 -1.4363 H 2.518899 1.986585 -1.98264 H -4.56837 -2.96001 -0.65021 H 4.313753 4.857035 1.148771 O -3.47155 -1.08191 -2.64647 H -3.25502 5.10631 2.475543 H -0.42599 3.016235 0.810056 C -3.94162 4.401642 1.979682 H -4.40467 4.931427 1.130763 C -3.21915 3.152665 1.519634 H -2.419 3.440618 0.823265 H -2.72206 2.672776 2.381904 C -4.06807 2.108059 0.802915 H -4.85358 1.691874 1.44887 H -4.56031 2.563233 -0.0742 C -3.18381 0.972669 0.318498
191
Table A-14. Mn(II) n-terminus water/oxalate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
20.9428 0.141794 593.646 12.6929 1126.5 1.17372 1481.29 3.55329 29.1625 0.38412 594.865 5.86349 1140.6 0.375899 1525.36 31.7687 44.4188 0.161524 598.99 2.42303 1142.55 1.39693 1556.76 6.01409 58.5013 0.486727 668.008 0.23669 1163.27 8.01134 1562.98 1.71962 59.0993 0.53409 682.951 0.234139 1175.83 4.92177 1575.64 3.32179 65.7062 0.335147 683.613 0.187524 1178.71 0.898638 1690.46 7.03805 77.2732 0.321393 686.996 0.674444 1184.2 3.60815 1776.6 41.0464 81.2277 0.052783 690.337 0.462745 1192.63 0.417298 1855.29 51.6043 87.5446 0.442153 692.526 0.381705 1193.85 1.33145 2935.27 2.30117 98.4864 0.070472 699.402 0.264493 1196.96 0.780225 2951.65 2.69994 105.435 0.73886 725.101 0.102108 1212.82 0.018993 2952.79 2.21777 120.126 0.311142 734.51 3.24081 1223.01 0.007869 2953.22 1.89587 131.081 0.359741 771.813 3.28538 1223.5 0.059904 2964.03 1.24585
138.28 0.301266 781.888 1.95747 1227.99 0.035582 2968.31 2.73746 147.243 0.767197 787.826 3.70541 1265.2 0.225249 2970.4 4.98666 159.301 0.694128 824.408 0.757887 1272.86 0.619642 2981.86 2.40643 165.183 1.36832 845.667 4.79574 1291.09 0.128879 2985.8 1.77227 183.775 0.366261 853.814 0.339535 1295.81 0.163293 2989.3 1.28628 196.845 5.359 862.385 0.362304 1297.95 0.232064 2998.18 4.95429 206.686 1.69305 863.11 0.218503 1298.92 0.049268 2999.11 3.0687 220.949 0.005817 864.665 0.608439 1303.05 0.064655 2999.96 3.43051 223.046 0.006168 865.154 1.02701 1308.09 1.17577 3000.49 3.0556 225.301 0.008516 865.784 0.043054 1313.81 0.375139 3001.08 4.50014 226.365 1.43788 866.482 1.6004 1373.39 0.535167 3002.24 3.31656 231.778 0.00161 868.104 3.43236 1375.44 3.24392 3005.97 1.15272 268.572 2.58419 869.741 0.045481 1386.85 1.28602 3008.1 1.43776 296.801 4.30256 869.779 0.100101 1407.85 0.219863 3009.35 5.70852 312.716 4.99627 870.49 5.02278 1415.29 77.202 3018.81 2.6304 314.848 5.29024 874.871 1.21447 1416.58 1.62021 3019.6 2.24876 349.257 3.11113 885.498 1.5058 1416.66 0.165084 3022.17 2.28991
419.66 6.24301 898.881 2.98368 1417.12 0.902641 3250.34 100 451.695 2.25708 918.897 2.43456 1442.91 0.663558 3261.49 9.42591
480.45 0.63368 924.86 23.8635 1447.64 0.965992 3275.03 0.176741 482.334 0.151772 958.837 21.9046 1449.5 0.921033 3285.21 0.206551 486.707 0.375536 988.961 0.253291 1449.86 0.813063 3286.36 0.563684 490.579 0.087118 997.44 0.369379 1451.69 0.112494 3292.25 4.67585 493.044 0.417116 999.655 0.422298 1452.67 0.314476 3312.78 3.92352 499.219 0.0984 1065.37 5.01372 1454.94 0.137157 3389.96 27.813 510.877 6.56022 1076.19 2.9286 1455.06 0.037396 3663.95 9.3516 573.026 6.52565 1081.52 5.84293 1462.12 0.972092 3669.18 9.69916 580.116 17.4903 1104.47 0.206744 1466.22 3.14326 3671.81 7.29621 584.524 15.8624 1118.61 0.222086 1477.96 3.00193 3823.54 6.75045
192
Table A-15. Mn(III) n-terminus water/oxalate coordination - atomic coordinates
Atom x y z Atom x y z H 5.197459 -3.10554 0.356688 H -5.04592 3.842924 2.646495 C 5.581866 -2.20767 -0.14761 H 3.011249 -2.89799 4.206968 H 6.670301 -2.3061 -0.25793 C 2.12217 -3.53938 4.103234 C 4.922895 -2.03113 -1.54794 H 2.166385 -4.30483 4.890462 H 5.359208 -1.15404 -2.05087 C 0.818966 -2.72556 4.223387 H 5.180386 -2.89938 -2.17498 H -0.0473 -3.39555 4.131715 C 3.427412 -1.87324 -1.51675 H 0.750307 -2.26643 5.222788 N 2.509175 -2.51258 -2.32385 C 0.670122 -1.65424 3.17481 H 2.709928 -3.26048 -2.97737 N 1.278932 -0.41596 3.246428 C 1.281659 -1.99482 -2.1019 H 1.85607 -0.08319 4.009369 H 0.376883 -2.34878 -2.58368 C 0.951869 0.297665 2.155068 N 1.336453 -1.03333 -1.19478 H 1.293093 1.307223 1.954975 C 2.668779 -0.95626 -0.82182 N 0.162948 -0.42247 1.374036 H 3.006488 -0.23475 -0.08431 C -0.02506 -1.64266 1.998435 H 5.383077 -1.3383 0.49651 H -0.64676 -2.40844 1.543821 H 4.746186 3.781254 0.114707 H 2.178642 -4.0437 3.127783 C 4.308852 4.777742 -0.04571 Mn -0.50156 -0.0094 -0.49311 H 5.113668 5.461003 -0.35101 O -1.02688 0.306499 -2.37103 C 3.21122 4.729167 -1.12335 H -1.30311 1.219888 -2.54134 H 2.827591 5.745382 -1.30991 H -1.85846 -0.31252 -2.571 H 3.647025 4.392655 -2.07538 O -1.24003 -1.89379 -0.71826 C 2.068052 3.82081 -0.80046 C -2.36254 -2.4957 -0.83404 N 0.996457 4.177977 -0.00719 O -2.77428 -3.45337 -0.19713 H 0.828624 5.097413 0.383623 C -3.29217 -2.01011 -1.96629 C 0.173209 3.114046 0.120432 O -4.40583 -2.67921 -2.01237 N 0.652675 2.070379 -0.53399 H -4.32186 -3.31534 -1.25622 C 1.831733 2.50489 -1.11413 O -3.01914 -1.12709 -2.77211 H 2.434142 1.846194 -1.73298 H -0.75955 3.124369 0.675647 H 3.913107 5.133307 0.918212 H -3.63014 4.873441 2.364013 C -4.27335 4.106691 1.906872 H -4.78855 4.570955 1.050744 C -3.46713 2.898288 1.476987 H -2.70572 3.230351 0.756862 H -2.917 2.487596 2.340653 C -4.24761 1.769732 0.81189 H -4.9804 1.291158 1.474047 H -4.79193 2.130044 -0.07687 C -3.28892 0.68558 0.351547 O -2.01129 1.033186 0.120433 O -3.6929 -0.45073 0.196059
193
Table A-16. Mn(III) n-terminus water/oxalate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
28.5264 0.226923 638.016 6.71156 1152.66 0.995014 1497.76 1.1253 30.2687 0.029104 638.398 1.28735 1155.43 0.671447 1527.66 14.7611
46.63 0.032039 675.241 0.098367 1165.12 1.74783 1553.59 3.25976 59.9246 0.213332 676.011 0.156157 1168.85 3.46683 1560.09 1.23381 74.0902 0.106473 679.975 0.082479 1181.37 5.88895 1574.62 2.2617 76.3795 0.073636 680.33 0.045438 1184.55 2.46608 1653.19 3.65815 85.6752 0.084367 685.878 0.072819 1192.7 3.00415 1771.49 5.32639 103.355 0.048444 689.516 0.036182 1194.88 0.352906 1819.76 51.1456
113.51 0.069005 696.6 0.050435 1196.26 0.217624 2292.54 100 120.815 0.073269 722.56 0.033777 1200.36 0.137273 2951.96 0.70993 144.095 0.482725 745.355 2.53451 1222.45 0.023867 2965.48 0.218667 149.851 0.102965 773.608 1.66397 1223.54 0.003903 2967.12 0.369944 165.358 0.247315 777.977 1.95471 1227.66 0.011708 2970.07 0.424456 174.738 0.284417 781.047 2.31359 1231.29 0.007965 2973.06 0.150831 182.119 0.078398 793.239 4.5358 1273.01 0.050586 2981.31 0.989735 199.739 0.352624 805.197 9.72191 1286.13 3.12213 2985.65 0.851603 211.902 0.319993 828.132 1.70631 1297.77 0.102594 2990.95 0.74862 219.795 0.002334 837.265 0.124137 1302.61 0.126677 2991.21 0.586939 221.326 0.006148 848.906 0.158721 1303.06 0.097841 2992.16 0.635205 224.125 0.000864 856.727 4.60753 1304.32 0.269844 3002.7 0.458844 230.862 0.000399 858.635 0.753255 1308.78 0.013174 3003.94 1.09693 232.974 0.098419 864.34 0.169697 1309.39 0.161873 3004.7 3.08709 250.824 0.390646 866.976 0.37086 1315.25 0.411381 3006.55 1.08213 262.787 0.190051 867.358 0.095587 1362.27 1.32857 3009.86 0.570253 271.446 2.91377 867.41 0.251937 1364.72 2.82007 3011.71 1.04432 297.356 0.500111 868.183 0.024187 1365.28 0.225322 3012.8 0.591885 324.821 4.27252 868.545 0.36739 1383.11 25.5477 3014.66 0.653789 337.536 3.5462 871.013 0.136202 1400.33 0.380929 3026.4 0.791963 374.144 1.11283 873.098 0.049914 1418.02 0.406403 3027.23 0.318039 435.326 0.360087 874.044 0.039629 1418.9 0.058231 3027.9 0.684945
476.45 3.80133 886.572 0.578026 1419.27 0.522316 3032.83 0.623248 487.467 0.080826 907.82 1.35072 1440.71 0.239792 3282.96 0.137207 492.555 1.60261 910.032 0.743322 1450.43 0.413039 3286.68 0.141096 494.502 0.119393 995.683 0.249725 1452.07 0.449939 3291.05 1.84192 494.988 0.18095 998.405 0.091355 1452.31 0.354765 3302.25 2.34048 500.142 0.572343 1014.19 0.091957 1453.2 0.404647 3313.04 0.856469 503.554 0.603114 1063.14 1.8902 1455.22 0.110968 3317.78 0.849213 508.442 2.65646 1066.77 1.19816 1455.27 0.177831 3446.89 9.19874 530.502 0.887841 1074.42 2.25805 1455.38 0.114885 3647.93 10.4417 582.002 7.30487 1108.35 0.04913 1462.5 0.193757 3648.41 1.93077 598.497 1.44603 1125.45 0.034604 1484.13 1.54682 3651.79 5.69046 635.049 4.15942 1144.95 0.39307 1485.73 1.5875 3818 5.36106
194
Table A-17. Mn(II) n-terminus bidentate oxalate coordination - atomic coordinates
Atom x y z Atom x y z H 5.786025 -1.15185 0.890517 H 3.125957 -3.23196 5.326974 C 5.856581 -0.19247 0.358074 C 1.333498 -2.28224 4.509462 H 6.912062 0.111715 0.318147 H 0.782754 -3.22934 4.416409 C 5.272614 -0.31587 -1.08068 H 1.039928 -1.8499 5.480724 H 5.400176 0.644058 -1.60824 C 0.873676 -1.37516 3.398029 H 5.869765 -1.0548 -1.63983 N 1.060736 -0.00666 3.439365 C 3.824086 -0.71754 -1.1323 H 1.466044 0.513528 4.205635 N 3.339733 -1.45668 -2.19106 C 0.565674 0.521165 2.294027 H 3.896369 -1.9329 -2.88832 H 0.581406 1.583534 2.066938 C 1.99165 -1.42645 -2.15577 N 0.081117 -0.422 1.5183 H 1.361383 -1.92851 -2.88574 C 0.262098 -1.60893 2.194716 N 1.583368 -0.71002 -1.12871 H -0.07276 -2.54354 1.752615 C 2.712419 -0.26854 -0.48156 H 3.147323 -3.03973 3.553003 H 2.650083 0.360384 0.400775 Mn -0.56992 -0.31624 -0.71886 H 5.308066 0.562172 0.941829 O -0.76146 -2.61887 -0.61807 H 2.871463 5.06987 0.256194 C -1.45784 -2.77595 -1.62875 C 2.119042 5.835959 0.017023 O -2.65948 -3.27803 -1.58398 H 2.644754 6.755307 -0.27998 C -0.96784 -2.15696 -2.94129 C 1.188331 5.352688 -1.10919 O -0.6857 -2.83021 -3.91991 H 0.477113 6.153626 -1.37156 O -0.82725 -0.89114 -2.76331 H 1.780099 5.164991 -2.01701 H -2.04488 2.403335 0.476366 C 0.437273 4.098512 -0.79503 H -2.98201 -3.02411 -0.64902 N -0.74028 4.041706 -0.07622 H -1.25493 4.832021 0.289383 C -1.13653 2.747089 -0.00859 N -0.27731 1.965047 -0.62658 C 0.703534 2.794628 -1.1216 H 1.535742 2.400444 -1.69815 H 1.556123 6.05198 0.938705 H -5.45286 3.088937 2.021138 C -5.73563 2.122016 1.575484 H -6.32647 2.333989 0.669223 C -4.51794 1.281202 1.252848 H -3.88221 1.83875 0.551256 H -3.91521 1.130259 2.165811 C -4.78658 -0.07619 0.611867 H -5.35538 -0.74879 1.267518 H -5.36221 0.050566 -0.3213 C -3.47052 -0.74646 0.259102 O -2.55322 0.003779 -0.20562 O -3.40646 -1.9792 0.443114 H -6.40765 1.616188 2.288195 H 3.431279 -1.63491 4.604334 C 2.848684 -2.56381 4.498343
195
Table A-18. Mn(II) n-terminus bidentate oxalate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
23.3102 0.409757 680.899 0.117731 1140.4 10.6768 1490.45 4.67874 40.0731 0.225164 682.663 0.147083 1170.95 0.722977 1509.86 13.1174 50.9361 0.302287 686.702 0.322569 1180.23 4.00121 1563.08 5.02582 56.6659 0.438545 690.239 0.430092 1182.86 1.16418 1569.87 3.48934 61.6154 0.205339 690.623 0.202035 1187.26 2.32965 1573.82 1.16046 80.0425 0.005032 693.364 0.404662 1189.85 1.41863 1716.18 55.4533 86.3448 0.064856 721.693 0.719646 1192.47 0.587016 1799.44 45.3281 93.2232 0.31425 723.233 5.35945 1197.48 0.531248 2930.35 100 117.102 0.119199 759.439 7.08447 1212.45 0.022422 2941.54 2.50809 123.985 0.041575 776.254 1.51493 1221.22 0.010181 2945.66 1.17665 133.582 0.241846 778.593 2.29214 1222.85 0.044539 2955.82 1.98752 142.881 0.412729 796.757 2.40096 1229.11 0.009635 2958.09 1.48076 150.405 0.278183 828.357 0.579583 1265 0.141594 2959.21 1.76608 160.393 0.337558 838.528 2.34948 1291.96 0.196197 2969.69 2.2566 168.258 0.113728 845.357 2.35512 1297.14 0.141495 2972.87 3.58278 183.355 1.94268 860.587 1.32461 1298.11 0.170454 2975.82 2.23774
186.24 1.39012 863.833 0.527138 1300.05 0.051595 2984.51 1.68416 199.429 0.568086 865.116 0.37154 1306.73 0.015786 2986.73 1.37373 219.941 0.000609 865.268 1.63015 1311.45 20.1456 2998.06 2.71675 224.191 0.00128 865.632 0.458701 1311.96 8.96657 2999.24 1.34552 226.207 0.000842 865.902 0.032576 1329.15 1.05079 2999.82 3.4548
232.25 0.000672 867.178 0.162628 1365.27 1.03054 3001.47 3.44418 237.908 3.26797 870.453 0.050699 1378.08 0.43148 3001.67 1.86732 264.952 3.78502 870.694 0.198677 1387.56 0.394776 3004.79 2.32913 296.648 2.19184 875.979 21.6664 1393.53 1.26074 3006.53 1.97859
305.01 2.62587 885.017 0.973419 1406.32 0.670416 3013.19 0.136803 323.404 4.60741 892.931 6.02685 1416.67 0.523951 3016.12 5.88358 384.284 1.15614 899.753 4.75563 1417.6 0.554977 3019.06 1.89661
473.87 2.77898 901.056 3.45851 1419.92 0.276804 3020.8 1.67617 482.4 0.104228 984.368 11.1202 1441.32 0.390048 3026.22 1.80036
482.878 0.310328 993.458 0.524837 1448.94 0.629942 3279.5 4.0557 487.206 0.056269 996.631 0.258411 1449.96 0.699517 3279.85 0.536155 490.916 0.235847 1002.44 0.360025 1450.62 0.634437 3281.91 0.064301 493.244 0.046634 1062.97 4.94076 1453.16 0.236626 3286.09 1.96707 508.933 0.087815 1065.96 3.18128 1454.06 0.235668 3287.21 1.10746 568.949 14.0668 1075.91 3.1262 1455.44 0.057048 3302.96 5.15517 579.167 8.30129 1099.69 1.27435 1456.33 0.128467 3664.35 8.13997
599.08 6.78403 1115.65 0.815869 1462.12 0.193305 3666.68 8.18979 603.585 6.79605 1132.45 0.296736 1474.94 3.06397 3668.52 5.8549 678.626 0.175128 1139.07 0.403354 1476.81 2.52118
196
Table A-19. Mn(III) n-terminus bidentate oxalate coordination - atomic coordinates
Atom x y z Atom x y z H 5.799178 -1.50923 0.398606 C 2.949164 -3.19212 3.956788 C 5.893585 -0.50209 -0.03154 H 3.220311 -3.95883 4.695829 H 6.960095 -0.25071 -0.09919 C 1.450596 -2.84987 4.068131 C 5.246232 -0.43941 -1.44692 H 0.850821 -3.75567 3.900041 H 5.375296 0.571475 -1.86695 H 1.214007 -2.51092 5.089337 H 5.788506 -1.1258 -2.11746 C 0.988982 -1.80561 3.0853 C 3.779411 -0.77089 -1.47582 N 1.139796 -0.45142 3.31951 N 3.110337 -1.0438 -2.65761 H 1.521921 -0.03632 4.160798 H 3.545084 -1.26303 -3.54647 C 0.652848 0.23089 2.268507 C 1.781079 -0.94661 -2.45728 H 0.636133 1.312207 2.184445 H 1.038998 -1.1005 -3.23509 N 0.204053 -0.61908 1.363455 N 1.520084 -0.62558 -1.20093 C 0.402527 -1.89308 1.859246 C 2.761761 -0.52166 -0.58127 H 0.098767 -2.76708 1.294781 H 2.845515 -0.24858 0.465271 H 3.189025 -3.57789 2.955363 H 5.420047 0.216851 0.653614 Mn -0.55916 -0.26941 -0.47056 H 3.164973 4.866003 0.5781 O -0.80654 -2.52667 -0.77113 C 2.433023 5.679099 0.463089 C -1.59182 -2.62658 -1.71091 H 2.98141 6.605524 0.241532 O -2.20831 -3.7218 -2.0492 C 1.435442 5.364395 -0.66562 C -1.79944 -1.44062 -2.66767 H 0.746783 6.21354 -0.80226 O -2.37919 -1.55096 -3.70933 H 1.979656 5.264551 -1.61638 O -1.19041 -0.35535 -2.22139 C 0.63976 4.115906 -0.45694 H -1.96255 2.485162 0.650888 N -0.59013 4.079164 0.168603 H -2.04683 -4.39296 -1.36136 H -1.11322 4.883082 0.494341 C -1.01607 2.799744 0.224627 N -0.12595 1.987684 -0.31897 C 0.907944 2.800978 -0.7491 H 1.777776 2.389935 -1.25288 H 1.920088 5.812081 1.428015 H -5.1668 3.047395 2.492861 C -5.51745 2.149251 1.962612 H -6.1379 2.483461 1.115724 C -4.35502 1.299145 1.494197 H -3.73121 1.912927 0.830072 H -3.7169 1.025354 2.350256 C -4.71296 0.034435 0.720993 H -5.27923 -0.69566 1.3158 H -5.31376 0.259594 -0.17465 C -3.44548 -0.64722 0.236763 O -2.30927 0.051137 0.284221 O -3.4989 -1.77492 -0.2273 H -6.17839 1.599097 2.651402 H 3.580141 -2.3088 4.141046
197
Table A-20. Mn(III) n-terminus bidentate oxalate coordination - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
40.2068 0.052859 662.728 2.3984 1153.87 2.95173 1498.78 4.04872 44.5943 0.712056 665.351 25.0725 1163.56 2.41913 1510.53 5.00069 46.9938 0.179385 676.779 0.134253 1169.64 8.16723 1556.92 9.79147 53.3384 0.711669 679.845 0.097637 1180.04 9.7414 1564.89 5.66213 60.5856 0.498281 684.005 0.993208 1189.01 0.886893 1583.07 9.4015 76.3475 0.493297 689.408 0.282216 1190.51 10.5565 1767.67 100 86.5257 0.305185 689.92 0.60608 1196.14 1.17185 1882.02 78.3408 104.501 0.327284 694.239 0.116864 1200.93 1.00031 2955.7 0.802031 134.566 0.042939 717.99 0.170626 1217.44 3.75922 2961.6 2.01908
149.44 1.13341 727.638 12.9285 1218.41 30.4923 2969.25 1.05397 156.781 0.23768 771.77 2.54056 1221.46 37.9763 2972.04 1.16629 166.679 0.427776 777.056 6.68555 1226.41 0.171046 2979.78 2.04319 180.856 0.788062 799.396 5.57047 1231.42 0.074925 2982.35 1.12912
193.05 0.481861 816.498 34.1701 1269.65 5.73282 2983.86 2.29778 198.523 0.153319 831.805 4.10208 1271.45 10.3768 2985.56 4.29689 220.235 0.000403 841.41 16.4614 1296.62 0.615775 2992.36 2.33241 221.683 0.179417 843.752 1.21806 1302.42 0.324977 2993.09 1.99177 224.247 1.84265 848.913 1.07517 1303.29 0.296104 3000.43 1.81918 225.731 0.005807 867.166 1.20864 1305.33 0.882667 3002.41 3.30641 231.598 0.00613 867.379 1.14743 1308.75 0.140656 3005.8 4.04027 233.132 0.053827 867.498 0.055988 1309.36 0.981062 3008.18 0.846744 251.119 4.78638 867.976 0.187095 1318.89 1.19966 3009 7.67711
291.41 10.735 868.149 0.989143 1350.8 0.59495 3010.4 3.53854 315.748 0.856455 870.62 0.341721 1365.32 0.524735 3014.3 0.716992 334.473 1.65912 873.331 0.208484 1379.58 1.38127 3015.76 1.8131
378.38 0.388079 874.021 0.158013 1396.92 1.63095 3016.66 8.09829 404.783 10.026 877.867 1.49681 1418.82 1.5983 3025.43 2.75598
431.13 1.2982 885.492 4.23829 1418.99 0.055004 3027.95 2.34154 482.368 0.459569 886.709 1.43828 1419.54 1.49683 3036.56 1.85941 492.547 0.685829 890.477 15.3791 1436.7 0.705024 3283.71 0.439406 494.099 0.033712 996.596 0.554512 1451.07 1.36374 3295.88 0.163561 496.711 0.059198 998.506 0.825065 1452.1 1.45592 3299.89 2.95306 498.688 0.486006 1018.94 0.687312 1452.48 1.76038 3309.92 1.4963 510.016 1.34693 1063.02 7.16057 1454.52 0.764464 3310.67 11.6626 529.005 11.2149 1064.06 5.00549 1455.48 0.46544 3317.1 4.86971 576.347 1.69394 1080.01 8.29587 1455.54 0.515767 3644.93 24.227 589.499 23.7134 1111.51 0.15698 1457.74 0.704588 3650.19 16.1032
635.5 13.1415 1117.34 0.203645 1461.83 0.654036 3651.22 22.2339 637.869 12.1266 1146.11 1.39818 1487.86 5.64525 3742.46 20.7072 639.159 11.731 1150.4 9.88128 1491.53 5.69568
198
Table A-21. Mn(II) n-terminus bidentate oxalate coordination with R92- atomic coordinates
Atom x y z Atom x y z C 6.069484 -1.13971 2.813349 C -2.06068 1.07012 3.804765 C 5.149901 -0.69454 1.666663 C -1.33586 0.280934 2.952921 C 5.904253 -0.78634 0.337966 N -3.04847 1.626684 3.014591 N 4.993471 -0.9032 -0.78826 C -2.8985 1.165491 1.753052 C 5.285991 -0.63542 -2.0528 N -1.86561 0.351272 1.681065 N 6.484188 -0.1174 -2.3927 H -1.43697 0.471401 5.721227 N 4.343984 -0.82794 -2.98247 H -2.93662 1.385161 5.715393 C 1.203858 5.487432 1.385507 H -3.78525 2.242716 3.333907 C 1.996396 4.649198 0.337267 H -3.55088 1.436641 0.927629 C 1.394826 3.361386 -0.08373 H -0.47357 -0.34947 3.153442 C 0.491069 2.498958 0.470582 C 1.141279 -2.37191 0.458567 N 1.817493 2.735497 -1.23888 O 0.625537 -1.45991 1.179644 C 1.181302 1.548588 -1.34127 C 1.555793 -1.9026 -0.94663 N 0.369743 1.376642 -0.32095 O 1.20805 -3.57996 0.73631 H 2.151045 5.276334 -0.55763 O 2.709423 -2.146 -1.38032 H 3.006045 4.457436 0.741601 O 0.660105 -1.26412 -1.56552 H 2.491788 3.104282 -1.89632 H -0.20375 -3.90246 1.195233 H 1.330916 0.826079 -2.13847 Mn -0.82296 -0.5241 -0.08323 H -0.06542 2.600632 1.397186 H -3.35291 4.651188 -2.51252 C -3.9401 4.246364 -3.35077 H -4.04124 5.038948 -4.1049 C -3.25624 3.015503 -3.97208 H -4.94274 3.998458 -2.97238 C -3.0226 1.836785 -3.0844 H 1.079302 4.930373 2.326035 C -2.3769 1.683774 -1.88852 H 1.750282 6.41331 1.607886 N -3.33912 0.549708 -3.47251 H 0.206157 5.752239 1.005974 C -2.88172 -0.30951 -2.53599 H -0.11855 2.568381 5.247047 N -2.29019 0.345137 -1.55899 H -1.62338 3.518552 5.217744 H -3.85515 2.681821 -4.83547 H -1.09992 2.733435 6.725717 H -2.28675 3.326727 -4.39624 H -6.13718 -4.53424 0.116383 H -3.82025 0.291306 -4.32485 H -6.37295 -3.31876 -1.15375 H -1.93517 2.448755 -1.25783 H -5.37763 -4.75695 -1.47062 H -2.9797 -1.38917 -2.60165 H 6.574222 -1.66454 0.363539 C -5.6357 -4.0038 -0.70889 H 6.53565 0.107971 0.209333 C -4.40272 -3.26232 -0.23326 H 4.269643 -1.35649 1.628664 C -3.36872 -4.18437 0.394811 H 4.774787 0.32828 1.821632 C -2.00728 -3.55511 0.520014 H 5.564597 -1.04659 3.784885 O -1.7319 -2.52143 -0.10265 H 6.366851 -2.19175 2.688494 O -1.18704 -4.20887 1.28133 H 6.986051 -0.52964 2.85479 H -3.93218 -2.74585 -1.08134 H 4.12185 -1.45834 -0.67733 H -4.67786 -2.47297 0.485976 H 3.502084 -1.35751 -2.64509 H -3.6754 -4.5575 1.383593 H 4.618654 -0.87913 -3.95472 H -3.22534 -5.08655 -0.22628 H 7.25972 -0.17647 -1.74785 C -1.14724 2.616815 5.633755 H 6.70742 0.030759 -3.36721 C -1.93122 1.341476 5.264669
199
Table A-22. Mn(II) n-terminus bidentate oxalate coordination with R92 - frequency tables
Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
15.952 0.048585 605.226 3.62181 1177.04 0.229387 1589.16 2.43553 22.9893 0.228476 639.489 1.40987 1180.42 0.060331 1605.35 24.888 29.3438 0.040539 658.945 0.889981 1195.85 0.020416 1651.26 0.928129 40.9457 0.055694 672.985 0.403301 1199.78 0.197844 1655.3 0.562798 49.1994 0.093943 680.483 0.066809 1204.74 0.004858 1664.52 0.028402 53.6499 0.06162 688.553 0.024782 1205.34 0.130276 1665.72 4.96518
56.131 0.187081 690.52 0.299447 1209.53 0.165855 1685.15 45.1161 59.1393 0.143965 692.167 0.125902 1212.79 0.055028 1732.65 0.981673 66.6088 0.077279 706.664 0.511648 1228.54 0.299964 1752.78 20.2673 78.1226 0.046868 706.939 0.265318 1233.52 0.412652 1760.25 53.1704 90.2789 0.032086 710.333 0.150264 1236.82 0.735037 2645.08 100 93.9417 0.035354 714.016 0.315055 1254.96 0.036215 2930.89 0.491377 98.8369 0.062374 732.297 0.046834 1262.6 0.11424 2946.76 0.371567 100.413 0.132239 736.957 0.045189 1269.09 0.093863 2960.36 0.612879
112.02 0.046851 759.922 4.1574 1277.86 0.184325 2960.72 0.585333 115.261 0.174719 779.707 1.3254 1283.89 0.023091 2963.26 0.919142 125.151 0.227768 792.956 0.464319 1284.99 0.081099 2969.37 0.088615 136.609 0.028589 817.675 0.346183 1286.3 0.137309 2974.9 0.782655 142.673 0.188276 843.342 0.542742 1300.47 1.76933 2979.61 1.06918 144.779 0.07953 844.426 1.50488 1301.06 0.377722 2984.73 0.915746 154.205 0.19697 853.11 0.308095 1303.26 0.045503 2986.26 0.793295 164.733 0.142638 856.068 0.160158 1304.08 0.075897 2988.29 0.645821 168.495 0.117182 856.313 0.635617 1309.74 0.148339 2992.99 27.7397 192.531 0.755186 860.065 0.059123 1310.67 0.361372 2996.92 0.348264 200.652 0.543175 864.447 0.816483 1311.31 0.05872 2997.3 0.877548 205.039 0.074515 867.158 0.018071 1367.47 11.6818 3000.83 0.448676 207.299 0.378693 867.46 0.226852 1374.51 1.28406 3001.84 0.681908 214.085 0.133795 868.748 0.262995 1397.06 0.04195 3003.01 0.999075 217.163 0.338917 870.604 0.236653 1400.88 0.0278 3005.03 1.11642 224.749 1.46564 871.03 0.259828 1404.49 0.048377 3007.34 0.746652 235.296 3.80269 871.824 0.065609 1419.99 0.201121 3009.86 1.32538 243.737 0.366904 876.576 0.166733 1420.24 0.422212 3011.35 0.668165 247.656 0.219739 878.634 0.022476 1421.71 5.98769 3016.98 1.16206 256.966 1.61257 878.866 0.051471 1424.81 0.381063 3023.83 0.832295
262.3 1.24875 889.568 1.4834 1433.81 0.450813 3025.78 0.580848 265.263 0.043438 905.28 0.734558 1434.48 0.184431 3028.02 0.781731 267.332 2.11987 915.536 2.67133 1439.58 0.892766 3030.84 2.80582 287.777 0.062811 938.364 0.191672 1449.6 0.300621 3034.55 0.643841 295.601 0.190129 956.569 2.07547 1452.15 0.564086 3059.21 0.318213 302.129 2.52913 958.227 0.905051 1453.23 0.406294 3086.98 1.54282 306.272 0.455743 963.607 0.559648 1454.83 0.237645 3118.45 0.27068 312.087 0.345607 967.822 1.61485 1455.68 0.096003 3197.47 69.757
333.03 0.261155 968.466 0.952033 1456.08 0.107627 3282.76 0.528567 341.504 0.95464 968.926 1.12231 1457.2 0.189081 3286.73 0.089043 345.941 0.434976 1012.77 0.499588 1458.36 0.1231 3287.62 0.203249
200
Table A-22. Continued Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
348.137 0.478041 1014.2 0.359545 1458.86 0.390512 3288.2 0.252342 359.918 0.181133 1015.09 0.33598 1460.27 0.32784 3293.71 1.22646 372.248 2.95719 1015.51 0.539864 1461.73 0.263419 3296.73 0.392555
387.2 1.49314 1040.01 3.14773 1476.45 0.233991 3622.56 3.78731 427.657 4.1724 1090.27 0.274046 1480.52 0.500423 3656.43 4.7124 484.262 1.39116 1129.77 1.01379 1484.28 0.599754 3658.49 4.18278 498.403 4.01723 1133.86 1.40983 1493.52 0.713604 3664.95 3.18851 541.533 0.510033 1134.4 1.80649 1531.34 4.83608 3670.66 4.74549 573.495 1.33567 1144.99 1.05201 1540.05 6.47336 3742.65 3.47069
580.43 2.61534 1146.09 1.40078 1553.34 3.87521
201
Table A-23. Mn(II) n-terminus monodentate oxalate coordination with R92- atomic coordinates
Atom x y z Atom x y z C -5.85153 2.337 -2.31266 N 2.394854 3.806119 0.109938 C -4.989 1.56815 -1.30093 C 1.990735 2.66081 0.71524 C -5.18605 0.065727 -1.5251 N 1.354747 1.881204 -0.13497 N -4.63773 -0.8272 -0.50785 H 2.238567 3.883911 -3.24621 C -5.32538 -1.23094 0.552462 H 3.698656 4.12811 -2.33641 N -6.42085 -0.55988 0.947015 H 2.941003 4.53987 0.542554 N -4.94915 -2.31572 1.234703 H 2.174543 2.43933 1.762791 C -0.19375 4.470722 3.464168 H 0.927583 2.111788 -2.22589 C -1.19873 3.282676 3.513739 Mn 0.826437 -0.33775 -0.40871 C -1.22666 2.386397 2.322892 O -0.39224 0.077329 -2.19645 C -0.95856 1.050243 2.192997 H -1.08012 -0.6203 -2.16261 N -1.60341 2.783965 1.054411 H 0.29196 -0.21629 -2.90124 C -1.52741 1.706843 0.235732 C -1.39468 -2.50536 -0.52487 N -1.13656 0.634735 0.890522 O -0.22455 -2.22245 -0.25141 H -0.97079 2.656853 4.388881 O -2.18389 -1.93191 -1.33888 H -2.20937 3.689593 3.690901 C -2.03743 -3.68707 0.237654 H -1.84745 3.72366 0.770159 O -1.61937 -4.2054 1.225169 H -1.68471 1.753072 -0.83868 O -3.24066 -4.01529 -0.31478 H -0.64598 0.366 2.975674 H 3.746914 0.163752 5.057212 C 4.232736 -0.81808 5.157069 H 4.47853 -0.9751 6.217454 C 3.314813 -1.93443 4.627976 H 5.181623 -0.78059 4.600289 C 2.98647 -1.69447 3.196934 H 1.680307 6.390725 -3.08615 C 1.839015 -1.2866 2.581264 H 3.186195 6.654294 -2.15112 N 3.94075 -1.68983 2.19766 H 3.271138 5.99112 -3.81162 C 3.35691 -1.28925 1.048227 H -5.58638 2.061731 -3.34492 N 2.081314 -1.02845 1.250211 H -6.92132 2.116933 -2.17271 H 3.79746 -2.91651 4.760417 H -5.71676 3.423003 -2.21325 H 2.377485 -1.96575 5.20083 H -3.92594 1.824477 -1.41042 H 4.909385 -1.96475 2.297116 H -5.26942 1.847007 -0.26973 H 0.850682 -1.18567 3.01569 H -4.72144 -0.22595 -2.47715 H 3.848284 -1.20311 0.083017 H -6.26212 -0.15232 -1.62492 C 4.904295 -4.0888 -3.01389 H -3.71357 -1.2547 -0.69895 C 3.850297 -3.06043 -2.66236 H -6.61043 0.371595 0.605553 C 3.682625 -1.77595 -3.47518 H -7.04886 -0.95493 1.63325 C 2.470183 -1.01149 -2.93819 H -4.37863 -3.03809 0.781964 O 2.468693 -0.74627 -1.68049 H -5.37576 -2.53011 2.126099 O 1.534125 -0.7194 -3.70875 H -0.39484 5.136987 2.610702 H 2.870934 -3.5733 -2.66713 H -0.27251 5.068401 4.382649 H 3.992275 -2.76279 -1.61104 H 0.840448 4.107403 3.374607 H 4.576117 -1.13548 -3.38365 H 5.925018 -3.67443 -2.96106 H 3.513863 -1.97927 -4.54145 H 4.851456 -4.93658 -2.3154 C 2.688045 6.00312 -2.8832 H 4.769855 -4.48858 -4.03202 C 2.633848 4.421138 -2.37173 H -3.27106 -3.46609 -1.13155 C 1.999111 3.753846 -1.21313 C 1.340364 2.560729 -1.32704
202
Table A-24. Mn(II) n-terminus monodentate oxalate coordination with R92 - frequency tables
Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
12.9347 0.054254 603.614 0.651657 1149.04 0.974552 1578.53 0.50658 22.5584 0.209889 608.046 1.27568 1151.21 0.304748 1643.61 1.56859 23.8886 0.166767 615.489 1.85423 1155.05 3.17085 1645.87 0.078961 36.0848 0.053106 630.018 0.715191 1163.83 0.10992 1646.23 0.276976 36.8542 0.173637 665.057 0.636907 1165.21 3.03371 1654.44 16.0167 42.8126 0.044367 681.314 0.029537 1178.01 0.375566 1663.9 0.991196
53.691 0.102078 683.404 0.820796 1181.64 0.084478 1668.57 5.38158 55.9322 0.254948 686.643 0.074683 1195.39 0.046069 1697.81 18.035 61.1991 0.026751 688.74 2.06354 1211.48 1.97341 1735.09 26.5218 66.3295 0.10597 689.542 0.289724 1212.2 0.140831 1765.56 35.4711 69.2511 0.208579 695.058 0.527728 1213.82 0.041592 1941.17 13.7402 76.6918 0.268829 701.491 1.08969 1227.01 0.358272 2768.03 100 80.3043 0.219963 710.171 0.204054 1231.26 0.282677 2929.55 0.888506 83.0871 0.030895 710.857 5.88534 1241.53 0.020418 2937.71 0.795 105.083 0.179788 713.281 0.00737 1262.75 0.244214 2943.69 1.30598 106.892 0.196196 718.587 0.137385 1264.42 0.140472 2947.62 0.981068 114.361 0.243414 722.06 0.478481 1274.85 0.155516 2964.2 0.681618 127.304 0.006951 736.773 3.99293 1281.85 0.313888 2969.83 1.6149
130.47 0.012479 753.068 0.447098 1289.31 0.841611 2979.61 0.484332 131.8 0.312176 755.364 3.16165 1291.33 0.097221 2987.19 3.11902
146.964 0.094397 777.434 12.942 1294.21 0.269698 2989.52 0.679916 152.581 0.038936 822.989 3.72762 1299.5 0.050637 2990.06 0.775838 155.435 0.131991 829.015 1.55699 1306.7 0.129549 2991.88 0.83371 161.046 1.07155 829.221 1.08623 1307.49 0.064082 2998.78 0.164598 175.644 0.170272 835.245 0.450715 1309.04 0.071242 3001.03 0.579958 183.604 0.168474 843.108 0.148109 1312.62 0.043637 3001.09 1.45213 190.925 0.578015 844.332 0.076873 1326.65 0.411345 3002.01 0.7487 199.028 1.41495 852.02 0.234364 1329.03 0.116833 3008.37 1.46938 200.537 0.361433 857.614 0.01818 1375.67 25.604 3010.14 1.00855 203.279 0.766708 859.053 1.3574 1382.55 0.138797 3012.59 1.39365
215.37 6.15517 863.547 0.272226 1387.71 1.13699 3015.74 0.993424 216.317 0.507578 867.393 0.268566 1391.01 0.07705 3016.91 1.09282 218.979 2.68559 868.03 0.071392 1397.73 0.067984 3021.35 0.998415 223.786 0.060894 868.629 0.467988 1424.12 0.37388 3025.74 0.703898
226.66 1.36329 869.405 0.618303 1426.99 0.548913 3027.31 0.897894 245.568 0.184179 872.031 0.264095 1429.74 1.23754 3028.43 0.923781 252.757 0.192885 875.204 0.471069 1431.43 1.22207 3050.09 0.737305 273.504 0.158458 878.282 2.21708 1437.13 0.667185 3062.62 0.939706 286.286 0.066513 882.828 0.511963 1437.62 0.383268 3068.06 1.73717 296.911 0.058548 886.681 0.264073 1443.87 7.09399 3135.92 0.280261 304.649 0.892655 903.896 1.13782 1443.97 10.8413 3136.17 0.985539 309.954 0.155683 906.352 3.30624 1449.85 0.377643 3228.35 53.3627 311.742 0.888968 918.793 0.002979 1450.87 8.0243 3283.67 0.054791 317.414 0.276542 944.692 1.47183 1451.24 7.12992 3286.05 0.230994 320.813 0.532354 948.255 0.799036 1452.63 0.030428 3286.81 0.51374
203
Table A-24. Continued Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
325.364 0.833001 954.592 0.143982 1453.25 0.12526 3289.55 0.501937 331.219 3.31393 962.799 1.11305 1454.78 0.300958 3293.83 1.93895 345.837 0.869108 974.304 2.13095 1457.25 0.651533 3304.3 0.504755 359.328 0.128293 1003.78 0.455959 1459.29 0.245824 3395.79 48.581 370.134 0.793513 1013.91 0.583571 1461.78 2.30318 3544.41 13.3584 382.224 7.43684 1016 0.319412 1466.88 5.01002 3591.08 20.3343 457.814 1.99019 1018.37 0.40996 1471.75 1.56941 3621.36 4.14545 466.257 0.237284 1080.13 0.046751 1472.57 0.451247 3662.37 2.7911
482.88 2.84223 1112.08 0.622082 1475.69 0.433019 3662.62 5.79818 505.711 3.0244 1122.04 0.747122 1482.37 0.849222 3662.96 5.42041 524.827 0.227671 1130.27 0.509935 1504.76 0.948622 3686.77 7.07351 579.874 6.94367 1131.62 2.14313 1540.97 0.706458 3746.98 5.85979 583.916 0.567635 1137.97 10.4094 1544.7 3.43077 601.983 3.95992 1144.29 1.1171 1553.17 2.5586
204
Table A-25. Mn(II) n-terminus water/water coordination with SMD - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-68.1702 0.529074 633.046 2.44859 1134.49 2.60935 1564.21 2.42798 -66.1533 0.088642 669.866 0.308798 1170.33 2.80703 1567.81 2.22754 -46.7356 0.196158 681.739 0.195042 1173.47 1.04442 1602.42 4.02672 -30.0754 0.523919 683.141 0.364701 1174.76 2.49613 1648.11 7.61994 57.8117 0.224554 683.409 0.191563 1189.76 0.103589 2530.66 100 72.2899 0.30172 687.278 0.282148 1191.57 0.167921 2941.59 1.69834
88.535 0.255417 692.57 0.413009 1192.27 0.117914 2966.29 0.885249 106.096 2.18406 698.021 0.251864 1216.54 0.01899 2966.97 0.85856 114.563 0.463162 725.482 0.078271 1219.3 0.030956 2968.35 0.712702 119.672 1.1192 777.393 1.62527 1223.2 0.00729 2970.34 0.533387 136.978 0.160635 781.812 1.1643 1224.91 0.007935 2977.84 2.16939
143.19 0.228952 782.265 1.71787 1269.33 0.052015 2985.19 1.67692 152.597 0.2896 834.788 1.21804 1274.59 0.047373 2991.31 1.18903 161.166 1.34884 835.776 2.43644 1281.27 0.056301 2992.13 1.17035 162.405 1.08701 847.513 1.13489 1283.5 0.056741 2994.88 0.80758 172.786 0.544897 852.671 0.374242 1286.6 0.039134 3002.02 0.834968 187.518 0.009804 856.98 0.084603 1299.79 0.274943 3002.47 1.61788 191.049 0.032755 858.838 0.137583 1304.61 0.304365 3004.13 2.41535 194.669 3.21724 859.071 0.490199 1305.22 0.378196 3004.51 1.10061 208.706 4.10344 862.469 0.497759 1376.76 0.076337 3004.87 1.83544 216.091 0.002456 862.827 0.276047 1377.67 0.036828 3007.08 2.00008 223.338 0.007309 864.38 0.884874 1379.31 0.139881 3009.71 1.77613 235.712 4.59059 864.662 0.017352 1389.67 0.348963 3011.08 1.86179 246.728 5.20699 868.165 0.200336 1398.75 0.28141 3012.61 1.31272 265.696 1.77864 873.268 0.5192 1400.28 0.300122 3024.32 1.48848 299.873 6.40855 887.366 15.9125 1401.82 0.321745 3026.02 1.42054 325.944 5.73458 892.594 0.802765 1422.8 0.35026 3030.31 1.33992 398.135 1.11723 995.659 0.199456 1424.37 0.095762 3287.21 0.076927 486.707 0.032261 997.728 0.214476 1425.68 0.362438 3295.61 0.019086 488.103 0.313246 1004.99 0.190224 1425.85 0.346341 3299.44 0.409804
491.83 0.011325 1067.61 5.69713 1428.11 0.195174 3301.2 0.080159 493.755 0.18866 1071.31 2.61219 1432.89 0.146538 3304.33 1.26182 495.422 0.211514 1074.45 3.21667 1433.02 0.129359 3304.5 0.347352
498.7 0.069603 1076.33 0.999437 1434.43 0.272194 3649.07 9.98388 533.364 8.77236 1095.16 1.26375 1438.79 0.108165 3657.37 9.28035 565.965 19.2649 1114 1.15962 1456.54 1.92862 3661.74 9.45617 573.968 6.09515 1119.71 4.1982 1460.42 1.78497 3674.55 9.17085 589.243 6.73008 1127.24 0.796618 1465.01 1.97776 3831.25 7.16056 596.105 5.84042 1129.28 1.10127 1559.46 4.05714 3860.83 4.71996
205
Table A-26. Mn(III) n-terminus water/water coordination with SMD - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-156.428 9.3447 656.515 0.480373 1139.61 7.54826 1543 2.02282 -49.6206 0.70624 661.951 0.815006 1145.87 4.05896 1547.86 5.48969 -36.3882 0.425876 675.116 0.42039 1157.22 3.98263 1558.42 5.76512
-16.678 0.734309 675.607 0.20371 1171.58 5.05099 1574.52 15.6669 7.8647 0.238461 679.918 0.747567 1181.08 0.314814 2527.62 100
39.7306 0.437604 680.143 0.46679 1192.96 0.090195 2958.79 1.32936 93.5428 1.75115 683.345 0.18 1196.22 0.111544 2965.3 0.52803 102.099 1.06441 690.742 1.43099 1211.2 0.006256 2974.38 0.723203
125.1 2.24862 730.293 0.399592 1222.82 0.01067 2975.27 0.371091 137.973 0.727729 740.012 35.7563 1223.1 0.010142 2979.05 0.60313 149.476 1.10359 766.831 3.53306 1227.53 0.009882 2984.32 2.52213 174.882 1.97717 776.933 1.55839 1275.44 0.044643 2992.01 1.19924 177.931 0.106675 796.093 1.86891 1279.84 0.084607 2993 1.78551 181.264 0.098361 810.632 2.32431 1280.54 0.078921 2996.25 1.36747 186.912 0.089286 825.669 1.62422 1283.3 0.070732 2998.76 1.3782 196.099 0.966626 828.917 2.10606 1288.22 0.20792 3003.53 1.08855 197.296 1.6098 849.437 0.685734 1288.82 0.540592 3004.14 1.77626 204.436 0.019198 857.599 0.372204 1291.29 0.78076 3007.12 2.95773 210.675 0.000556 859.771 0.223019 1306.84 1.01534 3007.7 2.41109 223.823 0.457544 860.329 0.614084 1333.02 0.126305 3009.49 1.22476 232.317 1.02541 862.35 0.360296 1355.81 0.106949 3013.09 1.75502 244.992 2.26953 862.917 0.374273 1363.82 0.056695 3016.95 1.5451 263.487 1.97821 863.827 0.195047 1372.58 0.561822 3020.49 1.69924 300.983 0.33123 865.094 0.03497 1398.31 0.31748 3022.2 0.877484
330.97 6.99252 866.802 0.258267 1398.88 0.410207 3029.88 1.85602 372.842 5.99897 869.837 0.876366 1400.09 0.404498 3031.71 1.71156 408.516 15.3032 880.373 0.7984 1423.49 0.201656 3038.84 1.41323 449.748 0.138352 994.184 6.19503 1423.72 0.416824 3287.16 0.133307
480.49 0.552691 995.433 0.781739 1426.82 0.520239 3301.62 0.395757 491.051 0.054919 1000.22 0.31787 1427.15 0.301046 3307.38 2.82765 491.953 0.0535 1016.63 0.197895 1429.86 0.335751 3312.32 1.37406 493.325 0.2806 1049.28 3.06513 1431.65 0.13097 3317.56 0.817561 495.969 0.088321 1059.1 2.78164 1431.83 0.299919 3324.48 2.01172 509.177 1.92064 1060.84 3.25318 1432.72 0.267444 3613 15.0236 591.886 23.407 1096.71 0.537444 1437.77 0.145758 3625.72 11.9513 604.996 8.30803 1122.58 1.20082 1470.95 1.9923 3627.81 15.94
622.4 7.67974 1127.74 1.34946 1474.33 2.20992 3728.83 10.2255 643.927 5.27517 1131.02 2.6621 1485.01 1.99679 3818.74 14.7225 646.469 4.65606 1139.36 0.888029 1538.87 8.15732 3842.87 9.37653
206
Table A-27. Mn(II) n-terminus formate/water coordination with SMD - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-98.1585 92.2087 640.9366 490.435 1173.777 44.9988 1560.633 133.2763 -59.8626 24.4518 678.114 17.4737 1176.509 153.6117 1565.45 20.4154 -50.1883 21.2918 680.2003 4.6599 1178.12 44.5679 1569.679 31.073
-29.554 6.8591 681.0103 6.195 1179.544 60.5333 1636.006 159.1462 12.6826 2.8601 684.6795 15.6892 1185.643 5.1964 1705.584 923.4824 48.8146 1.4239 687.3562 9.9705 1190.992 4.9653 2938.86 251.1104 61.1906 10.8215 689.1427 1.6058 1194.632 3.7233 2940.578 41.9329 67.3501 18.2528 704.9054 5.3746 1215.602 0.2545 2956.754 30.8778
80.408 5.7779 716.7549 2.5424 1219.624 0.7026 2959.522 26.2279 90.5531 6.051 757.7921 98.7386 1221.492 0.2455 2961.181 25.9012
111.4551 4.06 769.6388 39.6174 1227.047 0.116 2962.816 16.2957 130.163 5.4233 776.7473 37.5219 1271.333 2.9017 2969.89 56.9239
133.0957 31.0487 778.5247 33.1153 1277.145 0.9528 2975.47 52.9151 144.775 20.537 838.8818 8.6712 1280.942 1.4665 2985.833 31.3772
150.6387 38.8617 843.8929 76.9911 1287.028 1.5453 2987.836 24.4806 161.8242 42.7893 853.7258 6.0632 1292.02 0.9511 2988.306 27.4766 172.3376 23.5531 856.6994 18.0068 1296.656 27.3639 2998.247 30.5333 184.1781 13.7437 859.6593 8.7934 1299.318 20.633 2999.264 38.8213 187.6285 6.2636 861.64 6.1311 1306.739 10.0521 3000.018 50.9663 192.0868 31.9225 866.3104 38.6956 1314.338 521.6604 3000.104 58.1745 211.6195 89.7989 867.7395 3.8984 1383.832 2.9911 3001.326 45.6339 216.8812 0.55 867.8134 7.8171 1386.934 14.6054 3002.385 56.7421 217.2322 0.1729 868.3618 7.0695 1391.965 2.8039 3004.101 50.9981 228.9803 0.0945 868.4248 9.0335 1392.15 7.0824 3005.193 39.0465 233.1154 0.024 870.768 15.2052 1399.272 7.2859 3012.796 42.5621 288.4918 95.3219 873.0177 354.3762 1400.79 7.6961 3019.408 35.9292 317.5519 92.7424 885.4296 22.1481 1403.97 6.8063 3020.139 33.3078 322.6019 29.9884 901.0964 57.7176 1422.209 13.1647 3025.696 31.4403 373.6065 123.1974 988.9736 4.7552 1422.566 33.8243 3243.647 89.7121 482.2471 3.0018 995.5157 7.9437 1426.859 7.4756 3279.269 2.8441
485.702 0.7087 997.3101 5.5918 1427.224 10.1899 3288.825 2.7469 488.6956 14.5375 1069.665 71.3785 1427.689 8.5479 3304.046 35.7317 489.8002 1.4457 1072.479 83.1527 1432.391 3.4507 3304.852 7.7577 491.3032 8.7287 1074.608 24.5701 1434.131 3.0944 3308.02 25.1111 495.3429 4.1869 1094.61 1.6312 1434.904 3.0754 3606.269 397.5206 552.4259 155.8511 1099.341 23.0519 1435.829 10.2355 3665.98 238.3438 556.0639 155.6664 1111.61 21.2428 1437.669 8.3232 3672.708 239.8091 561.7751 109.056 1112.423 1.1328 1439.802 35.54 3679.322 237.2727 567.9178 231.7124 1114.266 22.4306 1446.648 49.8969 3681.144 371.0973 606.6262 156.7525 1128.558 9.4172 1450.686 47.6611
207
Table A-28. Mn(III) n-terminus formate/water coordination with SMD - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-72.3531 11.47 615.4151 171.9797 1151.856 109.7883 1555.111 128.2874 6.6883 3.9111 670.9033 6.3799 1156.613 143.4275 1558.832 52.3766 33.085 10.6731 676.5449 9.2503 1171.993 64.7451 1566.101 66.5156 43.921 5.5214 680.8314 10.5007 1182.93 23.4982 1572.49 138.1544 59.106 5.366 681.4765 5.3224 1186.554 161.8936 1770.052 777.6417
67.1567 2.5524 682.9494 3.9612 1188.354 494.0088 2951.291 34.1886 81.4797 12.6105 685.189 8.204 1202.01 1.6485 2966.524 12.5375 94.1069 63.5331 690.0818 7.3593 1208.908 261.0622 2969.428 20.5319
100.6576 16.203 732.3931 37.8685 1217.06 1.0352 2969.983 18.5734 110.4791 3.5009 735.5894 585.6431 1221.021 1.0049 2972.737 11.2529 135.7152 16.1948 746.6782 66.9712 1224.427 3.4259 2977.846 57.3715 142.7072 26.9989 764.6675 43.7156 1233.279 0.1935 2984.796 45.5462 157.0752 200.4712 772.6425 34.7754 1276.561 2.3804 2991.413 30.0794 164.8585 16.4472 785.6809 29.4247 1280.168 1.1202 2992.322 25.1862 173.5944 57.2855 823.4303 52.9931 1281.284 1.5557 2994.153 27.5613 196.7503 3.929 838.5156 34.5653 1284.485 1.4129 3003.135 24.6241
199.567 27.3815 859.1542 9.4997 1292.46 1.0294 3004.649 40.5404 205.6756 1.1564 862.229 10.8511 1299 9.7889 3005.227 24.5829 208.1229 32.9732 862.5795 7.4774 1300.124 5.646 3006.568 56.1053 209.3135 0.9146 863.5956 5.0452 1308.559 8.9506 3006.948 54.7142 213.8416 0.1298 864.9698 1.764 1349.988 3.2965 3007.627 59.2158 215.7839 0.3624 865.5561 4.481 1358.945 1.5175 3012.421 32.3417 225.1787 25.7712 867.0248 2.563 1372.296 9.8366 3014.187 39.1981
233.369 35.1269 870.5452 3.4921 1381.968 8.8712 3025.492 36.8052 238.1327 24.894 872.8999 4.7487 1395.007 7.5302 3026.265 27.0207 281.8589 112.2439 882.5541 20.7956 1400.987 28.9999 3027.069 37.1263 320.9239 246.029 883.3168 25.2579 1402.953 7.7017 3033.962 32.1388 338.6662 136.6997 885.4603 52.7871 1404.383 6.7754 3046.282 100.8905 365.8778 23.6534 992.7409 2.8908 1424.475 7.0279 3288.207 90.2094 456.0475 139.8887 995.9993 4.0926 1427.844 5.7773 3291.037 9.393
480.097 17.9182 1002.881 7.8747 1428.857 9.333 3292.001 3.7392 485.694 46.7132 1055.954 59.7199 1429.042 11.1294 3306.983 57.1442
487.0936 4.2577 1064.231 15.8032 1431.54 2.9893 3311.708 16.9767 493.6073 5.2509 1066.629 51.8656 1433.377 5.7727 3319.397 29.5204 500.4454 9.0333 1069.062 87.1826 1433.515 5.7734 3645.136 309.6675 502.8357 3.2743 1104.964 0.3991 1434.658 3.8188 3647.602 262.3633 560.1666 171.9591 1122.38 19.3023 1438.411 2.3208 3654.776 264.456 585.1248 169.9665 1128.347 19.2045 1458.976 47.5048 3721.85 137.322 602.3249 338.0963 1133.311 35.6734 1463.872 52.744 3851.157 211.0566 604.0904 52.2749 1136 3.749 1467.944 45.4667
208
Table A-29. Mn(II) n-terminus acetate/water coordination with SMD - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-58.9478 9.2435 621.5084 32.1578 1126.311 11.8407 1481.229 551.3404 -49.876 7.48 664.5186 113.8569 1166.663 145.8686 1562.881 95.1663
-28.3411 10.3997 678.2467 11.643 1173.985 58.2349 1569.419 27.735 -6.3858 8.1087 680.3479 11.4452 1180.451 44.7354 1575.089 140.7363 10.1491 0.5234 680.7537 2.5232 1184.874 69.1443 1590.707 947.7314 28.9635 8.4397 686.854 11.6078 1188.593 4.3913 1626.208 101.4857 45.4215 3.3705 687.6713 9.3508 1189.007 2.2284 2847.174 1981.327 59.2271 14.2905 689.639 4.4246 1193.965 3.2875 2938.884 43.4537 70.9997 4.8532 702.8607 6.2802 1217.531 0.8321 2954.967 31.9465 84.4109 8.4411 719.3421 3.4247 1219.349 0.3892 2957.794 18.1458
109.9053 31.5024 768.7371 41.5057 1225.001 0.1568 2959.499 25.6172 119.4644 1.368 779.4545 20.3079 1225.118 0.0776 2959.722 26.1916 125.0006 1.3868 780.6619 52.882 1269.844 2.4438 2968.7 56.9306 129.9414 3.4335 827.2273 65.611 1278.017 1.5287 2973.271 54.8948 141.2526 10.4687 844.5916 20.5313 1278.897 1.1379 2983.978 29.2101 152.0093 24.4333 854.1931 23.5017 1287.086 1.6006 2986.166 30.8416 165.1456 19.2027 858.5073 8.303 1290.4 0.9431 2988.618 27.668 168.5697 42.3463 860.8293 10.7358 1302.323 4.3389 2998.312 31.057
183.006 41.0802 862.2569 3.3274 1312.761 3.6678 2999.523 29.7282 199.9743 16.3941 864.0354 3.5012 1314.138 13.3011 2999.837 45.3126
207.757 35.5773 868.3588 3.6801 1342.113 12.1776 2999.984 44.7723 211.7135 0.0682 869.3888 1.5423 1382.294 12.2949 3000.33 61.4189
227.538 0.0607 869.8514 18.5199 1383.116 2.7809 3001.016 63.4391 236.6806 0.046 873.765 3.0244 1391.249 7.3145 3004.143 49.836 237.3821 0.0769 876.8438 12.4296 1396.108 5.3649 3004.95 39.5602 277.1062 155.295 879.6544 41.3318 1401.327 7.9979 3013.016 45.2224 291.3173 21.8936 884.4019 462.6241 1401.392 7.5466 3019.568 35.0629 295.1605 102.152 914.3427 24.945 1402.636 4.9043 3019.67 35.2416 352.1748 62.731 937.0635 63.9576 1412.749 87.3327 3025.33 32.2955 415.5026 149.3091 985.9216 5.2855 1424.219 12.5565 3069.04 2.9655 480.8923 2.7282 995.1109 6.1976 1427.356 3.027 3151.767 4.8439 483.2477 4.7241 999.9196 7.5914 1427.518 19.7565 3184.182 20.9411 485.1434 2.8668 1027.262 49.774 1427.881 5.6552 3259.684 36.4421 487.7741 0.8868 1050 181.2121 1429.852 7.144 3277.747 2.1798 489.2103 14.3223 1066.676 53.6568 1430.5 5.8179 3280.559 16.0134 493.4067 0.6102 1067.724 137.871 1433.271 2.9707 3282.217 6.1748 494.9861 2.102 1071.518 35.7157 1433.651 6.847 3296.38 9.1999 546.4157 140.5201 1074.048 41.0408 1436.069 43.3365 3302.604 33.526 555.1344 124.6946 1101.586 21.593 1438.46 8.9565 3665.034 240.5838 566.9596 145.7097 1106.192 1.5741 1440.815 4.8197 3667.281 229.0287 575.2573 212.2154 1115.295 26.1118 1446.414 53.4896 3678.588 242.218 588.2356 155.9395 1123.935 19.5519 1454.641 50.5996 3816.266 123.3891
209
Table A-30. Mn(III) n-terminus acetate/water coordination with SMD - frequency tables Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-64.5496 9.5734 613.0949 162.5079 1134.002 6.3918 1467.57 37.7984 -48.2928 1.1397 677.3746 9.2098 1154.725 83.1003 1556.764 174.377 -15.1134 13.1469 679.9882 44.3941 1159.82 136.945 1561.151 22.2012
22.802 2.0609 681.5123 2.6209 1171.712 75.6221 1574.764 82.0267 37.627 9.8516 682.6961 55.5573 1182.381 5.6575 1589.568 289.7531 69.659 2.7911 684.5513 24.2795 1187.452 1.7605 1678.062 554.4759
82.2232 2.5 685.8938 9.7097 1197.225 3.4609 2866.843 1772.732 87.54 0.913 687.3262 12.7918 1201.83 151.5254 2949.402 36.8185
109.7934 8.5357 689.6827 13.4046 1214.118 0.1375 2961.659 14.4212 129.0456 2.175 714.4945 3.0719 1217.196 0.5364 2970.492 18.4226 130.5516 8.8575 752.5743 569.2065 1222.173 0.4885 2970.707 13.0317 144.5349 11.3546 770.1029 41.5572 1229.156 0.1615 2972.657 15.552 172.9335 12.6218 771.4457 98.5303 1271.583 3.3841 2977.434 56.8517 184.1622 20.8888 783.2772 48.6572 1278.301 1.5546 2984.233 47.874 187.5038 2.7305 815.9269 49.1022 1279.721 1.3075 2988.718 29.7121 196.0992 22.6232 840.1154 38.6494 1285.636 1.6648 2991.982 30.002 214.2121 1.7752 840.6295 40.9744 1291.441 1.6125 2994.234 30.2104 214.5764 0.0215 857.8707 13.2535 1299.494 6.4475 3002.898 40.8581 215.8004 1.0249 859.7837 10.1257 1305.463 109.9547 3004.314 16.3342 217.1096 1.3672 860.8519 7.5753 1308.707 453.4064 3004.738 22.2524 222.4923 0.8936 862.5475 5.5689 1311.433 9.4099 3006.659 63.9075 232.4837 1.7043 865.2275 5.4471 1355.025 1.7858 3006.733 50.1749 246.6047 32.0136 865.3408 13.774 1359.082 3.7306 3008.394 59.5189 269.0005 41.2559 866.3042 5.1193 1368.465 9.9817 3012.317 31.699 292.8077 104.5153 870.246 1.5705 1381.358 506.0392 3013.923 38.2823 308.1543 81.5517 875.3081 6.8215 1384.418 6.7922 3025.567 37.7589 315.5724 63.4366 877.4691 18.8335 1401.795 7.6674 3025.61 30.263 355.1214 31.8279 880.0701 19.9994 1402.294 5.6224 3026.63 37.1895 366.2548 270.0405 941.3164 133.5415 1404.108 8.5397 3034.186 32.2513 381.4983 88.6657 996.9175 5.4561 1417.696 42.9197 3087.336 2.6336 475.7176 5.9574 999.4816 2.8563 1419.813 8.9821 3176.61 1.3691 486.7012 12.4621 1001.585 7.3757 1425.828 5.2853 3202.248 8.1898 490.4191 3.2496 1025.187 97.5003 1426.45 8.6711 3290.948 6.3427 492.1465 8.2887 1030.58 225.115 1428.734 9.5237 3295.021 6.6706 496.1129 5.077 1055.069 49.4949 1431.432 5.032 3298.448 7.2723 501.4485 2.566 1057.994 62.5804 1433.551 3.6928 3303.531 21.6692 549.6119 74.0171 1058.377 62.4237 1433.916 49.4522 3305.497 12.7502 560.4921 77.6695 1063.662 41.9677 1435 2.582 3308.708 70.6607 570.0184 309.4135 1102.178 0.2386 1435.624 5.1649 3640.69 297.2092 579.8977 150.3919 1123.696 14.9593 1438.934 2.8568 3650.259 367.6255 607.9312 173.7051 1132.608 35.9663 1462.007 49.1814 3650.578 175.1917 611.0946 55.3318 1133.336 14.0667 1465.721 60.1473 3834.93 159.8784
210
Table A-31. Mn(II) n-terminus monodentate oxalate/water coordination with SMD - frequency tables
Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-139.5251 68.5782 572.6556 116.6901 1120.414 18.3954 1454.244 50.1058 -67.7221 13.7861 581.7439 199.1363 1122.425 18.5052 1480.545 302.2696 -31.4144 6.9623 595.9757 27.7251 1128.668 15.861 1557.375 103.8002 -15.3494 4.4179 670.8806 9.3348 1159.058 152.0393 1562.815 31.0891
7.6717 5.2215 679.961 9.3493 1172.261 55.7585 1573.531 62.4532 34.0444 1.0366 680.9304 8.5109 1174.976 46.1142 1637.915 134.6474 45.3219 6.0935 683.7143 3.5485 1181.419 72.5695 1722.168 953.4558 55.2884 4.6905 685.503 8.0883 1191.396 2.796 1811.77 650.3832 67.8047 3.7939 689.5583 9.6307 1196.936 4.074 2939.163 44.7365 85.9292 15.6276 701.9842 5.9619 1197.482 4.3371 2955.972 29.5226 87.0245 4.153 723.5167 54.4937 1219.44 0.1305 2957.251 29.3682
104.2302 2.9419 725.0027 3.7597 1222.285 0.2238 2957.708 27.8278 114.4418 12.2066 768.8722 46.2038 1229.838 0.2433 2969.18 59.8628
122.917 29.3153 780.935 30.3104 1232.116 0.106 2969.467 16.076 140.1913 5.7338 781.8657 70.1256 1241.297 5.3982 2974.261 54.148 153.7047 6.5496 783.9057 122.4984 1268.895 2.4673 2986.627 30.683 159.9806 20.231 825.5056 298.2954 1273.251 1.0378 2988.401 29.6673 170.1529 15.8899 831.7864 62.858 1280.905 1.5631 2993.504 13.0595 183.0991 20.8799 845.5117 1.4238 1286.632 1.396 2999.068 48.7226 194.6714 43.0023 850.7004 8.1709 1290.584 1.1754 2999.218 38.8984 200.0226 29.4234 854.0893 20.7953 1301.484 6.9961 3000.493 45.2074 200.4236 12.6281 855.3504 7.7651 1303.279 13.0404 3001.119 37.3087 210.3948 0.3363 861.6542 6.9133 1307.129 7.0863 3001.959 63.2002 211.1122 0.114 863.0846 2.6505 1356.499 1248.5 3003.053 48.2499 227.4183 0.0381 863.8482 2.441 1381.634 6.8516 3003.417 50.2911 247.2344 158.0658 864.0707 4.9484 1382.664 2.7636 3005.703 39.8273 273.8708 66.29 865.1965 8.7079 1392.756 8.0049 3015.59 40.0559 280.7415 98.2543 866.4861 4.7706 1393.805 3.0952 3020.004 35.9761 291.0257 53.5649 867.067 16.0593 1399.285 6.2179 3020.37 33.773 322.2159 29.018 871.5497 6.0577 1400.794 7.2683 3023.079 32.7171 352.8773 8.2494 873.1969 32.9673 1402.391 7.8025 3246.113 1404.459 439.6407 25.34 879.1594 43.9905 1424.501 5.574 3260.252 19.6336 472.1146 193.2059 891.4529 40.831 1425.823 5.0055 3277.544 2.0947 482.2081 7.7915 896.2144 438.7943 1426.149 6.8023 3292.198 3.8725 484.2822 27.0331 989.889 4.086 1426.331 12.9112 3295.675 11.021
485.185 7.2939 998.0814 5.4454 1427.6 10.1868 3298.346 26.9618 488.4743 18.0131 1001.921 4.3473 1433.539 5.3718 3312.818 24.4462 489.9737 65.8861 1069.302 62.5519 1433.915 22.9023 3365.445 473.6875 491.8154 4.8743 1073.498 55.735 1434.627 21.8267 3656.822 236.6366 496.3743 9.4473 1077.749 36.0202 1435.053 7.9876 3669.159 248.5008 546.4725 139.3123 1092.161 1.0859 1438.365 3.5477 3671.967 233.019 568.3277 180.8888 1107.307 17.962 1448.373 52.1259 3817.241 142.9606
211
Table A-32. Mn(III) n-terminus monodentate oxalate/water coordination with SMD - frequency tables
Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-66.0932 9.9232 607.5724 176.363 1127.905 27.3129 1475.351 51.2997 -51.2867 15.927 610.2603 167.2487 1132.933 74.1832 1483.762 395.1039
-34.752 4.6123 669.5252 0.5231 1135.282 243.9528 1549.187 131.5254 21.353 5.0206 670.8508 9.6814 1160.798 84.3764 1557.849 78.2956
28.0481 2.3906 671.5992 6.7726 1163.787 90.9731 1565.056 74.3327 36.5217 1.7275 677.789 5.9249 1173.409 114.28 1598.981 156.0093 69.3604 3.8356 679.9297 6.5084 1186.35 7.2581 1750.399 1726.783 82.0823 15.7898 683.5946 3.4073 1187.595 165.7655 1759.764 409.0675 89.5174 4.8579 685.4399 8.7367 1192.768 2.4856 2271.717 3088.945
102.9639 18.9214 729.4087 6.2102 1195.963 2.1979 2955.921 31.4198 116.3742 18.7011 732.0609 167.352 1216 0.1682 2968.024 12.7145 141.1572 7.4441 754.1293 512.8312 1221.195 0.2365 2968.787 20.1605 152.8336 19.1511 759.6707 102.5161 1224.439 0.1563 2972.983 16.6724 159.8018 17.0657 773.2977 44.24 1226.941 0.1395 2975.039 7.2783 177.8775 18.4773 775.7814 81.3365 1260.219 130.3957 2978.273 65.0522 186.0632 23.5562 794.2526 41.5292 1265.813 1.2411 2985.497 46.6991 193.4009 0.2716 797.0673 165.43 1277.981 1.5903 2992.895 30.5866 195.8013 0.1128 831.5771 57.7618 1282.067 0.9696 2993.391 24.0611 213.9365 2.2743 847.0622 2.2936 1282.528 1.4827 2993.691 28.1029 226.9651 19.7522 852.4218 9.778 1290.721 0.9013 3001.815 23.8902 230.6706 0.0986 853.9039 11.2544 1298.33 8.4983 3004.742 70.2041 230.8446 0.0676 857.7056 4.7148 1303.745 10.1202 3004.779 30.7875 240.8748 41.3302 860.3728 6.7469 1306.647 11.9614 3006.885 36.5534 246.2605 15.8896 861.2997 1.4011 1339.13 1192.694 3009.539 62.511 254.8545 69.0378 861.5204 13.5453 1359.285 6.0055 3011.766 34.5214 292.1546 23.532 864.8431 12.7053 1364.564 2.5621 3012.42 40.4909
308.958 187.2982 864.893 29.8955 1371.22 6.3548 3013.892 31.0447 331.8995 112.2075 867.6965 1.8273 1379.451 8.9362 3024.258 25.8262 365.6019 74.4288 869.1487 10.1649 1397.572 8.5039 3025.49 37.6782 410.6225 219.7 870.9313 2.5911 1399.731 6.2046 3026.83 35.3929 434.5549 16.4575 874.686 13.9315 1401.432 8.1031 3032.502 31.8323 477.0142 30.7117 875.5226 59.4767 1421.572 5.2268 3285.629 3.1609 484.2073 31.3237 903.5293 32.8649 1425.801 6.3671 3292.976 36.1792 488.5895 104.1348 998.1837 6.836 1425.941 11.4361 3295.21 5.9912 491.4944 1.316 999.9959 3.2695 1429.831 9.8248 3310.738 48.2327 493.2871 2.9169 1011.233 3.6573 1430.406 4.6457 3320.954 26.5534 495.7508 27.7414 1062.088 73.5605 1431.598 7.2035 3322.321 38.5896 497.3114 2.9901 1064.927 35.8048 1435.855 3.1678 3422.461 428.6144 510.6021 62.9556 1065.722 76.8228 1436.018 2.7764 3640.041 301.2472 582.3312 250.2432 1099.208 0.8912 1438.636 3.7394 3651.513 245.9026 585.5801 176.3194 1125.413 2.3786 1457.851 44.6429 3656.941 319.1273 594.8396 41.2893 1126.884 12.9588 1459.953 51.9806 3808.902 255.6028
212
Table A-33. Mn(II) n-terminus bidentate oxalate coordination with SMD - frequency tables
Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-31.564 3.1656 676.3204 4.1465 1134.196 128.3986 1464.455 61.7674 -26.4836 17.1454 680 336.5037 1148.92 25.6613 1478.974 298.4986 -12.8288 12.937 682.6792 3.5991 1175.868 75.0373 1565.921 108.1004 29.0923 6.2133 684.4311 19.7028 1178.108 31.92 1570.298 64.9385 34.6791 5.8627 685.9221 5.7744 1183.492 75.5297 1576.967 32.0975 61.3843 17.5865 689.0266 62.295 1185.602 1.9337 1665.913 1073.731 72.8327 0.4047 691.2824 10.4529 1190.472 3.3323 1761.032 756.2488 82.6226 6.7619 705.9053 6.7631 1194.944 3.6924 2877.919 1797.98
102.5663 26.5691 725.2602 129.7837 1207.579 1.1959 2943.321 39.6875 116.4241 60.3718 773.6105 44.1679 1221.068 0.2073 2953.428 17.623 125.1736 6.7691 774.8567 32.5771 1226.877 0.311 2960.781 26.8971
127.415 22.7746 791.5733 63.4329 1231.361 0.1522 2961.802 23.8777 136.2865 12.3223 817.7165 295.1433 1259.655 1.6818 2962.112 24.5896 149.3833 7.5487 830.1305 52.6333 1272.596 628.8312 2970.051 57.417 152.7728 3.0085 837.7495 42.9864 1280.004 1.3977 2975.874 53.8456 173.8551 10.3845 850.4942 45.6659 1282.895 1.5719 2981.464 29.9253 181.9052 34.5118 855.4522 9.4079 1284.874 1.6134 2988.175 29.3871
202.501 19.4318 858.7724 1.8697 1287.022 1.3949 2989.125 29.0168 204.6478 13.2271 858.9 32.0314 1305.597 6.4821 2998.698 45.3889 206.5873 1.1577 861.9678 4.0395 1309.93 4.9388 2999.21 34.0356 211.1506 55.0587 865.1504 1.7431 1323.592 10.4706 3001.817 38.865 216.1894 0.1396 865.3204 10.656 1364.108 80.1416 3001.966 51.0796
239.612 0.0368 867.2286 3.5999 1378.187 4.937 3002.602 43.7369 258.3925 106.3135 869.2886 16.202 1385.592 10.782 3004.326 49.2994 270.8908 55.2239 869.6539 14.6631 1387.095 2.1943 3006.708 41.7305 287.7529 24.7104 872.7499 17.1744 1390.984 3.5992 3010.906 12.3312 311.7168 80.145 874.0153 7.7439 1396.106 7.3181 3012.08 73.0064 371.1105 24.5248 878.9637 18.4206 1399.802 8.2343 3020.158 36.1508 460.6808 57.0261 893.9191 52.7817 1403.437 6.2151 3021.737 33.651 483.7687 0.8486 940.4822 243.8153 1417.552 7.4406 3027.168 32.165 485.7362 3.7876 994.1471 6.7 1425.832 8.3409 3283.292 7.0621 488.2269 1.0036 996.4984 4.9422 1426.518 7.9238 3283.97 2.322 491.2892 3.1594 1006.204 5.3306 1429.605 3.174 3293.316 5.2139 491.6569 6.8379 1067.141 59.7261 1430.507 8.0879 3296.449 11.786 508.3026 1.262 1071.539 52.9388 1430.995 4.4636 3296.954 35.1497 553.4434 301.3666 1074.499 47.2185 1434.439 2.3922 3304.613 46.6178 570.0332 152.832 1093.626 17.5416 1436.254 3.9673 3664.425 249.6181 585.5567 158.4531 1109.07 23.4105 1436.62 4.764 3668.38 239.777 590.7227 150.9976 1117.981 42.289 1448.089 52.669 3672.14 241.9312 675.9465 17.8731 1119.799 19.9103 1449.642 49.6462
213
Table A-34. Mn(III) n-terminus bidentate oxalate coordination with SMD - frequency tables
Frequencies Intensities Frequencies Intensities Frequencies Intensities Frequencies Intensities
-70.656 11.5673 627.4718 295.6273 1141.537 62.3466 1472.741 41.5964 -65.8959 9.7563 652.1234 9.7316 1151.964 71.5066 1495.318 57.1182 -48.6827 5.148 671.861 4.8232 1165.385 110.3181 1554.745 118.5346 -42.1304 14.7866 673.4417 1.4778 1176.669 134.6878 1564.413 71.7987 -26.1122 11.2769 675.2886 4.1531 1176.778 283.6057 1575.152 110.7471 -19.2311 4.3462 682.3187 21.9683 1177.341 68.7128 1712.478 1179.983 43.8779 5.1691 682.7579 5.3746 1187.066 686.1156 1868.526 824.8641 87.7218 2.5989 683.4963 5.226 1189.28 4.9592 2959.313 13.0427 97.9309 17.854 725.3621 156.737 1190.752 16.6589 2964.492 22.0635 135.166 6.281 728.7562 3.311 1200.527 1.0745 2970.681 16.7486
137.6093 7.0971 768.5671 23.2787 1218.248 0.3793 2975.318 14.2211 144.3185 17.8189 772.228 48.6778 1220.478 0.0985 2977.183 57.487 167.7245 12.9278 796.2775 253.5759 1223.101 0.1518 2982.836 10.7874 183.4248 10.5528 800.3868 381.1584 1243.542 90.8821 2984.967 58.9274 190.4356 44.7587 823.8571 44.6667 1267.391 0.478 2986.491 29.6083
191.296 0.3787 843.2759 32.1036 1276.641 1.2729 2993.98 25.8881 197.7844 12.8519 847.2612 49.3572 1278.228 2.0571 2994.225 30.218 202.3962 1.5915 848.9705 13.8429 1281.255 1.5248 2999.644 29.3717 209.2278 0.2898 853.9825 5.9587 1291.249 1.3186 3003.067 38.0236
212.912 0.0224 857.5301 11.663 1300.811 8.2788 3004.349 56.7784 231.4606 31.7109 861.7775 7.7614 1308.919 11.1179 3006.808 23.5291 233.3163 0.816 861.9788 6.3268 1311.157 15.3197 3009.124 48.1447 269.2823 136.4042 863.1918 0.2354 1351.55 3.4882 3010.589 52.2766 295.4687 21.2208 863.2573 11.8334 1365.192 2.1828 3013.485 35.0837 305.9703 6.8902 863.9059 3.703 1373.273 12.1065 3013.806 18.4635 369.5964 2.8981 867.5562 2.7645 1383.612 7.2213 3017.243 53.7692 391.3917 160.1217 869.8198 21.886 1398.233 6.7236 3025.083 37.4989 424.5163 10.0167 876.985 28.9466 1399.297 8.4515 3027.464 35.7996 475.8566 6.3484 883.1894 16.9004 1402.475 8.2699 3035.289 29.9222 487.7835 1.6377 892.5841 224.3908 1423.736 9.157 3283.439 3.3348 488.5395 8.2665 996.375 4.8364 1425.493 11.1385 3296.71 7.0975 495.8841 0.7594 1000.037 7.95 1426.649 4.5449 3309.944 23.6907 498.8676 9.4141 1010.594 8.1402 1427.419 9.6387 3314.765 36.6563 503.4814 9.9181 1054.996 56.1353 1428.84 6.1069 3315.918 66.854
512.014 122.411 1064.865 61.9651 1431.193 3.3529 3322.243 33.5455 572.1828 4.3076 1078.071 86.9099 1431.465 2.3637 3646.871 303.1311 583.8362 157.1747 1106.971 0.6212 1433.486 3.8048 3650.488 308.3049 587.1345 274.9494 1112.892 4.5673 1437.827 4.1117 3651.879 266.6729 597.2144 155.6966 1124.233 15.3806 1465.297 52.9475 3738.936 288.4064 600.6625 168.9105 1129.176 24.7339 1469.566 57.9998
214
APPENDIX B SAMPLE CALCULATIONS FOR RFQ AGING TIMES AND REYNOLDS NUMBER
In order to determine the aging time of the RFQ system post-mixer, several
parameters need to be known: the syringe ram diameter, the hose diameters and the
syringe ram displacement velocity. Beginning with the syringe ram, the volume based
flow rate can be determined.
(B-1)
Where A is the cross-sectional area, and d is the inner diameter of the syringe barrel.
(B-2)
Where V is the total syringe barrel volume and l is the length.
Taking the cross sectional area of the syringe barrel in cm2 multiplied by the syringe ram
displacement speed in cm/s gives the flow rate for solution in the system in cm3/s or
mL/s.
(B-3)
Where FR is the flow rate of the solution and Sram is the linear displacement speed of the
syringe ram. For a 4 cm/s displacement speed and a 6.5mm internal diameter syringe
barrel, this results in a 1.33 mL/s flow rate.
The volume of each piece of tubing can be calculated in the same way as the volume of
the syringe. For an 85 mm aging hose with a 0.015" I.D. (0.038 cm), the volume is:
(B-4)
Taking the volume of the tubing and dividing by the flow rate induced in the syringe ram
gives a dwell time for the aging hose. For the same 85 mm aging hose with a syringe
ram displacement speed of 4 cm/s
215
(B-5)
Doubling the ram displacement speed would give halve the aging time using the same
hose.
The threaded reactor hose used to connect the grid mixer output to the spray
nozzle (or to the aging hoses themselves, which would then couple to the spray nozzle)
has a length dimension of 34.3 mm, resulting in an inherent time delay of 0.93 ms and
the coupling screw for connecting the reactor hose to additional delay tubing has an
internal distance of 5.87 mm between hose fittings, resulting in a cumulative dead time
post-reactor of 1.09 ms at a 4 cm/s ram displacement velocity.
Reynolds Number (Re) is a ratio of inertial to viscous forces based on the density
of the solution and is used to characterize a flow as either laminar or turbulent. If the
Reynolds number is high the flow of the system is characterized as turbulent. This
corresponds to ranges upwards of 2000. If Re < 1100, the flow is laminar. Under
laminar flow conditions mixing only occurs through diffusion, which is a slow process.
The Reynolds Number is defined in Equation B-6.
(B-6)
Where ρ is the density of the fluid, u is the fluid velocity, D is the pipe diameter, μ is the
kinematic viscosity of the solution, Q is the volumetric flow rate and A is the cross
sectional area of the pipe. The kinematic viscosity of water at 1 atm and 20⁰C is
9.7837E-7 m2/s.
Using the same example of a 0.015" I.D. tube and a 4 cm/s syringe ram
displacement speed (resulting in 1.33 mL/s flow rate), the linear flow velocity would be
the length of the tubing used over the dwell time. In this case, this is 36.76 mm/ms or
216
36.76 m/s. This results in a Reynolds Number of 14,278. The flow is therefore
turbulent.
217
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BIOGRAPHICAL SKETCH
Justin Lloyd Goodsell, son of Scott Goodsell and Cindy Goodsell, grew up in Los
Gatos, California, where he completed his primary and secondary education in 2006.
Justin started his undergraduate career in business administration in 2006 at San Jose
State University. From 2007-2009 Justin served as a missionary in the Dominican
Republic. Upon returning from the Dominican Republic, Justin spent two more years at
SJSU, eventually switching into the chemistry program, followed by a transfer to the
Rochester Institute of Technology in New York, from which he received his Bachelor of
Science Degree in chemistry in 2013 with Honors. In July of 2013, Justin started his
graduate career at the University of Florida, pursuing a Doctor of Philosophy in physical
chemistry under the advisement of Professor Alexander Angerhofer. His researched
focused on the enzymatic mechanism of Bacillus subtilis Oxalate Decarboxylase, as
studied by Electron Paramagnetic Resonance Spectroscopy, inorganic synthesis and
computational modeling. Justin received his Ph.D. in Chemistry in the spring of 2018.