multifrequency epr spectroscopy of solar energy harvesting ......metals in biology: applications of...

MultifrequencyEPR Spectroscopy of

Solar Energy harvesting Species

ACERT 2009Friday, February 26, 2010

Part 1: Multifrequency ESEEM of Metal

ClusterNitrogen Ligands

• the “exact cancellation condition” drives a need for multifrequency pulsed EPR

• applications to mitoNEET Fe-S, Mn Catalase, PSII OEC

Friday, February 26, 2010

Exact cancellation Limit

• 1970s Mims and Peisach, 1980s David Singel and coworkers

• For nuclei with significant isotropic hyperfine coupling (A), best results when the external field matches the hyperfine field

• for a given g-value, this field matching determines the desired microwave frequency...instrumentation has lagged theory


Exact Cancellation LimitS=1/2, I=1 Case N14

ElectronZeeman

NuclearZeeman Hyperfine

m = -1/2s

m = +1/2s

NuclearQuadrupole


Principal Investigator/Program Director(Last, First, Middle): Britt, R. David

Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims

The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •

Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.

B Progress Report

B.1 Publications during Review Period

Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.

Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.

Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.

Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.

Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.

Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.

Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.

B.2 EPR Instrumentation

During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.

Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.

PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page


Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims



B Progress Report














Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims



B Progress Report














Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims



B Progress Report














Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims



B Progress Report














Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims



B Progress Report














Research Planν−

ν0

ν+

νsq

νdq

Three frequencies ν0, ν−, and ν+ givequadrupolar parameters e2qQ and η

A Specific Aims



B Progress Report














Research Planν−

ν0

ν+

νsq

νdq

Three frequencies ν0, ν−, and ν+ givequadrupolar parameters e2qQ and η

Then νdq provides a good estimate of thehyperfine coupling A

A Specific Aims



B Progress Report



Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.

(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.








Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)



UC DavisCalEPR Center

Bruker ECS 106 X-Band (9 GHz) CW

X/Ku-Bands (8-18 GHz) Pulsed

Ka-Band (31 GHz) Pulsed

D-Band (130 GHz) Pulsed

Bruker E580 X/Q-Bands (9, 34 GHz) CW/Pulsed


lab-built X/Ku-band (8-18 GHz, 1kW pulsed)


Ka-band 31 GHz (100W pulse/cw power)


Bruker E580 X (9 GHz)and Q (34 GHz) bands


MitoNeet FeS Protein

Mark L. Paddock, Patricia Jennings, UCSD

Rachel Nechushtai, Hebrew University

Michelle Dicus, Stefan Stoll, UC Davis

FeS protein that interacts witha family of insulin sensitizing drugs

(thiazolidinedione (TZD))


MitoNEET: Mitochondrial Outer MembraneProtein with a dimer of 2Fe2S Centers

with 3Cys-1His Ligation


“Original” X-band Rieske ESEEM (spinach b6f complex)

Britt et al., (1991)Biochem. 30:1862-1901(from my grad school homebuiltwith Melvin Klein)

Reiske 2Fe-2S has two histidinenitrogen ligands, more strongly coupled than exact cancellation limit at X-band


Ka Band ESEEM of MitoNEET


N15



N15


N14


Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims



B Progress Report














Research Planν−

ν0

ν+

νsq

νdq

A Specific Aims



B Progress Report














CW X Band EPR


Ka Band EPR and ESEEM (inset)


Ka Band ESEEMN15N14


Ka Band ESEEM

Q Band ESEEM

N15

N15


ESEEM

Q Band HYSCORE

N15

N15


53

14N 15N Aiso (MHz) –6.25(7) +8.77(10) T (MHz) –0.94(7) +1.32(10) Ax (MHz) Ay (MHz) Az (MHz) ! (°) " (°) # (°)

–5.28(7) –5.35(7) –8.13(7) +105(180) +57(2) +54(2)

+7.4(1) +7.5(1) +11.4(1) +105(180) +57(2) +54(2)

e2Qq/h (MHz) –2.47(4) $ +0.38(4) Px (MHz) Py (MHz) Pz (MHz) ! (°) " (°) # (°)

+0.38(2) +0.85(2) –1.24(2) –22(2) +51(2) +66(2)

Table 1. Experimentally determined 14N and 15N hyperfine and nuclear quadrupolar

coupling parameters of the N! nitrogen of His87 in human mitoNEET. Euler angles

describe the tensor orientations relative to the frame of the g-tensor.

Q Band ENDORN15


53

14N 15N Aiso (MHz) –6.25(7) +8.77(10) T (MHz) –0.94(7) +1.32(10) Ax (MHz) Ay (MHz) Az (MHz) ! (°) " (°) # (°)

–5.28(7) –5.35(7) –8.13(7) +105(180) +57(2) +54(2)

+7.4(1) +7.5(1) +11.4(1) +105(180) +57(2) +54(2)

e2Qq/h (MHz) –2.47(4) $ +0.38(4) Px (MHz) Py (MHz) Pz (MHz) ! (°) " (°) # (°)

+0.38(2) +0.85(2) –1.24(2) –22(2) +51(2) +66(2)

Table 1. Experimentally determined 14N and 15N hyperfine and nuclear quadrupolar

coupling parameters of the N! nitrogen of His87 in human mitoNEET. Euler angles

describe the tensor orientations relative to the frame of the g-tensor.

ESEEM N14


Current tensor orientation model (JACS submitted)[want to extend to single crystal studies]


Mn Catalase

Jim Penner-Hahn, U of Michigan

Jim Whittaker, OHSU

Troy Stich, UC Davis


!"#$%&'()* +,)(-"(+./0001+./021(34#%.5%.'6'(!%-%7%6'

Stemmler, T.L., et al. J. Am. Chem. Soc. 899:, 119, 9215.

X;<%.=(>($?76'(@)@@+

!=A

!"

!#

!$

Dinuclear Mn catalaseStudying Mn(III)Mn(IV) form

-relevance to PSII Mn

!"#$%&'()* +,)(-"(+./0001+./021(34#%.5%.'6'(!%-%7%6'

Stemmler, T.L., et al. J. Am. Chem. Soc. 899:, 119, 9215.

X;<%.=(>($?76'(@)@@+

!=A

!"

!#

!$


Mn1 Mn2

K162

E155

H188E70H73

E36

Mn1 Mn2R147

E148

H181E66H69

E35

Figure 1.

LP MnCat TT MnCat

Now have crystal structures from two organismsBoth show one histidine ligand to each Mn

Lactobacillus plantarum Thermus thermophilus


MHzFrequency [MHz]1086420

µs

1.151.101.05Field [T]

T + ! [µs]

N1

N2

exp

Frequency [MHz]

N1

N2

exp

* *9.77 GHz 30.67 GHz

400350300Magnetic Field [mT]

121086420

sim

N1

N2

exp

sim

LP MnCat

T + ! [µs]

N1

N2

exp

sim

543210

20151050

sim

Figure 2.

Lactobacillus plantarum

-10 -5 0 5 100

2

4

6

8

10

12

!1 [MHz]

-10 -5 0 5 100

2

4

6

8

10

12

!1 [MHz]

! 2[MHz]

! 2[MHz]

(N2 !dq",N2 !dq# + !0)(N2 !dq",N2 !dq# + !$)(N2 !dq",N2 !dq# + !+)

(N2 !dq",N2 !dq#)

(!0, !dq)(!$, !dq)(!+, !dq)

Figure 3.

X and Ka Band ESEEM and sims

X Band HYSCORE and sims

Aiso = −5.75 MHzAaniso = [−0.2 −0.6 +0.8] MHze2qQ = 2.01 MHz, η = 0.79 Euler angles α = 75° β = 0° γ = −10°

Aiso = +2.67 MHzAaniso = [+0.41 +0.15 −0.57] MHze2qQ = 2.25 MHz, η = 0.58 Euler angles α = 110°, β = 15°, γ = 85°

N1

N2


T + ! [µs]

N1

N2

exp

sim

121086420

Frequency [MHz]

T + ! [µs]

N1

N2

exp

Frequency [MHz]

N1

N2

exp

N1

N2

exp

* *30.67 GHz

1.151.101.05Field [T]

9.77 GHz

Figure 4

400350300Magnetic Field [mT]

543210

sim

TT MnCat

1086420 20151050

sim

Figure 5.

-10 -5 0 5 100

2

4

6

8

10

12

-10 -5 0 5 100

2

4

6

8

10

12

!1 [MHz]!1 [MHz]

! 2[MHz]

! 2[MHz]

(!", !dq)(!+, !dq)

(N2 !dq#,N2 !dq$ + !+)

(N2 !dq#,N2 !dq$)

Thermus thermophilus

X and Ka Band ESEEM and sims

X Band HYSCORE and sims

N1

N2

Aiso = +2.28 MHzAaniso = [+0.42 +0.28 −0.70] MHz,e2qQ = 2.29 MHz, η = 0.50 Euler angles α = 0° β = 50° γ = 50°

Aiso = −5.2 MHzAaniso = [−0.3 −0.7 +1.0] MHze2qQ = 2.0 MHz, η = 0.4Euler angles α = 70° β = 60° γ = 80°


Photosystem IIOEC Manganese

Richard Debus, UC Riverside

Greg Yeagle, Troy Stich, UC Davis


D1-His332

D1-His337

Ca

MnD

MnC MnA

MnB

B

D1-Asp170

D1-His332

D1-His337

D1-Glu333

D1-Tyr161

CP43-Glu354

D1-Asp342MnA

D1-Glu189

D1-Asp170

MnD

A

D1-Ala344

Ca

MnB

MnC

Summary of current X-ray picture of ligands

Superposition ofdifferent structures


3-pulse multifrequency ESEEM of the Photosystem II S2-state Mn “multiline” signal in the X/Ku band instrument

specific labelling (with Bruce Diner)showed these to be histidine N transitions15


76543210T + ! [!S]

20151050Frequency [MHz]

sim

exp

sim

exp

sim

exp

simexp

Ka-band

Q-band

Ka-band

Q-band

14N-PSII from Synechocystis

sim

exp

Ka-band

Q-band

Ka-band

Q-band

76543210T + ! [!S]

sim

exp

20151050Frequency (MHz)

sim

exp

sim

exp


Ka and Q band ESEEM and Simulations

N and N15 14


76543210T + ! [!S]

20151050Frequency [MHz]

sim

exp

sim

exp

sim

exp

simexp

Ka-band

Q-band

Ka-band

Q-band


sim

exp

Ka-band

Q-band

Ka-band

Q-band

76543210T + ! [!S]

sim

exp

20151050Frequency (MHz)

sim

exp

sim

exp


14 Aiso(14N) = 6.89 MHzAaniso(14N) = [1.14 0.56 -1.70] MHz e2Qq/h = -1.98 MHz, eta = 0.84

N simulation parameters


D1-His332

D1-His337

Ca

MnD

MnC MnA

MnB

B

D1-Asp170

D1-His332

D1-His337

D1-Glu333

D1-Tyr161

CP43-Glu354

D1-Asp342MnA

D1-Glu189

D1-Asp170

MnD

A

D1-Ala344

Ca

MnB

MnC

Nearly identical spectra fromspinach PSII ( N)

(Yeagle et al, Inorg Chem 2008)

X-Ku results simulated with same parameters, no evidence for

multiple nitrogens

Which histidine is it?

14


Part 2: HF-EPR and Substrate/Cofactor Radical Electronic

Structure

• Single crystal study of biliverdin radical intermediate

• pterin radical of nitric oxide synthase


Biliverdin Radicals in Light Harvesting/

Sensing Pigment Synthesis

Stefan Stoll, Alex Gunn, Clark Lagarias, Andy Fisher, UC Davis

Andrew Ozarowski, NHMFL


C-phycocyanin

disk formed as a trimer of alpha/beta heterodimers

2.5nm


Phycocyanobilin:Ferredoxin Oxidoreductase

(pcyA)

Biliverdin

Phycocyanobilin

RadicalIntermediates

in Vinyl Reduction


D-band 130 GHz(100mW pulse power)

“Cryogen-free” 0-8 Tesla Magnet


PcyA: high-field EPR of crystals and powdersStoll et al, JACS 2009 131 1986


PcyA: DFT predictions of g tensor


Pterin Radicals in NO Synthesis

Stefan Stoll, Yaser NejatyJahromy UC Davis

Josh Woodward, Michael Marletta UC Berkley

Andrew Ozarowski, NHMFL


These structures inform the discussion of the mechanismsof NOS in a number of interesting ways. The hydrogen-bonding network seems to orient arginine and NHA rigidlywith respect to the heme as shown in Figure 3. It is imme-diately apparent that NHA is bound as the antistereoisomer, a fact that has not been previously discussed.This is noteworthy because the hydroxylimine oxygen andthe guanidinium carbon of the NHA are both distant fromthe heme iron (4.3 Å and 4.4 Å, respectively). Thisarrangement presents significant difficulties for some ofthe proposed mechanisms of NOS discussed below.

Characteristics of the NOS reactionsThe NOS reaction generates citrulline by oxidizing L-Argthrough the intermediacy of NHA. However, the identity ofthe nitrogen oxide product, NO, or the nitroxyl anion, NO–,was only determined recently. Several reports suggested thatthe nitroxyl anion, NO–, was generated by NOS and con-verted to NO under the catalysis of superoxide dismutase(SOD) [28,29]. Recently, however, NO from the NOS reac-tion was directly detected in the absence of SOD [30]. Therehas been a debate regarding the stoichiometry of NADPHand the amount of citrulline generated in the NOS reaction.The controversy arises because several NADPH-consumingreactions occur along with the NOS reaction. These sidereactions include the generation of superoxide ion (O2

–) bythe NOS heme [31•,32] and the oxidation of NADPH byperoxynitrite generated from NO and O2

– [28]. Hevel andMarletta [9] have correctly determined the NADPH/cit-rulline stoichiometry by conducting the reaction withH4B-saturated iNOS in the presence of SOD. Anotherimportant characteristic of the NOS reaction products is theoxygen source for NO and the urea-oxygen of citrulline.Using 16O-labeled NHA under 18O2 conditions, the ureaoxygen of citrulline was found to contain exclusively 18O,

whereas the NO product originated completely from the N-hydroxy group of NHA [33,34].

The N-hydroxylation of L-Arg consumes one equivalenteach of NADPH and O2, and is typical of a P450 oxygenasereaction. The generally accepted P450 reaction mechanismcan account for the stoichiometry and the product forma-tion [35]. Marletta et al. [36] have proposed anon-P450-type mechanism to account for the oxidation ofL-Arg in which O2 activation by an H4B and a non-hememetal ion was proposed to furnish the intermediate oxidiz-ing L-Arg to generate NHA. This mechanism is analogousto that of the pterin-dependent aromatic amino acid hydro-genases [37]. However, this notion has been challenged bytwo recent discoveries. First, the 5-methyl H4B analogsupports the NOS reaction, but it does not support O2 acti-vation [38]. Secondly, catalytic activity analysis of amutated NOS, in which the histidine residues near theheme group were substituted, and metal ion analysis bothsuggest that there is no catalytic role for a non-heme redox-active metal in NOS [39].

In the second step of the NOS reaction, the heme group acti-vates O2 by recruiting only one reducing equivalent fromNADPH and, apparently, one from NHA. A key questionthat needs to be answered is the order of the redox eventsinvolved in the oxidation of NHA. Particularly, it is unclearwhether the NOS Fe(III)-heme is reduced by NHA or the

Nitric oxide synthase: models and mechanisms Groves and Wang 689

Table 1

Redox potentials of NOS cofactors, NHA derivatives andvarious iron porphyrin oxidation states.

Redox couples Redox potentials* Reference

OxoFe(IV)(P+•)/oxoFe(IV)P !1.4~1.6 V [67]

OxoFe(IV)P/Fe(OH)P !1.0~1.3 V [67]

NHA/NHA-derived iminoxy radical !710 mV (pH 7.5) [8]

H4B/qH2B 150~180 mV [68]

SuperoxoFe(III)/peroxoFe(III)heme 10 mV [69]

NHA–/NHA•† !–200 mV [57]

NOS Fe(III)/Fe(II)heme –248~–263 mV [70]

FAD/FAD+• –270~–290 mV [71]

NADPH –324 mV [72]

*E1/2 versus NHE, at pH 7 aqueous solution except where otherwisecited. †Estimated from the redox potential of the amidoximate/amidoxyliminoxy radical couple. NHA–, oximate derived from the N-hydroxygroup of NHA; NHA•, the iminoxy radical derived from NHA;qH2B, quinonoid dihydrobiopterin.

Figure 3

Schematic structure of the active site of NHA-bound murine iNOS.NHA is shown in bold.

NN

NNFe

OHO

O

O

S

N

N

NH

O

N OH

H2N

H

HO

Cys194

N

N

N

O2CH

H2N

ON

NO

O

N O

R

OH

H

H

H

O

N

N

O

III

Trp366

Glu371

H

Gly365

4.0Å

R!

HOH

H

OH

H

H

H

H

H

Current Opinion in Chemical Biology


X band CW EPR


High Field CW EPR


Q Band Davies ENDOR


g = [2.0043 2.00355 2.00215]

A_H6 = [42 45 51]A_N5 = [0.1 0.1 65]A_N8 = [0.1 0.1 15]A_H5 = [-9 -45 -30]A_H8 = [-3 -15 -10]

EPR Parameters Constrained by Simultaneous Fit to All Spectra


DFT Calculations SupportsCation Radical Assignment:H6 resonance for example


Stefan StollStefan Stoll

Michelle Dicus Greg YeagleKa band, mitoNEET Ka band, PSII

Catalase, PSII mitoNEET, RadicalsDr. Troy Stich Dr. Stefan Stoll

Yaser NejatyJahromyNOS

Alex GunnpcyA


The End


multifrequency epr spectroscopy of solar energy harvesting ......metals in biology: applications of...

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