biophysics of metalloenzymes
TRANSCRIPT
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Biophysics of Metalloenzymes
Topics and Themes:
1) (Metallo-) Proteins and Enzymes in the Cell
2) Some Principles of Coordination Chemistry
3) Methods for Investigation at Molecular Level
4) Overview on Metal Cofactors in Biology
5) Cofactor Assembly and Maturation
6) Excitation-Energy and Electron Transfer
7) Proton Transfer
8) Metal centers in Photosynthesis and Water Oxidation
9) Biological Hydrogen Catalysis
10) Metal Cofactors in Nitrogen Fixation
11) Carbon Oxide Conversion at Metal Sites
12) Molybdenum Enzymes
13) Oxygen Reactions
14) Metal Centers in Human Diseases
15) Bioinspired Materials
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Nitrogenase protein x107
x108
Fe3O4 catalyst (ferrit)
300 bar
500 °C
~20 % Ausbeute
Turnover 10 s-1 => 150 Mt / 50t(enzyme) year
Ammonia-plant (Haber-Bosch) x10-3
150 Mt / year
Nitrogen for Industry
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Prof. Gerhard Ertl (78)
Fritz-Haber Institut Berlin
Chemie Nobelpreis 2007
Fritz Haber Nobelpreis 1918, Carl Bosch Nobelpreis 1931
Detailed mechanism of N2 catalysis at catalyst surface
Ertl hat die Grundlage der modernen Oberflächenchemie geschaffen, die so unterschiedlichen Vorgängen wie
dem Rosten von Eisen oder der Wirkung eines Katalysators im Auto nachgeht. Ertl wurde 1936 in Stuttgart
geboren. Er promovierte 1965 in physikalischer Chemie an der Technischen Universität München und arbeitete
nach verschiedenen Stationen - auch in den USA - seit 1986 am Fritz-Haber-Institut in Berlin.
Haber-Bosch Process
ca. 1.5 % of world energy demand !
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Hydrogen from N2 Fixation
http://esraa-chemist.blogspot.de/2010/12/biohydrogen-produced-in-air.html
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N2 Synthetic Chemistry
Arashiba et al. Nature Chemistry: 3, 120–125 (2011)
Low TON and TOF !
N2 binding energy 945 kJ/mol
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Biological Nitrogen Cycle
http://en.wikipedia.org/wiki/Nitrogen_fixation#mediaviewer/File:Nitrogen_Cycle.svg
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N2-fixing Organisms
Free-living bacteria: Diazotrophs are cyanobacteria, e.g. trichodesmium, green
sulfur bacteria, azotobacteraceae, rhizobia, and Frankia, e.g. in soil.
Plants that contribute to nitrogen fixation include the legume family – Fabaceae –
with taxa such as beans, lupines, and peanuts. They contain symbiotic bacteria
called Rhizobia within nodules in their root systems.
Others: of 122 genera in the Rosaceae, only 4 are capable of fixing nitrogen.
Root nodules with billions of N2-fixing bacteria (Knöllchenbakterien)
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Nitrogenase
Crystal structure (1 Å)
Oliver Einsle (Göttingen)
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Organisation
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Genes for Nitrogenase
Oldroyd, Current Opinion in Biotechnology 2014, 26:19–24
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Biophysics of Metalloenzymes M. Haumann SS2014
Overall Reaction Cycle
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Metal Cofactors
P-cluster 8Fe7S
FeMo cofactor (M-cluster)
1Mo7Fe9S1C
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Substrates
FeMoco
Val70
Mo
substrate
Mutagenesis broadens substrate specificity
(Markus Ribbe, UC-Irvine)
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Biophysics of Metalloenzymes M. Haumann SS2014
What is X?
x
Spatzal et a. Science
2011; 334, 940.
Crystallography:
X is carbon, C
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Fig. 3. (A) Comparison of the calculated V2C
XES spectra of FeMoco with an interstitial C4–
(black), N3– (blue), and O2– (red) and of the
spectra of the P clusters (gray). (B) Calculated
V2C XES spectra of FeMoco with an interstitial
C4– (black) and the P clusters (gray). (C)
Experimental difference spectrum of FeMoco
with the P clusters (gray), as well as calculated
difference spectra of the P clusters with FeMoco
containing interstitial C4– (black), N3– (blue), and
O2– (red).
What is X ff
Lancaster et al. Science 334, 974 (2011)
X-ray emission spectroscopy
XES: X is carbon, C
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Biophysics of Metalloenzymes M. Haumann SS2014
P-cluster Assembly
Fig. 4. Stepwise assembly of P-clusters in NifDK (A) and EPR features of the assembly intermediates in the dithionite-reduced (B)
and IDS-oxidized (C) states. (A) The different conformations of P-cluster during assembly are represented by ΔnifH NifDK (left),
which contains two [Fe4S4] cluster pairs (or P*-clusters); ΔnifBΔnifZ NifDK (middle), which contains one P-cluster and one [Fe4S4]
cluster pair (or P*-cluster); and ΔnifB NifDK (right), which contains two P-clusters. Maturation of the “first” P-cluster requires NifH,
whereas maturation of the “second” P-cluster requires both NifH and NifZ. Formation of the P-cluster at the α/β-subunit interface also
induces a conformational change of the α-subunit, which “opens” up the M-cluster site. (B and C) The P*-cluster in ΔnifH NifDK (B,
left) displays a characteristic S=1/2 signal at g=2.05, 1.93, and 1.90 in the dithionite-reduced state; the P-cluster in ΔnifB NifDK (C,
right) displays a characteristic g=11.8 parallel-mode signal in the IDS-oxidized state; the ΔnifBΔnifZ NifDK (B and C, middle) displays
both P*- and P-specific signals at ~50% intensity.
Hu & Ribbe,
Biochimica et
Biophysica Acta 1827
(2013) 1112–1122
Markus Ribbe, UC
Irvine
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Hu & Ribbe, J Biol Chem 288, 13173–13177, 2013
Formation of an 8FeC core
M-cluster Assembly
C-atom insertion
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Mo Exchange
Hu & Ribbe, J Biol Chem 288, 13173–13177, 2013
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Cluster Transfer
Hu & Ribbe, J Biol Chem 288, 13173–13177, 2013
Key AS residues
FeMo protein
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Energetic Bottleneck
Current Opinion in Chemical Biology 2006, 10:101–108
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Putative Intermediates
Barney et al. Dalton Trans. 2006, 2277-2284
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Rate Constants
Duval, Proc Natl Acad Sci U S A 2013, 110(41):16414-9
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Protein association
Conformational gating of ET
Interplay of ET/PT and
Chemistry
PCET ?
Gating of ET
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Where does Hydride Bind?
Hoffman, Acc Chem Res 2013
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Biophysics of Metalloenzymes M. Haumann SS2014
N2 Mechanism
Seefeldt, Annu. Rev. Biochem. 2009. 78:701–22
distal
mechanism
alternating
mechanism
still speculative
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Pathways from DFT
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Summary
Global nitrogen cycle
Biological N2 fixation
Haber Bosch Process
H2 from N2
Nitrogenase
Crystal structure
Genes
Reaction cycle
Cofactors, P- and M-clusters
Nature of X in FeMoco
Cluster assembly
Intermediates
Reaction mechanism
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Literature
Einsle, Nitrogenase FeMo cofactor: an atomic structure in three simple
Steps. J Biol Inorg Chem 2014
Rees, Structural basis of biological nitrogen fixation, Phil. Trans. R. Soc. A 2005 363,
2005
Hoffman, Nitrogenase: A Draft Mechanism, Acc Chem, Res 46, 587–595, 2013
Lancaster, X-ray Emission Spectroscopy Evidences a Central Carbon in the Nitrogenase
Iron-Molybdenum Cofactor, Science 334, 974, 2011
Peters, Exploring new frontiers of nitrogenase structure and mechanism, Current
Opinion in Chemical Biology 2006, 10:101–108
Hu & Ribbe, Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase, J. Biol.
Chem. 2013, 288:13173-13177
Hu & Ribbe, Nitrogenase assembly, Biochimica et Biophysica Acta 1827 (2013) 1112–
1122
Seefeldt, Electron transfer in nitrogenase catalysis, Current Opinion in Chemical Biology
2012, 16:19–25