Biophysics of Metalloenzymes Topics and Themes: (Metallo-) Proteins - - PowerPoint PPT Presentation

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Biophysics of Metalloenzymes Topics and Themes: (Metallo-) Proteins - - PowerPoint PPT Presentation

Jan 06, 2023 355 likes •641 views

Biophysics of Metalloenzymes Topics and Themes: (Metallo-) Proteins and Enzymes in the Cell 1) 2) Some Principles of Coordination Chemistry 3) Methods for Investigation at Molecular Level 4) Overview on Metal Cofactors in Biology 5)

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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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SLIDE 13

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