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) Cofactor Assembly and Maturation Excitation-Energy and Electron Transfer 6) Proton Transfer 7) 8) Metal centers in Photosynthesis and Water Oxidation 9) Biological Hydrogen Catalysis 10) Metal Cofactors in Nitrogen Fixation Carbon Oxide Conversion at Metal Sites 11) Molybdenum Enzymes 12) Oxygen Reactions 13) Metal Centers in Human Diseases 14) Bioinspired Materials 15)
Nitrogen for Industry Fe 3 O 4 catalyst (ferrit) 300 bar 500 ° C ~20 % Ausbeute Nitrogenase protein x10 7 Ammonia-plant (Haber-Bosch) x10 -3 150 Mt / year x10 8 Turnover 10 s -1 => 150 Mt / 50t(enzyme) year
Haber-Bosch Process Fritz Haber Nobelpreis 1918, Carl Bosch Nobelpreis 1931 Prof. Gerhard Ertl (78) Fritz-Haber Institut Berlin Detailed mechanism of N 2 catalysis at catalyst surface Chemie Nobelpreis 2007 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. ca. 1.5 % of world energy demand !
Hydrogen from N 2 Fixation http://esraa-chemist.blogspot.de/2010/12/biohydrogen-produced-in-air.html
N 2 Synthetic Chemistry N 2 binding energy 945 kJ/mol Arashiba et al. Nature Chemistry : 3, 120 – 125 (2011) Low TON and TOF !
Biological Nitrogen Cycle http://en.wikipedia.org/wiki/Nitrogen_fixation#mediaviewer/File:Nitrogen_Cycle.svg
N 2 -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 N 2 -fixing bacteria (Knöllchenbakterien)
Nitrogenase Crystal structure (1 Å) Oliver Einsle (Göttingen)
Organisation
Genes for Nitrogenase Oldroyd, Current Opinion in Biotechnology 2014, 26:19 – 24
Overall Reaction Cycle Biophysics of Metalloenzymes M. Haumann SS2014
Metal Cofactors P-cluster 8Fe7S FeMo cofactor (M-cluster) 1Mo7Fe9S1C
Substrates FeMoco Mo substrate Val70 Mutagenesis broadens substrate specificity (Markus Ribbe, UC-Irvine)
What is X? Spatzal et a. Science 2011; 334, 940 . x Crystallography: X is carbon, C Biophysics of Metalloenzymes M. Haumann SS2014
What is X ff X-ray emission spectroscopy 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). XES: X is carbon, C Lancaster et al. Science 334 , 974 (2011)
P-cluster Assembly Hu & Ribbe, Biochimica et Biophysica Acta 1827 (2013) 1112 – 1122 Markus Ribbe , UC Irvine 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 N ifDK (C, right) displays a characteristic g=11.8 parallel-mode signal in the IDS- oxidized state; the ΔnifBΔnifZ NifDK (B and C, middle) d isplays both P*- and P-specific signals at ~50% intensity. Biophysics of Metalloenzymes M. Haumann SS2014
M-cluster Assembly C-atom insertion Formation of an 8FeC core Hu & Ribbe, J Biol Chem 288, 13173 – 13177, 2013
Mo Exchange Hu & Ribbe, J Biol Chem 288, 13173 – 13177, 2013
Cluster Transfer FeMo protein Key AS residues Hu & Ribbe, J Biol Chem 288, 13173 – 13177, 2013
Energetic Bottleneck Current Opinion in Chemical Biology 2006, 10:101 – 108
Putative Intermediates Barney et al. Dalton Trans. 2006, 2277-2284
Rate Constants Duval, Proc Natl Acad Sci U S A 2013, 110(41):16414-9
Gating of ET Protein association Conformational gating of ET Interplay of ET/PT and Chemistry PCET ?
Where does Hydride Bind? Hoffman, Acc Chem Res 2013
N 2 Mechanism still speculative alternating distal mechanism mechanism Seefeldt, Annu. Rev. Biochem. 2009. 78:701 – 22 Biophysics of Metalloenzymes M. Haumann SS2014
Pathways from DFT
Summary Global nitrogen cycle Biological N 2 fixation Haber Bosch Process H 2 from N 2 Nitrogenase Crystal structure Genes Reaction cycle Cofactors, P- and M-clusters Nature of X in FeMoco Cluster assembly Intermediates Reaction mechanism
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
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