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Electron Bifurcation in C1-Fermentations C1net - Conference 4 – Chemical from C1 gas East Midland Conference, University of Nottingham, 20-23 January 2019 Electron Bifurcation in C1-Fermentations Wolfgang Buckel Laboratorium für Mikrobiologie Fachbereich Biologie Flavin-Based Electron Bifurcation (FBEB) Low potential ̶ Energy + Reduction potential Buckel & Thauer (2018 ) Chemical Reviews & Frontiers in Microbiology Ferredoxin or Flavodoxin, the 'energy rich' electron carriers in anaerobes. Clostridial ferredoxin with two [4Fe-4S] clusters E°' = −60 mV E°'1 = −340 mV 1 E°' = −420 mV E°'2 = −405 mV 2 Ferredoxin (oxidized) + e − = Ferredoxin − (reduced) Flavodoxin (semiquinone) + e − = Flavodoxin ̶ (hydroquinone) 3 Acetogenesis requires reduced ferredoxin H2 or CoA NADPH ATP + THF Ferredoxin − CO Formate CH -THF CO CO 2 2 NADH 3 2 Acetyl-CoA Ferredoxin − CO 2 Acetate + ATP Pyruvate Cell carbon + − H2 + 2 Ferredoxin ox = 2 H + 2 Ferredoxin red ; ∆G°' ≈ 0. Reduction potential E°' (H 2) = −414 mVolt E°' (Ferredoxin) = −405 mVolt Bifurcating Hydrogenases NAD + reductase in acetogens Heterodisulfide reductase in methanogens > 250 Pa H2; E' = −370 mV > 10 Pa H2; E' = −320 mV E'/ mV 5 E'/ mV − 10 Pa H2 2 Fd /Fd -500 2 Fd /Fd − -500 2 × e− -400 90 mV -400 2 × e− 2 H2 180 mV 2 H2 -300 -300 2 × e− NAD +/NADH 2 × e− -200 -200 Heterodisulfide/CoMSH+CoBSH -100 -100 Schut and Adams 2009 Thauer, Buckel et al. 2008 Schuchmann and Müller 2012 Thauer et al. 2013 Butyrate norishes the mucosa cells. Food Colon Mucosa cells Fibers Blood vessel Monosaccharides Bacteria Amino acids Butyrate O2 CO 2 anaerobic aerobic Clostridial butyrate synthesis Carbohydrates and amino acids Amino acids 7 Flavin-based Electron Bifurcation in Clostridia Electron transferring flavoprotein (EtfAB) and Butyryl-CoA dehydrogenase (Bcd) _ 2 Ferredoxin − → 2 reduced Fd 2- Low potential acceptor EtfB − E' = -500 mV FAD 2 × e Reduction EtfA Potential 2 NADH FAD (Energy) Two-electron Bcd Bcd donor FAD FAD E' = -300 mV 2 × e- High potential acceptor E' = -10 mV Crotonyl-CoA → Butyryl-CoA + 8 Buckel and Thauer 2008 Flavin-based electron confurcation catalyzed by the NfnAB complex NADH-Dependent Ferredoxin: NADP Reductase _ 2 reduced Fd −→ 2 Ferredoxin Low potential donor E' = -500 mV 2 × e− NfnB Reduction FAD 2 NADP + → 2 NADPH Potential 2 × e− NfnA Two-electron (Energy) FAD NADH → NAD + acceptor High potential donor E' = -370 mV + E' = -280 mV 9 Thauer et al. 2010, 2012; Ermler, Thauer et al. 2015; BETCy group: Lubner et al. 2017 Electron transferring flavoprotein (Etf) – Butyryl-CoA dehydrogenase (Bcd) Acidaminococcus fermentans Clostridia Megasphaera elsdenii Gram-positive Firmicutes Gram-negative Firmicutes 10 EtfAB from Acidaminococcus fermentans Domain II α-FAD Domain III Hindge region α−subunit β−subunit β-FAD Domain I NADH and ferredoxin binding sites 11 Amr M. Mowafy and Ulrich Ermler Interaction of NADH with EtfAB from Acidaminococcus fermentans the in the bifurcation-like state 80° •− β-FAD 10° α-FAD 18 Å NADH Ferredoxin binding site Nilanjan Pal Chowdhury & Ulrich Ermler EtfAB-Bcd Complex from Clostridium difficile Bifurcation-like state 13 EtfAB-Bcd Complex from Clostridium difficile Dehydrogenase-State Etf domain II α-FAD δ-FAD Bcd2 Etf domain I β-FAD Etf domain III Bcd1 Demmer, J. K., Chowdhury, N. P., Selmer, T., Ermler, U., Buckel, W. Nature Commun. 2017, 8, 1577 Leonor Michaelis 1875 - 1949 Theory of Oxidation-Reduction: “The task of the protein be ( a) to establish the proper geometric configuration between the different prosthetic groups, and ( b) to aid the formation and stabilization of free radicals.” in The Enzymes, Chemistry and Mechanism of Action (eds Sumner, J. B. & Myrbäck, K.) vol. 2, part 1, pp. 1- 54 (Academic Press , New York, 1951 ). Stepwise oxidation of flavin hydroquinone Main interactions of FAD semiquinone anions in EtfAB Stable α-FAD •− Extremely unstable β-FAD •− +4 −15 Ks = 10 Ks ≈ 10 [SQ] 2 Stability constant Ks = [Q] × [HQ] Electron bifurcation requires a very unstable semiquinone Nernst equation: −1 Reduction potential: E = ± [2.3 RT (2 F) × lg Ks + Eflavin ] = ± [0.030 lg Ks − 0.28] −1 ∆E = E1 − E2 = 2.3 RT × F × lg Ks = 0.059 lg Ks Region of electron bifurcation Region of most flavoproteins -1000 -800 -600 E (SQ/QH ) Free flavin 1 2 -400 E (mV) b-Nfn? -200 β-Etf ? Flavodoxin 0 +200 E (Q/SQ) Ubiquinone O α-Etf 2 in bc complex +400 1 -20 -15 -10 -5 0 +5 +10 +15 +20 lg K ‘crossed-over‘ ‘nomal‘ s Adapted from Nitschke and Russel 2011 Proposed electron flow in the EtfAB-Bcd complex. −800 Q/SQ 6 Å Reduction potential Em (mV) − −600 e 40 − Ferredoxin 0 NADH H ̶ Q/QH 2 β-FAD α-FAD −200 SQ/QH 2 e − Crotonyl-CoA Q/SQ + SQ/QH 2 2e − 0 e− Q/SQ δ-FAD Butyryl-CoA 14 Å +200 SQ/QH 2 Electron-transferring flavoprotein Butyryl-CoA dehydrogenase The eleven established flavin-based electron-bifurcating systems System Donor High pot. Low pot. Organism Established by Acceptor Acceptor Butyryl-CoA 2 NADH Crotonyl-CoA Ferredoxin Clostridia Buckel & Thauer 2008 dehydrogenase/EtfAB Caffeyl-CoA 2 NADH Caffeyl-CoA Ferredoxin Acetobacterium Müller 2010 reductase/EtfAB Lactate 2 NADH Pyruvate Ferredoxin Acetobacterium Müller 2012 dehydrogenase/EtfAB FixABCX 2 NADH Rhodoquinone Ferredoxin Rhodospirillum rubrum Nordlund 2004 Ubiquinone Aztobacter vinelandii Ledbetter et al. 2017 Transhydrogenase, Nfn 2 NADPH NAD + Ferredoxin Clostridia, Acetogens Thauer 2010 Pyrococcus furiosus Lubner et al. 2017 HdrABC-[NiFe]- 2 H 2 CoM-SS-CoB Ferredoxin Methanogens Thauer & Buckel 2008 hydrogenase HdrABC-[W/Se]- 2 CoM-SS-CoB Ferredoxin Methanogens Leigh 2010 formate deydrogenase Formate HdrABC-F420 H2 2 F 420 H2 CoM-SS-CoB Ferredoxin Methanosarcina acetivorans Ferry 2017 + NADH dehydrogenase: 2 H 2 NAD Ferredoxin Thermotoga, Acetobacterium Adams ,Müller ,Thauer [FeFe]-hydrogenase Morella thermoacetica 2009-10-11 + NADPH dehydrogenase: 2 H 2 NADP Ferredoxin Clostridium Thauer 2013 [FeFe]-hydrogenase autoethanogenum NADH-dehydrogenase- 2 NAD + Ferredoxin Clostridium acidi-urici Thauer 2015 21 [W/Se]-formate-dh Formate EtfAB Transhydrogenase HdrABC NuoF (FMN) Crystal structure of the heterodisulfide reductase/hydrogenase complex from Methanobacterium wolfei CoM-S-S-CoB + 2 H+ → CoM-SH + CoB-SH + 2 H 2 → 4 H FAD − 2 Ferredoxin ox → 2 Ferredoxin red Tristan Wagner, Ulrich Ermer & Seigo Shima, Science 2017 Bifurcating Hydrogenase 4 H+ HydA [FeFe] [4Fe4S] 2 H 3 [4Fe4S] 2 [2Fe2S] 2 Ferredoxin − ? [2Fe-2S] HydB ∼ NuoF 2 Ferredoxin HydC ∼ NuoE 3 [4Fe4S] [2Fe2S] FMN NADH NAD + + H + Hydrogenase with a Bifurcating H-Cluster ? HydA 3x[4Fe4S] − 2 Ferredoxin [2Fe2S] 4 H + [2Fe-2S] [FeFe] 2 Ferredoxin [4Fe4S] 2 H HydC ∼ NuoE 2 [FeFe] [4Fe4S] 3 [4Fe4S] HydB ∼ NuoF [2Fe2S] FMN Fe II ---- Fe II ∼ FAD Hydroquinone Fe III ---- Fe II ∼ FAD Semiquinone NADH NAD + + H + Fe III ---- Fe III ∼ FAD Quinone Peters, Beratan, Schut & Adams (2018) Chem Com. Formate Dehydrogenase with a Bifurcating Molybdenum Cofactor ? HydA FdhF2 2 Ferredoxin − 3 [4Fe4S] [2Fe2S] 2 CO 2 Mo-Cofactor [2Fe-2S] 2 Ferredoxin 2 Formate − HydC ∼ NuoE 3 [4Fe4S] [2Fe2S] Molybdate IV ∼ FAD hydroquinone 3 [4Fe4S] Molybdate V ∼ FAD semiquinone [2Fe2S] FMN VI HydB ∼ NuoF Molybdate ∼ FAD quinone NADH NAD + + H + Nitschke, W. and Russell, M. J. 2011 Wang, S., Huang, H., Kahnt, J., and Thauer, R. K. 2013 Acknowledgements Coworkers Collaborations Ulrich Ermler and Julius Demmer, MPI für Biophysik, Frankfurt Thorsten Selmer, FH Aachen Rudolf K. Thauer, MPI Marburg Nilanjan Pal Chowdhury Wolfgang Nitschke, CNRS Marseilles Amr Mohammed Mowafy 26.
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