C1net - Conference 4 – Chemical from C1 gas East Midland Conference, University of Nottingham, 20-23 January 2019

Electron Bifurcation in C1-

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

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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

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 , 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 ?

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

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