Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review

Abstract

Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F₄₂₀H₂, H₂, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E₀' = -420 mV) with NADH (-320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (-10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H₂-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F₄₂₀H₂-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms-in which they have been found-are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO₂ fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD⁺ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered.

Keywords: Ech-complex; Rnf-complex; crossed-over reduction potentials; electron bifurcation; electron-transferring flavoproteins (EtfAB); energy conservation; ferredoxin; flavodoxin.

Figure 1

Energy metabolism of Clostridium kluyveri growing in batch culture on crotonate. For simplification, acetyl-CoA and reduced ferredoxin used in biosyntheses are not considered. The yellow dots represent the electron-bifurcating butyryl-CoA dehydrogenase-EtfAB (B/E) complex and the NfnAB (Nfn) complex. Acac-CoA, acetoacetyl-CoA; β-Hbu-CoA, beta-hydroxybutyryl-CoA; Crot-CoA, crotonyl-CoA; acetyl-P, acetyl-phosphate. C. kluyveri contains an NAD-specific and an NADP-specific (orange arrow) β-hydroxybutyryl-CoA dehydrogenase (Madan et al., 1973). The metabolic scheme is compatible with the finding that H₂-formation by cell suspensions of C. kluyveri is inhibited by the protonophore tetrachlolorosalicylanilide (TCS) and the inhibition is relieved by dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton-translocating membrane ATPase (Pfeiff, 1991). In the presence of TCS, the F₁F₀-ATPase hydrolyzes ATP to prevent the collapse of the electrochemical proton potential and as a consequence of ATP hydrolysis also the acetyl-CoA and acetyl-phosphates pools are depleted inhibiting acetyl-CoA reduction to butyryl-CoA. In the presence of DCCD, ATP hydrolysis via the F₁F₀ ATPase is stopped.

Figure 2

Energy metabolism of Ruminococcus albus growing in batch culture on glucose. Glucose used in anabolism is not considered. The yellow dot represents the electron-bifurcating hydrogenase HydABC. The pink arrow represents the non-bifurcating hydrogenase HydA2 (Zheng et al., 2014). G-6-P, glucose-6-phosphate; GAP, glyceraldehyde phosphate; PGA, 1, 3-bisphosphoglycerate; Pyr, pyruvate; Acetyl-P, acetyl-phosphate; AcH, acetaldehyde.

Figure 3

Scheme of the electron-bifurcating [FeFe]-hydrogenase HydABC. The electron-bifurcating hydrogenase from Acetobacterium woodii contains a fourth subunit without a prosthetic group (Poehlein et al., ; Schuchmann and Müller, 2012). As drawn, the FMN in HydB cannot be the site of electron bifurcation The presence of a second flavin was proposed (Buckel and Thauer, 2013).

Figure 4

Electron-bifurcating reactions catalyzed by flavoenzyme complexes (yellow dot) and ferredoxin re-oxidizing reactions. The reduction potential E₀′ of ferredoxin (Fd) is generally given as −420 mV, but it can be significantly lower (see Introduction). The E₀′ of the other electron donors or acceptors are given in parenthesis. Note that the reduction potential E′ of the electron donors or acceptors under in vivo conditions (E′) may be quite different from E₀′. Thus, E′ of ferredoxin can be lower than −500 mV, that of H₂ as high as −300 mV and that of NADH is higher than that of NADPH. The electron-bifurcating flavoenzymes are cytoplasmic except for the enzyme complex that uses ubiquinone as a high potential electron acceptor. In the case of protons, or NAD(P)⁺ or pyruvate as high-potential electron acceptors, the electron-bifurcating reactions proceed reversibly: the back reaction is confurcating.