Molecular Basis of the Electron Bifurcation Mechanism in the [FeFe]-Hydrogenase Complex HydABC

Abstract

Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO 2 , but the molecular mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating [FeFe]-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H 2 ). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experiments, functional studies, infrared spectroscopy, and molecular simulations, we show that HydABC from the acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui employ a single flavin mononucleotide (FMN) cofactor to establish electron transfer pathways to the NAD(P) + and Fd reduction sites by a mechanism that is fundamentally different from classical flavin-based electron bifurcation enzymes. By modulation of the NAD(P) + binding affinity via reduction of a nearby iron−sulfur cluster, HydABC switches between the exergonic NAD(P) + reduction and endergonic Fd reduction modes. Our combined findings suggest that the conformational dynamics establish a redox-driven kinetic gate that prevents the backflow of the electrons from the Fd reduction branch toward the FMN site, providing a basis for understanding general mechanistic principles of electron-bifurcating hydrogenases.

The Moon‐Forming Impact and the Autotrophic Origin of Life

“…Though only discovered in 2008, [60] flavin‐based electron bifurcation is essential and apparently universal in the physiology of strictly anaerobic prokaryotes [61–62] . The mechanisms of flavin‐based electron bifurcation have been studied in some detail [63–64] …”

“…[61][62] The mechanisms of flavin-based electron bifurcation have been studied in some detail. [63][64] Although not required for nonenzymatic CO 2 reduction by H 2 in alkaline conditions, electron bifurcation allows cells, both modern and ancient ones, to exploit the reductive potential of environmental H 2 at pH 6~7 even at low H 2 partial pressures near 10 À 5 atm [65] (compare Table 1), forging a link between metabolism and environment. [48] The issue of ferredoxin reduction with electrons from H 2 intuitively leads to thoughts about early evolution.…”

“…Environmentally produced H 2 from serpentinization could directly reduce CO 2 made accessible by the Moon-forming impact in metal-catalyzed geochemical reactions under hydrothermal conditions. The transition to an enzymatically catalyzed protometabolism, shown here, involved a gradual decrease in dependency on vent conditions, whereby H 2 became the electron donor for ferredoxin reduction catalyzed by hydrogenases, which employ flavin-based electron bifurcation in order to couple the endergonic reduction of ferredoxin with the exergonic reduction of a higher potential acceptor [61,[63][64]. CO 2 is fixed in the acetyl-CoA pathway, with ferredoxin as the electron donor in reactions of both the methyl and the carbonyl branch [39].…”

An essential role of the reversible electron-bifurcating hydrogenase Hnd for ethanol oxidation in Solidesulfovibrio fructosivorans

“…It contains, in addition to the hydrogenase catalytic subunit (HndD) which is closely related to the CpI hydrogenase from Clostridium pasteurianum , a [2Fe-2S] subunit (HndA), a subunit homologous to the NuoF flavin subunit of complex I (HndC), and a fourth subunit (HndB) which probably does not contain any redox center ( Malki et al, 1995 ; Kpebe et al, 2018 ). The recent structures obtained by cryo-EM of trimeric hydrogenases of this class has allowed to propose a mechanism of electron bifurcation for these hydrogenases ( Furlan et al, 2022 ; Katsyv et al, 2023 ).…”

Correlating Conformational Equilibria with Catalysis in the Electron Bifurcating EtfABCX of Thermotoga maritima

“…Thus far, BF enzymes have been split into four phylogenetically unrelated, albeit independently evolved groups. These groups include [FeFe] hydrogenases containing HydABC, heterodisulfide reductases containing HdrA, transhydrogenases containing NfNAB, and electron transfer flavoproteins (ETFs) containing EtfAB. − The HydABC-type hydrogenases were recently shown to be representative of the diverse and ubiquitous so-called Bfu family with a noncanonical FMN/FeS cluster-based BF mechanism . Some BF-enzymes can also catalyze the reverse of electron bifurcation, or confurcation, such as the Acetomicrobium mobile NiFe-HydABCSL hydrogenase that reduces NAD + and ferredoxin using electrons donated from H 2 as well as the reverse reaction in which NADH and reduced ferredoxin provide electrons for proton reduction …”