An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism

Abstract

Microorganisms utilize electron bifurcating enzymes in metabolic pathways to carry out thermodynamically unfavorable reactions. Bifurcating FeFe-hydrogenases (HydABC) reversibly oxidize NADH (E′∼−280 mV, under physiological conditions) and reduce protons to H2 gas (E°′−414 mV) by coupling this endergonic reaction to the exergonic reduction of protons by reduced ferredoxin (Fd) (E′∼−500 mV). We show here that HydABC homologs are surprisingly ubiquitous in the microbial world and are represented by 57 phylogenetically distinct clades but only about half are FeFe-hydrogenases. The others have replaced the hydrogenase domain with another oxidoreductase domain or they contain additional subunits, both of which enable various third reactions to be reversibly coupled to NAD+ and Fd reduction. We hypothesize that all of these enzymes carry out electron bifurcation and that their third substrates can include hydrogen peroxide, pyruvate, carbon monoxide, aldehydes, aryl-CoA thioesters, NADP+, cofactor F420, formate, and quinones, as well as many yet to be discovered. Some of the enzymes are proposed to be integral membrane-bound proton-translocating complexes. These different functionalities are associated with phylogenetically distinct clades and in many cases with specific microbial phyla. We propose that this new and abundant class of electron bifurcating enzyme be referred to as the Bfu family whose defining feature is a conserved bifurcating BfuBC core. This core contains FMN and six iron sulfur clusters and it interacts directly with ferredoxin (Fd) and NAD(H). Electrons to or from the third substrate are fed into the BfuBC core via BfuA. The other three known families of electron bifurcating enzyme (abbreviated as Nfn, EtfAB, and HdrA) contain a special FAD that bifurcates electrons to high and low potential pathways. The Bfu family are proposed to use a different electron bifurcation mechanism that involves a combination of FMN and three adjacent iron sulfur clusters, including a novel [2Fe-2S] cluster with pentacoordinate and partial non-Cys coordination. The absolute conservation of the redox cofactors of BfuBC in all members of the Bfu enzyme family indicate they have the same non-canonical mechanism to bifurcate electrons. A hypothetical catalytic mechanism is proposed as a basis for future spectroscopic analyses of Bfu family members.

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

“…Our resolved cryoEM structures of HydABC Tk and HydABC Aw show that the H-clusters in the respective protomers, HydA and HydA′, are connected via bridging [4Fe–4S] clusters A5 and A5′ (Figures f and S3c) (cf. also refs , , ). Although the short distance between these clusters could support electron transfer between the protomers (Figures f and S3e), their rare histidyl ligands will shift their redox potentials relative to the [2Fe–2S] center (A4) along the main chain .…”

Gut bacteria impact host uric acid burden and its association with atherosclerosis

“…8 , filled green circles), although we have not observed copious gas accumulation in these cultures and the predicted fermentation balance with UA does not require overt H2 metabolism. However, these systems have been recognized to interact with an array of substrates other than H + 42 .…”

Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima

“…Electron bifurcation is an energy-coupling mechanism that combines exergonic and endergonic electron-transfer events to efficiently utilize enzymatic energy to maximize cellular yields. , Electron-bifurcating enzymes are found in most anaerobic microorganisms where they are used to generate the low-potential reducing equivalents (e.g., reduced ferredoxin) required in fundamental pathways such as H 2 , methane, and butyrate metabolism as well as fixation of both nitrogen and carbon; ,− they are also part of some aerobic respiratory chains . An electron bifurcation mechanism is essential for autotrophic microbes and important for heterotrophs grown on certain substrates. , Electron bifurcation is referred to as a primary energy-conservation mechanism along with oxidative phosphorylation and substrate-level phosphorylation. , …”

“…6 An electron bifurcation mechanism is essential for autotrophic microbes and important for heterotrophs grown on certain substrates. 2,5 Electron bifurcation is referred to as a primary energy-conservation mechanism along with oxidative phosphorylation and substrate-level phosphorylation. 7,8 Bifurcating enzymes contain a flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) that splits electron pairs from a midpotential donor and transfers them to separate high-and low-potential acceptors in a tightly coupled manner.…”

“…A key element of all bifurcating flavins is that they possess highly crossed half-potentials, with the semiquinone/hydroquinone couple being much higher than the quinone/semiquinone couple. The first, high-potential electron out of the fully reduced bifurcating flavin once it has been reduced is sent along a high-potential pathway to reduce the high-potential acceptor, while the remaining low-potential and highly thermodynamically unstable electron on the bifurcating flavin is used to reduce, e.g., ferredoxin. ,, Thus, with each bifurcating event, one electron is transferred to the high-potential pathway and another to ferredoxin. Moreover, the enzyme need not become fully reduced before ferredoxin reduction occurs.…”