Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction

Abstract

In methanogenic archaea, the carbon dioxide (CO) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

Methods for Expression, Purification, and Characterization of PqqE, a Radical SAM Enzyme in the PQQ Biosynthetic Pathway

“…What distinguishes PqqE from other SPASM proteins is the fact that it contains a unique Cx 2 Cx 5 Dx 21 C motif with the ligand aspartate (D) in AuxII, while other SPASM proteins contain a conserved Cx (2–4) Cx 5 Cx (21–22) C, with four cysteines in their AuxII site. Additionally there is a CxC motif present in AuxI of PqqE, a feature observed in both [4Fe–4S] and [2Fe–2S] containing proteins (Banci et al, 2013; Wagner, Koch, Ermler, & Shima, 2017; Fig. 3).…”

Electron Bifurcation Makes the Puzzle Pieces Fall Energetically into Place in Methanogenic Energy Conservation

“…[2] This initial activation and reduction of CO 2 to the formyl group requires low-potential electrons that are carried by as malli ron-sulfur protein called ferredoxin. [12] Biochemical assays have shown that Hdr receives two electrons from the oxidation of H 2 through MvhAGD [NiFe]-hydrogenase, [7] althoughi tw as not clear which subunits interacto rt hrough which cofactors electrons are transferred. Am ethyl-CoM reductase catalyzes the reductive resolution of methyl-CoM to form methane and ad isulfide CoM conjugate of coenzyme B( CoM-S-S-CoB).…”

“…These reactions are coupled through ar ecently realizedb iochemical process called flavin-based electron bifurcationt hat maximizes the energy efficiency in hydrogenotrophic methanogenesis. [12] Structures, although extremely informative, can sometimes leave al ot to be desired, especially when mechanistic insight into challenging chemical reactions is sought. [4] Flavinbased electron bifurcationf unctions similarly in the coupling of endergonic and exergonic electron-transferr eactions to maximize thermodynamic efficiency in metabolism.T he bestcharacterized model of flavin-based electron bifurcation is the NADH-dependent reduced ferredoxin:N ADP + oxidoreductase (Nfn).…”

“…[12] Biochemical assays have shown that Hdr receives two electrons from the oxidation of H 2 through MvhAGD [NiFe]-hydrogenase, [7] althoughi tw as not clear which subunits interacto rt hrough which cofactors electrons are transferred. [12] Biochemical assays have shown that Hdr receives two electrons from the oxidation of H 2 through MvhAGD [NiFe]-hydrogenase, [7] althoughi tw as not clear which subunits interacto rt hrough which cofactors electrons are transferred.…”

“…In the proposed mechanism of bifurcation, the bifurcation-site flavin would receive two electrons from as ingle donor,a nd then bifurcate one electron to reduce the heterodisulfide and one electron to reduce ferredoxin. [12] Biochemical assays have shown that Hdr receives two electrons from the oxidation of H 2 through MvhAGD [NiFe]-hydrogenase, [7] althoughi tw as not clear which subunits interacto rt hrough which cofactors electrons are transferred. Over the years, Hdre nzymes have been implicated in receiving reducinge quivalents from several enzymes and electron carri- [7] F 420 -reducing hydrogenase, [7] Fdh, [8] Flx, [9] F 420 H 2 , [10] methanophenazine [10] ), although the binding sites and mechanisms of energy transfer have remained largely unidentified.…”