Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH.

syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in are not well understood. The genome of contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The genes for the multimeric [FeFe]-hydrogenase (; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in , and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD and oxidized NADH without the presence of ferredoxin. The HydB subunit of the multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in , demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms.