A novel hexameric NADP⁺-reducing [FeFe] hydrogenase from Moorella thermoacetica

Acetogenic bacteria such as the thermophilic anaerobic model organism Moorella thermoacetica reduce CO2 with H-2 as a reductant via the Wood-Ljungdahl pathway (WLP). The enzymes of the WLP of M. thermoacetica require NADH, NADPH, and reduced ferredoxin as reductants. Whereas an electron-bifurcating ferredoxin- and NAD(+)-reducing hydrogenase HydABC had been described, the enzyme that reduces NADP(+) remained to be identified. A likely candidate is the HydABCDEF hydrogenase from M. thermoacetica. Genes encoding for the HydABCDEF hydrogenase are expressed during growth on glucose and dimethyl sulfoxide (DMSO), an alternative electron acceptor in M. thermoacetica, whereas expression of the genes hydABC encoding for the electron-bifurcating hydrogenase is downregulated. Therefore, we have purified the hydrogenase from cells grown on glucose and DMSO to apparent homogeneity. The enzyme had six subunits encoded by hydABCDEF and contained 58 mol of iron and 1 mol of FMN. The enzyme reduced methyl viologen with H-2 as reductant and of the physiological acceptors tested, only NADP(+) was reduced. Electron bifurcation with pyridine nucleotides and ferredoxin was not observed. H-2-dependent NADP(+) reduction was optimal at pH 8 and 60 degrees C; the specific activity was 8.5 Umg(-1) and the K-m for NADP(+) was 0.086 mm. Cell suspensions catalyzed H-2-dependent DMSO reduction, which is in line with the hypothesis that the NADP(+)-reducing hydrogenase HydABCDEF is involved in electron transfer from H-2 to DMSO.