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

Acetogenic bacteria such as the thermophilic anaerobic model organism Moorella thermoacetica reduce CO 2 with H 2 as reductant via the Wood-Ljungdahl pathway (WLP). The enzymes of the WLP of M. thermoacetica require NADH, NADPH and reduced ferredoxin as reductant. 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, contained 58 mol of iron and one mol FMN. The enzyme reduced methylviologen with H 2 as reductant and of the physiological acceptors tested only NADP + was reduced. Electron bifurcation to pyridine nucleotides and ferredoxin was not observed. H 2 -dependent NADP + reduction was optimal at pH 8 and 60°C; the specific activity was 8.5 U/mg and the K m for NADP + was 0.086 mM. Cell suspensions catalysed 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.