A hexameric NADP+-reducing hydrogenase from Moorella thermoacetica: First insights into a unique hydrogenase

Introduction: Moorella thermoacetica reduces CO₂ with H₂ as reductant via the Wood-Ljungdahl pathway (WLP). The enzymes of the WLP in 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 encoded by M. thermoacetica.

Goals: In order to clarify the electric connectivity between the oxidative and reductive branches in acetogenesis in M. thermoacetica, we attempted to isolate the NADP⁺-reducing hydrogenase. Since cells grow poorly on H₂ + CO₂ and the activity was not present in glucose-grown cells we first identified conditions under which the enzyme was produced and cell mass could be obtained easily.

Materials & Methods: Growth experiments, enzyme purification, enzyme assays, genome-wide expression analyses and bioinformatic analysis were performed.

Results: Cell-free extract of H₂ + CO₂-grown cells catalyzed H₂-dependent NADP⁺ reduction whereas this activity was not observed in glucose-grown cells. Cells grown on glucose + DMSO also produced the NADP⁺-reducing hydrogenase. We have purified the hydrogenase to apparent homogeneity from cells grown on glucose + DMSO. The enzyme had six subunits encoded by hydABCDEF, contained 58 mol iron and one mol FMN. The enzyme reduced methyl viologen with H₂ as reductant and of the physiological acceptors tested only NADP⁺ was reduced. Electron bifurcation to pyridine nucleotides and ferredoxin was not observed. Hydrogen-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.

Summary:

The NADP⁺-dependent hydrogenase HydABCDEF from M. thermoacetica has been purified and characterized. Despite its similarity to the electron-bifurcating hydrogenase HydABC, the HydABCDEF hydrogenase did not bifurcate electrons and NADP⁺ is the only physiological electron acceptor. The enzyme provides the WLP with NADPH.