Subforms and in vitro reconstitution of the NAD-reducing hydrogenase of Alcaligenes eutrophus

The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer, HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation, A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most of hoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide, While the hydrogenase dimer, produced by a strain deleted of hoxF and hoxU, displayed H-2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H-2, although nickel was incorporated into HoxH. Deletion of hoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity, Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.