THE NADP-REDUCING HYDROGENASE OF DESULFOVIBRIO FRUCTOSOVORANS - EVIDENCE FOR A NATIVE COMPLEX WITH HYDROGEN-DEPENDENT METHYL-VIOLOGEN-REDUCING ACTIVITY

The NADP-reducing hydrogenase of Desulfovibrio fructosovorans represen ts a novel class of [Fe] hydrogenases which is encoded by the well-cha racterized hndABCD operon containing the genes hndA, hndB, hndC, and h ndD. Expression of this operon, monitored by measuring the NADP-reduci ng activity, was found to be maximum during the exponential phase of g rowth on fructose and then decreased when the concentration of the car bon and energy source became limiting. The optimum pH for the H-2-driv en NADP reduction was 8, and the apparent K-m and V-max were determine d to be 0.09 mM and 13.10(-3) u/mg, respectively. Heterologous express ion of the hnd genes in Escherichia coli was carried out to raise anti sera against the different subunits of the NADP-reducing hydrogenase. The antisera were used to detect the four subunits in cell extract of D. fructosovorans after separation by SDS- and native PAGE. The four s ubunits of the NADP-reducing hydrogenase were demonstrated to be assoc iated in a complex which exhibited Hz-driven methyl viologen reduction . Furthermore, on native gel, a form lacking HndD, with no hydrogen-de pendent methyl viologen reductase activity was also shown to be presen t in D. fructosovorans. (C) 1998 Academic Press.