Isolation, purification, and some properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome C2 reductase from Rhodopseudomonas spheroides.

A method has been described for the isolation and purification of NADPH-cytochrome cZ reductase from light-grown Rhodopseudomonas spheroides. The enzyme is a nonmetalloflavoprotein with flavin adenine dinucleotide as the prosthetic group, and it catalyzes the reduction of R. spheroides cytochrome c2, 2,6dichloroindophenol, and K,Fe(CN), with NADPH as the electron donor. It does not reduce R. spheroides cytochrome 5.53 but reduces Rhodospirillum rubrum cytochrome c2 and mammalian cytochrome c very slowly. There is no NADH or NADPH oxidase activity, NADP+ reductase activity, or transhydrogenase activity associated with the enzyme. The pH optimum was found to be 7.5; and K, values of 3.7 X 10M5 M, 1.25 X 10d5 M, and 1.24 X 10M4 M were calculated for cytochrome c2, 2,6-dichloroindophenol, and K3Fe(CN)G reduction, respectively. NADH functioned as a competitive inhibitor, and the Ki was calculated to be 5.5 X 1o-5 M. The enzyme was inhibited by p-chloromercuribenzoate, N-ethyhnaleimide, iodoacetate, and thyroxine; and the inhibition by sulfhydryl reagents was reversed by reduced glutathione. Preincubation with NADPH protected the enzyme against the poisoning effects of sulfhydryl reagents but not thyroxine. The behavior of the enzyme on Sephadex G-100 and in the analytical ultracentrifuge indicates the presence of several molecular forms of the enzyme.