Oxidation-reduction components of a reduced diphosphopyridine nucleotide dehydrogenase

Abstract

DPNH dehydrogenase, solubilized from a particulate DPNH oxidase by treatment of the latter with ethanol at pH 4.8 and 43°, has been further purified by precipitation with ammonium sulfate at pH 7.8 and by filtration through Sephadex G-25. The enzyme migrates as a single band when subjected to electrophoresis on starch gel or polyacrylamide. Based upon a molecular weight of 70,000 (determined by filtration through Sephadex G-200), each molecule of protein contains 1 FMN, 2 nonheme irons, 1 labile sulfide group, and 7 thiol groups. The latter are determined by titration of the protein with DTNB in the presence of 4 M urea. When the enzyme is reduced by DPNH under anaerobic conditions and in the absence of an external electron acceptor, the flavin appears to be fully reduced rather than at the semiquinone level; under these conditions, each mole of enzyme oxidizes approximately 3 moles of DPNH. Removal of the bound FMN by passage of the enzyme through Florisil or Bio-Gel does not affect the DPNH-ferricyanide activity while completely abolishing activity with cytochrome c or 2,6-dichlorophenolindophenol; cytochrome c activity can be largely restored by the addition of FMN. p-Chloromercuribenzoate (pCMB) inhibits the dehydrogenation of DPNH coupled to ferricyanide, indophenol, or cytochrome c; inhibition of the DPNH-ferricyanide activity is a biphasic function of pCMB concentration. Preincubation of the enzyme with DPNH increases the sensitivity toward pCMB when ferricyanide and cytochrome c are used as acceptors.