Reduced Nicotinamide Dinucleotide Phosphate Diaphorase from Bacillus subtilis

NADPH‐diaphorase was extensively purified from extracts of Bacillus subtilis N. Y.. FMN is most probably the prosthetic group of this enzyme. NADPH is oxidized by oxygen as the terminal acceptor only in the presence of catalytic amounts of free flavins (Km for FMN was 1.6 × 10−6 M at pH 8.0). NADPH was directly oxidized in the presence of various electron acceptors such as quinones, ferricyanide and tetrazolium salts. Cytochrome c was a very poor acceptor in this system. Km for NADPH was 2.0 × 10−4 M and Ki for NADP+ as a competitive inhibitor was 1.1 × 10−3 M at pH 7.2. Mn++ or Mg++ at mM concentration showed some stimulation of enzyme activity. The enzyme was partially inhibited by various chelating agents and p‐hydroxymercuribenzoate but not by several typical inhibitors of the respiratory chain. It is suggested that this flavoprotein can serve as a useful tool for NADP+ regeneration in coupled enzyme reactions.