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NADH oxidase from Amphibacillus xylanus is a potent alkyl hydroperoxide reductase in the presence of the small disulfide-containing protein (AhpC) of Salmonella typhimurium. In the presence of saturating AhpC, kcat values for reduction of hydroperoxides are approximately 180 s-1, and the double mutant flavoprotein enzyme C337S/C340S cannot support hydroperoxide reduction (Niimura, Y., Poole, L. B., and Massey, V. (1995) J. Biol. Chem. 270, 25645-25650). Kinetics of reduction of wild-type and mutant enzymes are reported here with wild-type enzyme; reduction by NADH was triphasic, with consumption of 2.6 equivalents of NADH, consistent with the known composition of one FAD and two disulfides per subunit. Rate constants for the first two phases (each approximately 200 s-1) where FAD and one disulfide are reduced are slightly greater than kcat values for AhpC-linked hydroperoxide reduction. The rate constant for the third phase (reduction to the 6-electron level) is too small for catalysis. Only the first phase of the wild-type enzyme occurs with the mutant enzyme. These results and the stoichiometry of NADH consumption indicate Cys337 and Cys340 as the active site disulfide of the flavoprotein and that electrons from FADH2 must pass through this disulfide to reduce the disulfide of AhpC.

The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide. The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the alkyl-hydroperoxide reductase from Salmonella typhimurium. AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein. In the presence of the 22-kDa colorless component (AhpC) of the Salmonella alkyl-hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water. Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene hydroperoxide. Both enzymes' Km values for hydrogen peroxide, cumene hydroperoxide, and NADH are so low that they could not be determined accurately. Vmax values for hydrogen peroxide or cumene hydroperoxide reduction are > 10,000 min(-1) at 25 degrees C. These values are almost the same as the reduction rate of the flavoprotein component by NADH. The involvement in catalysis of a redox-active disulfide of the A. xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C40SC337S/C340S. Very little activity for hydrogen peroxide or cumene hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S). Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional alkyl-hydroperoxide reductase system within these catalase-lacking bacteria.

A flavoprotein from Amphibacillus xylanus catalyzes the reduction of oxygen to hydrogen peroxide. Each polypeptide chain in the tetrameric enzyme contains 5 cysteine residues. The complete reduction of enzyme by dithionite requires 6 electrons. Such behavior indicates the presence of redox centers in addition to the FAD, and these could be disulfides. In order to assess the catalytic role of disulfide in the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high degree of homology with alkyl hydroperoxide reductase F52a protein and thioredoxin reductase, have been changed to serines by site-directed mutagenesis of the cloned flavoprotein gene (individually and in a double mutant). Titration of the three mutant enzymes, lacking Cys-337, Cys-340, or both cysteines, requires only 2 electron eq to reach the reduced flavin state. These results indicate the absence of a redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide. The catalytic activity of the three enzymes was examined by steady-state analysis. The Km for NADH and oxygen and the kcat value of these mutant enzymes were essentially the same as those of wild type. The NADH oxidase activities were also accelerated markedly in the presence of free FAD, which is the case for wild-type enzyme. The NADH:5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) oxidoreductase activities of all mutant enzymes were less than 3% of the activity of wild-type enzyme. The weak DTNB reductase activities in the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and nitrothiobenzoate by FADH2. However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH2 can reduce either DTNB or another disulfide directly, albeit inefficiently. These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.