A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of ox ygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which fan act as red ox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T, Nishino, J, Biol, Chem, 270 :5812-5817, 1995), The NADH-FAD reductase activity was directly dependent o n the FAD concentration, with a second order rate constant of approximately 2.0 x 10(6) M-1 s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted fro m reduction of peroxide by free FADH(2) reduced via enzyme bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylan us ahpC gene was cloned and overexpressed in Escherichia coli. When the NAD H oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V-max values for hydrogen peroxide and cumene hyd roperoxide reduction were both approximately 150 s(-1). The K-m values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate d etermination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.