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

Abstract

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen 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 can act as redox 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 on 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 from 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. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination 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.

FIG. 1

Oxygen consumption during the catalysis of A. xylanus NADH oxidase in the presence of FAD or AhpC. The reaction mixture (2.5 ml) contained 50 mM sodium phosphate buffer (pH 7.0), 0.5 mM EDTA, 0.02% bovine serum albumin, 2% ammonium sulfate, and 600 μM NADH, with 50 μM FAD (B), 30 μM AhpC (C), or neither (A). The reaction was started at 25°C by the addition of 52.8 μg (A), 5.3 μg (B), or 105.6 μg (C) of NADH oxidase, as indicated by arrows a. During oxygen consumption, 60 μg of catalase was added, as indicated by arrows b.

FIG. 2

Steady-state kinetics of NADH-FAD reductase activity. Initial rates of reduction of FAD were monitored at 450 nm on mixing 1.0 μM NADH oxidase with the concentrations of NADH and FAD shown in the figure in a stopped-flow spectrophotometer under anaerobic conditions (the concentrations given are those after mixing). The enzyme was made anaerobic and stored under argon in a tonometer, and the NADH-FAD mixtures were made anaerobic by bubbling for 15 min with argon before introduction into the stopped-flow apparatus. The reaction conditions were 0.05 M sodium phosphate (pH 7.0), 0.5 mM EDTA, and 25°C.

FIG. 3

Proposed reaction mechanism of the NADH oxidase in the presence of FAD and oxygen.

FIG. 4

Proposed pathway for pyruvate metabolism in A. xylanus. The NADH oxidase-AhpC system is enclosed by the solid rectangle. CoA, coenzyme A.