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Volume 271, Number 48, Issue of November 29, 1996 pp. 30459-30464
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Reaction Mechanism of Amphibacillus xylanus NADH Oxidase/Alkyl Hydroperoxide Reductase Flavoprotein

(Received for publication, May 14, 1996, and in revised form, August 21, 1996)

Youichi Niimura ^§ and Vincent Massey ^§

From the  Department of Food Science and Technology, Tokyo University of Agriculture, Abashiri-shi, Hokkaido 099-24, Japan and the ^§ Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

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, k_cat values for reduction of hydroperoxides are approximately 180 s¹, 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 s¹) where FAD and one disulfide are reduced are slightly greater than k_cat 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.

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