REACTION-MECHANISM OF AMPHIBACILLUS-XYLANUS NADH OXIDASE ALKYL HYDROPEROXIDE REDUCTASE FLAVOPROTEIN

NADH oxidase from Amphibacillus xylanus is a potent alkyl hydroperoxid e 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 s( -1), and the double mutant flavoprotein enzyme C337S/C340S cannot supp ort hydroperoxide reduction (Niimura, Y., Poole, L. B., and Massey, V. (1995) J. Biol. Chem. 270, 25645-25650). Kinetics of reduction of wil d-type and mutant enzymes are reported here with wild-type enzyme; red uction by NADH was triphasic, with consumption of 2.6 equivalents of N ADH, consistent with the known composition of one FAD and two disulfid es per subunit, Rate constants for the first two phases (each approxim ately 200 s(-1)) where FAD and one disulfide are reduced are slightly greater than k(cat) values for AhpC-linked hydroperoxide reduction, Th e 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 en zyme occurs with the mutant enzyme. These results and the stoichiometr y of NADH consumption indicate Cys(337) and Cys(340) as the active sit e disulfide of the flavoprotein and that electrons from FADH(2) must p ass through this disulfide to reduce the disulfide of AhpC.