Oxygen reactivity of an NADH oxidase C42S mutant: Evidence for a C(4a)-peroxyflavin intermediate and a rate-limiting conformational change

The flavoprotein NADH oxidase (O-2 --> 2H2O) from Enterococcus faecalis 10Cl contains a cysteinyl redox center, in addition to FAD. We have proposed a cysteine-sulfenic acid (Cys-SOH) structure for the oxidized form of Cys42; the presence of this redox center is consistent with the stoichiometries reported for earlier reductive titrations of wild-type oxidase, and we have proposed that Cys42-SH plays a key role in the overall four-electron reduction of O-2 --> 2H2O. To test these proposals, we provide in this report an analysis of the oxidative half-reaction of an oxidase mutant in which Cys42 is replaced by Ser. NADH titrations lead to direct flavin reduction with 1.05 equiv of NADH/FAD and give rise to the formation of a very stable E-FADH(2.)NAD(+) complex. Kinetic analyses indicate that this species is catalytically competent, and its reactivity with O-2 has been analyzed in detail by stopped-flow spectrophotometry using both single-wavelength and diode-array modes of data acquisition. The combined results of this analysis demonstrate that replacement of Cys42 with Ser provides for an altered O-2 reduction stoichiometry in which H2O2, not 2H2O, is the product. The two subunits of the reduced enzyme NAD+ complex react with O-2 in an asymmetric mechanism, consistent with an alternating sites cooperativity model such as that proposed [Miller, S. M., Massey, V., Williams, C. H., Jr., Ballou, D. P., and Walsh, C. T. (1991) Biochemistry 30, 2600-2612] for mercuric reductase. An FAD C(4a)-hydroperoxide is identified as the primary oxygenated intermediate in reoxidation of the complex, but the reaction of O-2 with the complementary subunit does not proceed until full reoxidation has occurred at the primary subunit. To our knowledge, this is the first report of a C(4a)-peroxyflavin intermediate outside the flavoprotein monooxygenase class.