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

Abstract

The flavoprotein NADH oxidase (O2 --> 2H2O) from Enterococcus faecalis 10C1 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 O2 --> 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-FADH2.NAD+ complex. Kinetic analyses indicate that this species is catalytically competent, and its reactivity with O2 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 O2 reduction stoichiometry in which H2O2, not 2H2O, is the product. The two subunits of the reduced enzyme.NAD+ complex react with O2 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 O2 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.