Equilibrium Analyses of the Active-Site Asymmetry in Enterococcal NADH Oxidase: Role of the Cysteine-sulfenic Acid Redox Center†

Recent studies [Mallett, T. C., and Claiborne, A. (1998) Biochemistry 37, 8790−8802] of the O2 reactivity of C42S NADH oxidase (O2 → H2O2) revealed an asymmetric mechanism in which the two FADH2·NAD+ per reduced dimer display kinetic inequivalence. In this report we provide evidence indicating that the fully active, recombinant wild-type oxidase (O2 → 2H2O) displays thermodynamic inequivalence between the two active sites per dimer. Using NADPH to generate the free reduced wild-type enzyme (EH2‘/EH4), we have shown that NAD+ titrations lead to differential behavior as only one FADH2 per dimer binds NAD+ tightly to give the charge-transfer complex. The second FADH2, in contrast, transfers its electrons to the single Cys42-sulfenic acid (Cys42-SOH) redox center, which remains oxidized during the reductive titration. Titrations of the reduced NADH oxidase with oxidized 3-acetylpyridine and 3-aminopyridine adenine dinucleotides further support the conclusion that the two FADH2 per dimer in wild-type enzyme ca...