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 O-2 reactivity of C42S NADH oxidase (O-2 --> H2O2) revealed an asymmetric mechanism in which the two FADH(2). NAD(+) per reduced dimer display kinetic inequivalence. Tn this report we provide evidence indicating that the fully active, recombinant wild-type oxidase (O-2 --> 2H(2)O) displays thermodynamic inequivalence between the two active sites per dimer. Using NADPH to generate the free reduced wildtype enzyme (EH2'/EH4), we have shown that NAD(+) titrations lead to differential behavior as only one FADH(2) per dimer binds NAD(+) tightly to give the charge-transfer complex. The second FADH(2), 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 FADH(2) per dimer in wild-type enzyme can be described as distinct "charge-transfer" and "electron-transfer'' sites, with the latter site giving rise to either intramolecular (Cys42-SOH) or bimolecular (pyridine nucleotide) reduction. The reduced C42S mutant is not capable of intramolecular electron transfer on binding pyridine nucleotides, thus confirming that the Cys42-SOH center is in fact the source of the redox asymmetry observed with wild-type oxidase. These observations on the role of Cys42-SOH in the expression of thermodynamic inequivalence as observed in wild-type NADH oxidase complement the previously described kinetic inequivalence of the C42S mutant; taken together, these results provide the overlapping framework for an alternating sites cooperativity model of oxidase action.