ANALYSIS OF THE KINETIC MECHANISM OF ENTEROCOCCAL NADH PEROXIDASE REVEALS CATALYTIC ROLES FOR NADH COMPLEXES WITH BOTH OXIDIZED AND 2-ELECTRON-REDUCED ENZYME FORMS

Anaerobic titrations of the two-electron-reduced NADH peroxidase (EH(2)) with NADH and 3-acetylpyridine adenine dinucleotide (AcPyADH) yield the respective complexes without significant formation of the four-electron-reduced enzyme (EH(4)). Further analysis of the EH(2)/EH(4) redox couple, however, yields a midpoint potential of -312 mV for the free enzyme at pH 7. The catalytic mechanism of the peroxidase has been evaluated with a combination of kinetic and spectroscopic approaches, including initial velocity and enzyme-monitored turnover measurements, anaerobic stopped-flow studies of the reactions of both oxidized enzyme (E) and EH(2) with NADH and AcPyADH, and diode-array spectral analyses of both the reduction of E --> EH(2) by NADH and the formation of EH(2) . NADH. Overall, these results are consistent with rapid formation of an E NADH complex with distinct spectral properties and a rate-limiting hydride transfer step that yields EH(2), with no direct evidence for intermediate FADH(2) formation. The EH(2) . NADH complex described previously [Poole, L. B., & Claibome, A. (1986) J. Biol. Chem. 261, 14525-14533] is not catalytically competent and reacts relatively slowly with H2O2. Stopped flow analyses do, however, support the very rapid formation of an EH(2) . NADH* intermediate, with spectral properties that distinguish it from the static EH(2) . NADH form, and yield a first-order rate constant for the conversion between the two species that is smaller than K-cat. The combined rapid-reaction and steady-state data are best accommodated by a limiting type of ternary complex mechanism very similar to that proposed previously [Parsonage, D., Miller, H., Ross, R. P., & Claiborne, A. (1993) J. Biol. Chem. 268, 3161-3167].