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-el ectron-reduced enzyme (EH(4)). Further analysis of the EH(2)/EH(4) red ox couple, however, yields a midpoint potential of -312 mV for the fre e enzyme at pH 7. The catalytic mechanism of the peroxidase has been e valuated with a combination of kinetic and spectroscopic approaches, i ncluding initial velocity and enzyme-monitored turnover measurements, anaerobic stopped-flow studies of the reactions of both oxidized enzym e (E) and EH(2) with NADH and AcPyADH, and diode-array spectral analys es of both the reduction of E --> EH(2) by NADH and the formation of E H(2) . NADH. Overall, these results are consistent with rapid formatio n of an E NADH complex with distinct spectral properties and a rate-li miting hydride transfer step that yields EH(2), with no direct evidenc e for intermediate FADH(2) formation. The EH(2) . NADH complex describ ed previously [Poole, L. B., & Claibome, A. (1986) J. Biol. Chem. 261, 14525-14533] is not catalytically competent and reacts relatively slo wly with H2O2. Stopped flow analyses do, however, support the very rap id formation of an EH(2) . NADH intermediate, with spectral propertie s that distinguish it from the static EH(2) . NADH form, and yield a f irst-order rate constant for the conversion between the two species th at is smaller than K-cat. The combined rapid-reaction and steady-state data are best accommodated by a limiting type of ternary complex mech anism very similar to that proposed previously [Parsonage, D., Miller, H., Ross, R. P., & Claiborne, A. (1993) J. Biol. Chem. 268, 3161-3167 ].