The active-site histidine-10 of enterococcal NADH peroxidase is not essential for catalytic activity

In order to test the proposal [Stehle, T., Claiborne, A., & Schulz, G.E. (1993) Eur. J. Biochem. 211, 221-226] that the active-site His10 of NADH peroxidase functions as an essential acid-base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala. The k(cat) values for both H1OQ and H10A peroxidases, and the pH profile for k(cat) with H1OQ, are very similar to those observed with wild-type peroxidase. Both mutants, however, exhibit K-m(H2O2) values much higher (50-70-fold) than that for wild-type enzyme, and stopped-flow analysis of the H2O2 reactivity of two-electron reduced H1OQ demonstrates that this difference is due to a 150-fold decrease in the second-order rate constant for this reaction with the mutant. Stopped-flow analyses also confirm that reduction of the enzyme by NADH is essentially unaffected by His10 replacement and remains largely rate-limiting in turnover; the formation of an E . NADH intermediate in the conversion of E-->EH(2) is confirmed by diode-array spectral analyses with H10A. Both H10Q and H10A mutants, in their oxidized E(FAD, Cys42-sulfenic acid) forms, exhibit enhanced long-wavelength absorbance bands (lambda(max) = 650 nm and 550 nm, respectively), which most likely reflect perturbations in a charge-transfer interaction between the Cys42-sulfenic acid and FAD. Combined with the 50-fold increase in the second-order rate constant for H2O2 inactivation (via Cys42-sulfenic acid oxidation) of the H1OQ mutant, these observations support the proposal that His10 functions in part to stabilize the unusual Cys42-sulfenic acid redox center within the active-site environment.