CHEMICAL AND KINETIC EVIDENCE FOR AN ESSENTIAL HISTIDINE RESIDUE IN THE ELECTRON-TRANSFER FROM AROMATIC DONOR TO HORSERADISH-PEROXIDASE COMPOUND-I

Horseradish peroxidase, when incubated with diethyl pyrocarbonate (DEP C), a histidine-specific reagent, shows time-dependent inactivation to oxidize aromatic electron donor, guaiacol. The inactivation follows p seudo-first order kinetics with a second order rate constant of 0.67 m in-1 M-1. The pH dependence of inactivation shows an inflection point at 6.02, indicating histidine derivatization by DEPC. A difference spe ctrum of modified versus native enzyme shows a peak at 244 nm for N-ca rbethoxyhistidine that is diminished by hydroxylamine. Stoichiometric studies indicate that out of 2 histidine residues modified, one is res ponsible for inactivation. A plot of log reciprocal half-time of inact ivation against log DEPC concentration suggests that only 1 histidine is essential. From the computer-stimulated structure of horseradish pe roxidase, we tentatively suggest that this critical histidine is most likely distal histidine 42. Binding studies show that this histidine i s not involved in guaiacol binding. Modified enzyme forms compound I w ith H2O2 but not compound II, suggesting a block of electron transfer process. Modified compound I cannot oxidize guaiacol as evidenced by t he absence of donor-induced spectral shift from 408 nm, suggesting a b lock of electron transfer from bound donor to compound I. We suggest t hat this tentatively identified distal histidine controls aromatic don or oxidation by regulating electron transport without affecting donor binding or compound I formation.