Effects of the location of distal histidine in the reaction of myoglobin with hydrogen peroxide

To clarify how the location of distal histidine affects the activation proc ess of H2O2 by heme proteins, we have characterized reactions with H2O2 for the L29H/H64L and F43H/H64L mutants of sperm whale myoglobin (Mb), designe d to locate the histidine farther from the heme iron. Whereas the L29H/H64L double substitution retarded the reaction with H2O2, an 11-fold rate incre ase versus wild-type Mb was observed for the F43H/H64L mutant. The V-max va lues for 1-electron oxidations by the myoglobins correlate well with the va ried reactivities with H2O2. The functions of the distal histidine as a gen eral acid-base catalyst were examined based on the reactions with cumene hy droperoxide and cyanide, and only the histidine in F43H/H64L Mb was suggest ed to facilitate heterolysis of the peroxide bond. The x-ray crystal struct ures of the mutants confirmed that the distal histidines in F43H/H64L Mb an d peroxidase are similar in distance from the heme iron, whereas the distal histidine in L29H/H64L Mb is located too far to enhance heterolysis. Our r esults indicate that the proper positioning of the distal histidine is esse ntial for the activation of H2O2 by heme enzymes.