On the formation and reactivity of compound I of the His-64 myoglobin mutants.

Myoglobin (Mb) catalyzes various two-electron oxidations; however, ferryl porphyrin cation radical equivalent to peroxidase compound I has not been identified yet. Distal histidine mutants of sperm whale Mb (His-64 --> Ala, Ser, and Leu) afford an apparent intermediate followed by the formation of a ferryl heme (Mb-II) in the reaction with m-chloroperbenzoic acid. Because the intermediate exhibits characteristic absorption spectrum of compound I and bears two electron oxidizing equivalents above the ferric state, we have assigned the species as compound I of myoglobin (Mb-I). Although we have recently observed compound I of the F43H/H64L Mb mutant, F43H and wild type Mb react with m-chloroperbenzoic acid to give Mb-II without any accumulation of Mb-I. The results unambiguously indicate that His-64 plays a key role in destabilizing wild type Mb-I. Furthermore, Mb-I is found to be capable of performing two-electron oxidation of styrene, thioanisole, and H2O2.