Revised Manuscript Received September 23, 1992 abstract: Histidine-93 (F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted'H-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic 0-0 bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for 0-0 bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of Y. TV-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.

Oxo-ferryl porphyrin x-cation radical species (Por+, Fe-(IV)= 0, compound I) have been postulated to be highly active reaction intermediates responsible for the catalytic reactions of heme enzymes such as P-450, peroxidase, and catalase (Ortiz de Montellano, 1986; White & Coon, 1980; Dunford & Stillman, 1976; Hewson & Hager, 1979; Dolphin etal., 1971). In fact, the formation of compound I has been observed in various heme enzymes (Dunford, 1982; Chance, 1943, 1949;