The roles of His-64, Tyr-103, Tyr-146, and Tyr-151 in the epoxidation of styrene and beta-methylstyrene by recombinant sperm whale myoglobin.

Previous studies demonstrated that at least two mechanisms are involved in the epoxidation of styrene and stilbene by myoglobin and H2O2 (Ortiz de Montellano, P. R., and Catalano, C. E. (1985) J. Biol. Chem. 260, 9265-9271). One mechanism, reaction of the olefin with the ferryl oxygen, preserves the olefin stereochemistry and incorporates an oxygen from H2O2 into the epoxide. The second mechanism, proposed to be a protein-mediated co-oxidation process, results in loss of stereochemistry and incorporation of an atom of molecular oxygen. To examine the role of individual residues in olefin epoxidation, we have examined the catalytic activities of the possible Tyr-->Phe mutants, the His-64-->Val mutant, and a protein combining all the tyrosine and histidine mutations. The latter protein is less stable than the other mutants and is the only one for which a protein radical is not detected in the reaction with H2O2. The Km and Vmax for styrene epoxidation range, respectively, from 0.3-8 mM and 12-35 pmol/min/nmol of protein. Incubation with H(2)18O2 results in 20-30% incorporation of labeled oxygen into the epoxide with all the mutants except Y103F/Y151F, Y146F/Y151F, H64V, and H64V/Y103F/Y146F/Y151F, for which 52, 58, 89, and 96% of the epoxide oxygen, respectively, is labeled. Oxidation of cis-beta-methylstyrene by wild-type myoglobin yielded a 54:46 ratio of cis- and trans-beta-methylstyrene oxides. The cis-isomer accounts for 47-100% of the epoxide produced by the mutant hemoproteins, with the two H64V mutants yielding almost exclusively the cis-epoxide. The oxygen in the cis-epoxide derives primarily or exclusively from H2O2 and that in the trans-epoxide from an alternative source. These results indicate that tyrosine residues may participate in, but are not essential for, protein-mediated epoxidation. In contrast, His-64 appears to be essential for co-oxidative epoxidation because in its absence olefin epoxidation is mediated almost exclusively by ferryl oxygen transfer.