Requirement of a second oxidation equivalent for ferryl oxygen transfer to styrene in the epoxidation catalyzed by myoglobin-H2O2.

Ferric myoglobin (Mb) is oxidized by H2O2 to a ferryl (FeIV = O) species and a protein radical, whereas ferrous Mb is similarly oxidized to the ferryl species without the protein radical. The protein radical is unstable and decays within 5 min, but the ferryl species is stable for more than 1 h. Previous studies have shown that styrene is oxidized to styrene oxide and benzaldehyde by ferric Mb and H2O2. We demonstrate here that the ferryl species produced from ferrous equine Mb and H2O2 does not epoxidize styrene. Furthermore, the EPR signal intensity of the protein radical formed from ferric equine Mb and the ability to oxidize styrene decrease in parallel as a function of the time separating the addition of H2O2 and styrene. The ability to oxidize styrene as a function of time after addition of H2O2 is lost much more rapidly with the H64V/K102Q/Y103F/Y146F/Y151F mutant of sperm whale Mb than with the native protein or the Y146F/Y151F mutant. The results indicate that styrene epoxidation requires a two-electron oxidized species of Mb in which the ferryl (FeIV = O) complex is coupled to a protein or highly transient (undetectable) porphyrin radical. Benzaldehyde formation appears to be catalyzed by the same oxidizing species. Styrene oxidation thus differs from linoleic acid oxygenation, which is catalyzed by the ferryl species alone.