PROTEIN RADICALS IN MYOGLOBIN DIMERIZATION AND MYOGLOBIN-CATALYZED STYRENE EPOXIDATION

Two mechanisms have been identified for the H2O2-dependent epoxidation of styrenes by sperm whale myoglobin (Mb) [S. Rao, A. Wilks, and P.R. Ortiz de Montellano, J. Biol. Chem. 268, 803-908 (1993)]: (a) ferryl (Fe-IV = O) oxygen transfer with retention of stereochemistry and inco rporation of an oxygen from H2O2, and (b) protein peroxy radical cooxi dation with loss of stereochemistry and incorporation of an oxygen fro m O-2. As shown here, cis-beta-methylstyrene is preferentially oxidize d to the trans-epoxide when the H2O2:Mb ratio is <0.5 but increasingly to the cis-isomer as the ratio increases to and above 1. At a high (4 :1) H2O2:Mb ratio, both the absolute yield and the cis:trans-epoxide r atio increase in proportion to the cis-beta-methylstyrene concentratio n. A protein radical formed in the Mb-H2O2 reaction also causes dimer and trimer formation, maximum dimer formation (similar to 30%) being o btained with 1 equivalent of H2O2. At low H2O2:Mb ratios, the oxidatio n equivalents utilized for protein oligomerization and styrene oxidati on account for the available H2O2. Previous studies have shown that Hi s-64 is important for protein-mediated olefin cooxidation and Tyr-151/ Tyr-103 for Mb dimerization. The W7F, W14F, and W7F/W14F Mb mutants ha ve now been prepared and the W14F, but not W7F, mutation shown to mode stly decrease cooxidation of cis-beta-methylstyrene to the trans-epoxi de. Neither tryptophan mutation alters dimer formation. Dimer formatio n is modestly increased rather than decreased by styrene, suggesting t hat styrene cooxidation and dimerization do not compete. The results i ndicate that (a) cis-beta-methylstyrene cooxidation and protein dimeri zation, both of which are mediated by protein radicals, are favored at low H2O2:Mb ratios, (b) as the H2O2:Mb ratio increases, the ferryl ep oxidation pathway surpasses the cooxidation mechanism, (c) Trp-14 but not Trp-7 influences olefin cooxidation, and (d) different, possibly n onequilibrating, radicals mediate olefin cooxidation and protein dimer ization. (C) 1996 Academic Press, Inc.