Reaction of human myoglobin and H2O2 -: Electron transfer between tyrosine 103 phenoxyl radical and cysteine 110 yields a protein-thiyl radical

The sequence of human myoglobin (Mb) is similar to that of other species except for a unique cysteine at position 110 (Cys(110)). Adding hydrogen peroxide (H2O2) to human Rib affords Trp(14)-peroxyl, Tyr(103)-phenoxyl, and Cys(110)-thiyl radicals and coupling of Cys(110)-thiyl radicals yields a homodimer through intermolecular disulfide bond formation (Witting, P, K,, Douglas, D, J,, and Mauk, A. G, (2000) J, Biol, Chem, 275, 20391-20398); Treating a solution of wild type Mb and H2O2 with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) at DMPO:protein less than or equal to 10 mol/mol yields DMPO-Cys(110) adducts as determined by EPR, At DMPO:protein ratios (25-50 mol/mol), both DMPO-Tyr(103) and DMPO-Cys(110) adducts were detected, whereas at DMPO:protein greater than or equal to 100 mol/mol only DMPO-Tyr(103) radicals were present. The DMPO-dependent decrease in DMPO-Cys(110) was matched by a near 1:1 stoichiometric increase in DMPO-Tyr(103). In contrast, reaction of the Y103F human Mb with H2O2 gave no DMPO-Cys(110) at DMPO:protein less than or equal to 10 mol/mol, and only trace DMPO-Cys(110) at DMPO:protein greater than or equal to 100 mol/mol (i.e. conditions that consistently gave DMPO-Tyr(103) in the case of wild type Mb), No detectable homodimer was formed by incubation of the Y103F variant with H2O2. However, the homodimer was detected in a mixture of both the Y103F and C110A variants of human Mb upon treatment with H2O2 (C110A:Y103F:H2O2 2:1:5 mol/mol/ mel); the yield of this homodimer increased with increasing ratios of C110A:Y103F, Together, these data suggest that addition of H2O2 to human Mb can produce Cys(110)-thiyl radicals through an intermolecular electron transfer reaction from Cys(110) to a Tyr(103)-phenoxyl radical.