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 (H(2)O(2)) to human Mb 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 H(2)O(2) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) at DMPO:protein </= 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 >/= 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 H(2)O(2) gave no DMPO-Cys(110) at DMPO:protein </= 10 mol/mol, and only trace DMPO-Cys(110) at DMPO:protein >/= 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 H(2)O(2). However, the homodimer was detected in a mixture of both the Y103F and C110A variants of human Mb upon treatment with H(2)O(2) (C110A:Y103F:H(2)O(2) 2:1:5 mol/mol/mol); the yield of this homodimer increased with increasing ratios of C110A:Y103F. Together, these data suggest that addition of H(2)O(2) to human Mb can produce Cys(110)-thiyl radicals through an intermolecular electron transfer reaction from Cys(110) to a Tyr(103)-phenoxyl radical.
Witting
P K
PK
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Mauk
A G
AG
D019943:Amino Acid Substitution; D000818:Animals; D003497:Cyclic N-Oxides; D003545:Cysteine; D019281:Dimerization; D004220:Disulfides; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D005914:Globins / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D010636:Phenols; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels; D014443:Tyrosine
D003497:Cyclic N-Oxides; D004220:Disulfides; D005609:Free Radicals; D009211:Myoglobin; D010636:Phenols; D011994:Recombinant Proteins; D013113:Spin Labels; C042329:phenoxy radical; D014443:Tyrosine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D005914:Globins; D006861:Hydrogen Peroxide; D003545:Cysteine