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J Biol Chem. 2001 May 11;276(19):16540-7. Epub 2001 Feb 13.

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

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  • 1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.


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.

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