Reaction of Human Myoglobin and H2O2: ELECTRON TRANSFER BETWEEN TYROSINE 103 PHENOXYL RADICAL AND CYSTEINE 110 YIELDS A PROTEIN-THIYL RADICAL

Paul K Witting,A Grant Mauk

doi:10.1074/jbc.m011707200

Paul K Witting, A Grant Mauk

Open Access

https://doi.org/10.1074/jbc.m011707200

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Abstract

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.

Highlights

The sequence of human myoglobin (Mb) is similar to that of other species except for a unique cysteine at position 110 (Cys110)
(25), we investigated the reaction of human Mb and its C110A variant to evaluate the role of the reactive sulfhydryl group in the reaction of this protein with H2O2
(25), we investigated the reaction of human Mb and its C110A variant to evaluate the role of the unique sulfhydryl group in the reaction of this protein with H2O2 and demonstrated by low temperature EPR spectroscopy that the human protein undergoes radical formation at Trp14, Tyr103, and Cys110 in this reaction

Summary

The abbreviations used are

Myoglobin; DMPO, 5,5-dimethyl1-pyrroline N-oxide; DMPO-Cys110, DMPO adduct of cysteine 110; DMPO-Tyr103, DMPO adduct of tyrosine 103; DTPA, diethylenetriaminepentaacetic acid; globin1⁄7, protein radicals; H2O2, hydrogen peroxide; MNP, 2-methyl-nitrosopropane; mT, millitesla; metMb, metmyoglobin; PAGE, polyacrylamide gel electrophoresis. Spin trapping agent DMPO readily reacted with both Tyr103phenoxyl and Cys110-thiyl radicals where present in reactions of human Mb and H2O2. The precise mechanism for the formation of the Cys110-thiyl radical on wild type human Mb was not elucidated. We report further studies of the mechanism of Cys110-thiyl radical formation in the reaction of human Mb and H2O2 by the combined use of site-directed mutagenesis, gel electrophoresis, and EPR spectroscopy

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION