Characterization of adducts of ethanol metabolites with cytochrome c.
Cytochrome c (cyt c) is found in the mitochondria of all mammalian cells where hydrogen peroxide is produced as a byproduct of the electron transport chain. In the presence of peroxide cyt c generates a ferryl heme and radicals at Tyr residues (Barr et al., 1996). These radicals can be transferred to Trp residues within the protein or to Tyr- and Trp-containing peptides (Deterding et al., 1998). We report that addition of ethanol to this system of cyt c plus peroxide results in replacement of the Tyr/Trp radicals by 1-hydroxyethyl radicals (HER), and covalent binding of up to 10 mol of ethanol per mol of cyt c. In the absence of exogenously added peroxide, ethanol incorporation to cyt c is attained also with a reconstituted system of the ethanol-inducible cytochrome P-4502E1 isozyme. Comparative studies with myoglobin and apomyoglobin suggest that the heme is necessary for ethanol adduction of the protein to occur. Structural analysis by mass spectrometry of the tryptic digestion fractions of adducted cyt c is consistent with several peptides bearing one-to-three acetaldehyde moieties on Lys residues, and three distinct Tyr/Trp-containing peptides: P[28-53], P[56-73], P[73-91] carrying one-to-two HER. The x-ray crystallographic structure of cyt c shows that the Tyr/Trp residues in the adducted peptides are in close proximity to the heme. In conclusion, our data show that ethanol metabolites alkylate cyt c under oxidative stress and point to HER-Tyr/Trp adducts as plausible markers of alcoholism.
Thomas Jefferson University, Jefferson Medical College, Department of Pathology-Anatomy and Cell Biology, Alcohol Research Center, Philadelphia, Pennsylvania 19107, USA.
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D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002492:Central Nervous System Depressants / Q000378:metabolism*; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D000431:Ethanol / Q000378:metabolism*; D006736:Horses; D006801:Humans; D013058:Mass Spectrometry
D002492:Central Nervous System Depressants; D003574:Cytochrome c Group; C065424:1-hydroxyethyl radical; D000431:Ethanol