Protein oxidation of cytochrome C by reactive halogen species enhances its peroxidase activity.
Reactive halogen species (RHS; X(2) and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOCl) at pH 7.4, the peroxidase activity of cyt c was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into the protein as indicated by molecular ions of m/z = 12,360 (cyt c), 12,376 (cyt c + O), and 12,392 (cyt c + 2O). Using tandem mass spectrometry, a peptide (obtained from the tryptic digests of HOCl-treated cyt c) corresponding to the amino acid sequence MIFAGIK, which contains the methionine that binds to the heme, was identified to be involved in the oxygen incorporation. The location of the oxygen incorporation was unequivocally determined to be the methionine residue, suggesting that the oxidation of heme ligand (Met-80) by HOCl results in the enhancement of peroxidase activity of cyt c. ESR spectroscopy of HOCl-oxidized cyt c, when reacted with H(2)O(2) in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), yielded more immobilized MNP/tyrosyl adduct than native cyt c. In the presence of H(2)O(2), the peroxidase activity of HOCl-oxidized cyt c exhibited an increasing ability to oxidize tyrosine to tyrosyl radical as measured directly by fast flow ESR. Titration of both native cyt c and HOCl-oxidized cyt c with various amounts of H(2)O(2) indicated that the latter has a decreased apparent K(m) for H(2)O(2), implicating that protein oxidation of cyt c increases its accessibility to H(2)O(2). HOCl-oxidized cyt c also displayed an impaired ability to support oxygen consumption by the purified mitochondrial cytochrome c oxidase, suggesting that protein oxidation of cyt c may break the electron transport chain and inhibit energy transduction in mitochondria.
Chen
Yeong-Renn
YR
Laboratory of Pharmacology and Chemistry and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. chen6@niehs.nih.gov
Deterding
Leesa J
LJ
Sturgeon
Bradley E
BE
Tomer
Kenneth B
KB
Mason
Ronald P
RP
D003574:Cytochrome c Group / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006219:Halogens / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D003574:Cytochrome c Group; D006219:Halogens; D014443:Tyrosine; D010544:Peroxidases; D014357:Trypsin