Involvement of protein radical, protein aggregation, and effects on NO metabolism in the hypochlorite-mediated oxidation of mitochondrial cytochrome c

Cytochrome c (cyt c)-derived protein radicals, radical adduct aggregates, and protein tyrosine nitration have been implicated in the pro-apoptotic event connecting inflammation to the development of diseases. During inflammation, one of the reactive oxygen species metabolized via neutrophil activation is hypochlorite (HOC1); destruction of the mitochondrial electron transport chain by hypochlorite is considered to be a damaging factor. Previous study has shown that HOC1 induces the site-specific oxidation of cyt c at met-80. In this work, we have assessed the hypothesis that exposure of cyt c to physiologically relevant concentrations of HOC1 leads to protein-derived radical and consequent protein aggregation, which subsequently affects cyt c's regulation of nitric oxide metabolism. Reaction intermediates, chemical pathways available for protein aggregation, and protein nitration were examined. A weak ESR signal for immobilized nitroxide derived from the protein was detected when a high concentration of cyt c was reacted with hypochlorite in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane. When a low concentration of cyt c was exposed to the physiologically relevant levels of HOC1 in the presence of 5,5-dimethyl-pyrroline N-oxide (DMPO), we detected DMPO nitrone adducts derived from both protein and protein aggregate radicals as assessed by Western blot using an antibody raised against the DMPO nitrone adduct. The cyt c-derived protein radicals formed by HOC1 were located on lysine and tyrosine residues, with lysine predominating. Cyt c-derived protein aggregates induced by HOC1 involved primarily lysine residues and hydrophobic interaction. In addition, HOC1-oxidized cyt c (HOC1-cyt c) exhibited a higher affinity for NO and enhancement of nonenzymatic NO synthesis from nitrite reduction. Furthermore, HOC1-mediated cyt c oxidation also resulted in a significant elevation of cyt c nitration derived from either NO trapping of the cyt c-derived tyrosyl radical or cyt c-catalyzed one-electron oxidation of nitrite. (C) 2004 Elsevier Inc. All rights reserved.