On the Oxidation of Cytochrome c by Hypohalous Acids

Oxidation of cytochrome c, a key protein in mitochondrial electron transport and a mediator of apoptotic cell death, by reactive halogen species (HOX, X2), i.e., metabolites of activated neutrophils, was investigated by stopped-flow. The fast initial reactions between FeIIIcytc and HOX species, with rate constants (at pH 7.6) of k > 3 × 106 M−1 s−1 for HOBr, k > 3 × 105 M−1 s−1 for HOCl, and k = (6.1 ± 0.3) × 102 M−1 s−1 for HOI, are followed by slower intramolecular processes. All HOX species lead to a blue shift of the Soret absorption band and loss of the 695-nm absorption band, which is an indicator for the intact iron to Met-80 bond, and of the reducibility of FeIIIcytc. All HOX species do, in fact, persistently impair the ability of FeIIIcytc to act as electron acceptor, e.g., in reaction with ascorbate or O•−2. I2 selectively oxidizes the iron center of FeIIcytc, with a stoichiometry of 2 per I2, and with k(FeIIcytc + I2) ≈ 4.6 × 104 M−1 s−1 and k(FeIIcytc + I•−2) = (2.9 ± 0.4) × 108 M−1 s−1. Oxidation of FeIIcytc by HOX species is not selectively directed toward the iron center; HOBr and HOCl are considered to react primarily by N-halogenation of side chain amino groups, and HOI mainly by sulfoxidation. There is some evidence for the generation of HO• radicals upon reaction of HOCl with FeIIcytc. Chloramines (e.g., NH2Cl), bromamine (NH2Br), and cyclo-Gly2 chloramide oxidize FeIIcytc slowly and unselectively, but iodide efficiently catalyzes reactions of these N-halogens to yield fast selective oxidation of the iron center; this is due to generation of I2 by reaction of I− with the N-halogen and recycling of I− by reaction of I2 with FeIIcytc. Iodide also catalyzes methionine sulfoxidation and thiol oxidation by NH2Cl. The possible biological relevance of these findings is discussed.