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 Respiratory burst during phagocytosis of bacteria by neutrophils is a physiological response (1). It is well known that during the respiratory burst, a number of enzymatic processes are activated, which then act to produce oxygen metabolites. For example, the hexose monophosphate shunt is activated to increase glucose oxidation and production of CO 2 and NADPH (1). Superoxide anion is generated by activation of NADPH oxidase; superoxide then dismutates to hydrogen peroxide (1).However, the respiratory burst of neutrophils or monocytes can also mediate the metabolism of halide and generate reactive halogen species (RHS) 1 such as HOX and X 2 (where X represents Cl, Br, or I) (1, 2). In the presence of H 2 O 2 , a halide anion is a substrate in the peroxidative reaction catalyzed by myeloperoxidase (MPO) released from the primary granule (1, 3, 4). The halide that is commonly most abundant is Cl Ϫ (physiological concentration of 100 mM), and the reactive MPO system is unique in oxidizing chloride ion (Reactions 1 and 2). MPO(Fe III

The reaction of purified bovine mitochondrial cytochrome c oxidase (CcO) and hydrogen peroxide was studied using the ESR spin-trapping technique. A protein-centered radical adduct was trapped by 5,5-dimethyl-1-pyrroline N-oxide and was assigned to a thiyl radical adduct based on its hyperfine coupling constants of a N ‫؍‬ 14.7 G and a ␤ H ‫؍‬ 15.7 G. The ESR spectra obtained using the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (