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17. Change of Redox Property of Cytochrome C by Peroxynitrite
Hidehiko Nakagawa, Yukiko Ohshima*, Nobuo Ikota and Toshihiko Ozawa
(*Kitasato University)
Keywords: peroxynitrite, cytochrome c, oxidation, 5-methoxytryptamine, lipoic acid, photospectrometry
Reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) are considered to be involved in the pathogenesis of various diseases. Nitric oxide and superoxide, ROS/RNS species, are endogenous and important compounds in physiological reactions such as vasodilatation, signal transduction, and protection from infection. However, when overproduced, these compounds also have toxic effects on various endogenous biological substances. Peroxynitrite (PN) is a product from a rate-limiting reaction between nitric oxide and superoxide. It is considered to be produced not only in vitro, but also in vivo systems. PN production proceeds oxidative reactions with various biological components, so that PN is considered to cause various types of oxidative damage to tissues and cells, such as LDL oxidation, lipid peroxidation, DNA strand breakage, and so on. One of the most characteristic reactions of PN is the nitration of free tyrosine and protein tyrosine residues. The presence of nitrotyrosine in tissues or cell cultures is often used as a marker for the production of PN. In this study, the effect of peroxynitrite on cytochrome c, which is one of the components of the mitochondrial electron transfer system, was examined. The inhibitory effect of 5-methoxytryptamine and lipoic acid, which we previously reported are peroxynitrite scavengers, was also tested.
Peroxynitrite was synthesized from NaN3 and ozone. Briefly, NaN3 was dissolved in an alkaline solution adjusted to pH 12, followed by bubbling of an oxygen stream containing ozone while the solution was kept in an ice bath. After bubbling, the solution was frozen by cooling in a dry ice-acetone bath and stored at -20°C until use. The concentration of the peroxynitrite solution was determined spectrophotometrically by measuring of the absorbance at 302nm (
=1670M-1cm-1). Hypoxanthine and xanthine oxidase were added to a solution of cytochrome c (0.02mM) or peroxynitrite-pretreated cytochrome. The redox status of cytochrome c was monitored by measuring absorbance at 550nm (the characteristic absorption band in the reduced form of cytochrome c). Cytochrome c was reduced once by superoxide, and then oxidized by hydrogen peroxide, which had been produced from the dismutation of superoxide, using the hypoxanthine/ xanthine oxidase system. The oxidation rate of the reduced form of cytochrome c was increased, dose-dependently, for pretreatment with peroxynitrite. This acceleration of the oxidation in cytochrome c was depressed by pretreatment with 5-methoxytryptamine or lipoic acid prior to the peroxynitrite treatment, but the rate was not changed by treatment with the oxidized form of glutathione. Because the treated peroxynitrite was considered to be completely decomposed until addition of the hypoxanthine/ xanthine oxidase system, the change of the oxidation rate was due to the modification of cytochrome c by peroxynitrite. This acceleration was effectively reduced by the pretreatment with 5-methoxytryptamine. Since the oxidation rate of the intact cytochrome c was not affected by 5-methoxytryptamine which is a selective inhibitor of the nitration of tyrosine residue, the modification of cytochrome c was assumed to be due to the nitration of tyrosine residues (Fig. 12).
Fig.12. Oxidation rate of peroxynitrite-treated cytochrome c. Oxidation rate (change of absorbance at 550nm per minute) was determined from the reduction and oxidation reaction of cytochrome c with hypoxanthine and xanthine oxidase system. A) Oxidation rate of peroxynitrite-treated cytochrome c. B) Oxidation rate of 5MT (5MT: 5-methoxytryptamine) and peroxynitrite-treated cytochrome c.
Publications:
Nakagawa H., Sumiki E., Ikota N., Matsushima Y., Ozawa T.: Antiox. Redox Signaling, 1, 239-244, 1999.
