13. Functional Modification of Cytochrome c by Peroxynitrite in an Electron Transfer Reaction
Hidehiko Nakagawa, Yukiko Ohshima, Mitsuko Takusagawa, Nobuo Ikota and Toshihiko Ozawa
Keywords: cytochrome c, peroxynitrite, tyrosine nitration, functional modification, mitochondria, membrane potential
Reactive oxygen species (ROS) such as superoxide and reactive nitrogen species such as nitric oxide are suggested to be involved in the pathogenesis of various diseases. Although nitric oxide and superoxide are both endogenous and important compounds for physiological responses, they become toxic and pathogenic when overproduced. Peroxynitrite, which is derived from nitric oxide and superoxide by a diffusion rate-limiting reaction, is one of the toxic intermediates produced under the ROS and RNS overproduction conditions. A typical reaction of peroxynitrite for the biological components is the nitration of protein tyrosine residues. In various diseases associated with oxidative stress, nitrotyrosine formation has been reported, suggesting the involvement of peroxynitrite in the pathogenesis. The effect of the nitration of tyrosine residue in proteins, however, is not clear. In this report, we investigated the effect of tyrosine nitration in cytochrome c on its electron transfer reaction in the mitochondrial energy production. Cytochrome c was treated with a bolus of synthetic peroxynitrite at a sub-millimolar concentration under physiological conditions, and then subjected to reduction by superoxide and oxidation by hydrogen peroxide, that is the redox reaction of cytochrome c. The ability for the membrane potential formation in the mitochondrial respiratory chain was also evaluated. After the treatment with peroxynitrite, it was revealed that the cytochrome c molecule was mono-nitrated mainly at a tyrosine residue, by liquid chromatography-electrospray ionizing mass spectrometry (LC-ESIMS) and HPLC analysis. No obvious changes were observed in the circular dichromism (CD) spectra and the absorption spectra from peroxynitrite-treated cytochrome c. These results suggest that the sub-millimolar peroxynitrite treatment did not obviously affect the cytochrome c molecule except for mono-nitration of the protein. Although the redox capacity of cytochrome c was not changed by the treatment under the conditions in this study, it was found that the oxidation of ferrocytochrome c to ferricytochrome c by hydrogen peroxide was accelerated depending on the concentration of peroxynitrite used. When cytochrome c was treated with peroxynitrite in the presence of 5-methoxytryptamine, a selective inhibitor for the tyrosine nitration by peroxynitrite, the acceleration of hydrogen peroxide-mediated oxidation was suppressed. This result suggests that the tyrosine nitration induced the acceleration of the oxidation mediated by hydrogen peroxide, which is a non-physiological oxidation pathway of cytochrome c. Furthermore, it was also found that the formation of membrane potential in the rat liver mitochondria was suppressed when peroxynitrite-treated cytochrome c was used instead of the intact cytochrome c in vitro. From these results, we concluded that the sub-millimolar peroxynitrite treatment induced nitration of cytochrome c at a tyrosine residue, and that the resulting mono-nitrated cytochrome c became more susceptible to oxidation by hydrogen peroxide, concomitantly losing the ability to transfer electrons in the mitochondrial respiratory chain. We would suggest that the peroxynitrite-induced modification of cytochrome c increases the susceptibility to non-physiological oxidants, and may cause dysfunction of mitochondria by suppressing membrane potential.
Publications:
Nakagawa, H., Ohshima, Y., Takusagawa, M., Ikota, N., Takahashi, Y., Shimizu, S. and Ozawa, T.: Chem. Pharm. Bull. (Tokyo), 49, 1547-1554, 2001.
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