Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from cytochrome c-derived tyrosyl radical.
Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.
Chen
Yeong-Renn
YR
Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Chen
Chwen-Lih
CL
Chen
Weiguo
W
Zweier
Jay L
JL
Augusto
Ohara
O
Radi
Rafael
R
Mason
Ronald P
RP
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D045304:Cytochromes c / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals / Q000737:chemistry*; D006736:Horses; D006861:Hydrogen Peroxide / Q000737:chemistry; D015151:Immunoblotting; D008280:Magnetics; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry; D009573:Nitrites / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry; D051381:Rats; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
D005609:Free Radicals; D009573:Nitrites; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D009113:Muramidase; D010100:Oxygen