Biochemical properties of cytochrome c nitrated by peroxynitrite.
Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.
Jang
Byungki
B
Department of Biochemistry, Kangwon National University, Chunchon 200-701, South Korea.
Han
Sanghwa
S
D000818:Animals; D003486:Cyanides / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D005452:Fluoresceins / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013481:Superoxides / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000138:chemical synthesis
D003486:Cyanides; D005452:Fluoresceins; C065013:2',7'-dichlorodihydrofluorescein; D013481:Superoxides; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D045304:Cytochromes c; D010544:Peroxidases