Electron spin resonance investigation of the cyanyl and azidyl radical formation by cytochrome c oxidase.
Cyanide (CN(-)) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heme a(3) of cytochrome c oxidase (CcO). As-isolated CcO oxidized cyanide to the cyanyl radical ((.)CN) that was detected, using the ESR spin-trapping technique, as the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(.)CN radical adduct. The enzymatic conversion of cyanide to the cyanyl radical by CcO was time-dependent but not affected by azide (N(3)(-)). The small but variable amounts of compound P present in the as-isolated CcO accounted for this one-electron oxidation of cyanide to the cyanyl radical. In contrast, as-isolated CcO exhibited little ability to catalyze the oxidation of azide, presumably because of azide's lower affinity for the CcO. However, the DMPO/(.)N(3) radical adduct was readily detected when H(2)O(2) was included in the system. The results presented here indicate the need to re-evaluate oxidative stress in mitochondria "chemical hypoxia" induced by cyanide or azide to account for the presence of highly reactive free radicals.
Chen
Y R
YR
Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. chen6@niehs.nih.gov
Sturgeon
B E
BE
Gunther
M R
MR
Mason
R P
RP
D000818:Animals; D001386:Azides / Q000737:chemistry*; D002417:Cattle; D003198:Computer Simulation; D003486:Cyanides / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry*; D005609:Free Radicals / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D008929:Mitochondria, Heart / Q000201:enzymology; D013113:Spin Labels; D019097:Spin Trapping; D013997:Time Factors
D001386:Azides; D003486:Cyanides; D003497:Cyclic N-Oxides; D005609:Free Radicals; D013113:Spin Labels; C069977:2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy; D006861:Hydrogen Peroxide; D003576:Electron Transport Complex IV