Electron spin resonance investigation of the cyanyl and azidyl radical formation by cytochrome c oxidase

Abstract

Cyanide (CN-) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heine α3 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 (N3/-). 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/·N3 radical adduct was readily detected when H2O2 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.