Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical

Previous studies established that the cyanyl radical (.CN), detected as 5,5 -dimethyl-1-pyrroline N-oxide (DMPO)/.CN by the electron spin resonance (ES R) spin-trapping technique, can be generated by horseradish peroxidase (HRP ) in the presence of hydrogen peroxide (H2O2) and by mitochondrial cytochro me c oxidase (CcO) in the absence of H2O2 TO investigate the mechanism of i nhibition by cyanyl radical, we isolated and characterized the iron protopo rphyrin IX and heme a from the reactions of CN- with HRP and CcO, respectiv ely. The purified heme from the reaction mixture of HRP/H2O2/KCN was unambi guously identified as cyanoheme by the observation of the protonated molecu le, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ioni zation (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferr ous cyanoheme complex revealed that one of the four meso protons was missin g and had been replaced with a cyanyl group, indicating that the single, he me-derived product was meso-cyanoheme. The holoenzyme of HRP from the recon stitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no de tectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoH RP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, t he heme a isolated from partially or fully inhibited CcO did not show any c hange in the structure of the protoporphyrin IX as indicated by its MALDI m ass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridin e hemochromogen spectrum. However, a protein-centered radical on the CcO ca n be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in he me structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the differ ent inhibition mechanisms of HRP and CcO by cyanyl radical.