Ferric alpha-hydroxyheme bound to heme oxygenase can be converted to verdoheme by dioxygen in the absence of added reducing equivalents.

Whether or not reducing equivalents are indispensable for the conversion of ferric alpha-hydroxyheme bound to heme oxygenase-1 to verdoheme remains controversial (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-Saito, M. (1996) J. Biol. Chem. 271, 6618-6624; Liu, Y., Moënne-Loccoz, P., Loehr, T. M., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 6906-6917). To resolve this controversy, we have prepared a ferric alpha-hydroxyheme-heme oxygenase-1 complex and titrated the complex with O2 under strictly anaerobic conditions. The formation of verdoheme was monitored by optical and electron spin resonance spectroscopies. Electron spin resonance spectra of the complex showed that alpha-hydroxyheme exists as a mixture of resonance structures composed of the iron(III) porphyrin and the iron(II) porphyrin pi neutral radical. Upon addition of CO the latter species becomes dominant. The results obtained from these titration experiments indicate that alpha-hydroxyheme can be converted to verdoheme by an approximately equimolar amount of O2 without any requirement for exogenous electrons. The verdoheme formed from alpha-hydroxyheme was shown to be in the ferrous oxidation state by the addition of CO or potassium ferricyanide to the resultant verdoheme-heme oxygenase-1 complex.