Cryogenic absorption spectra of hydroperoxo-ferric heme oxygenase, the active intermediate of enzymatic heme oxygenation

Using radiolysis with P-32 enriched phosphate as an internal source of ionizing radiation, the formation of hydro-peroxo-ferric complex from oxy-ferrous precursor with a high yield was monitored at 77 K in heme oxygenase (HO) by means of optical absorption spectroscopy. Well-resolved absorption spectra (maxima at 421 nm, 530 ran, 557 nm) of hydroperoxo-ferric intermediate of this heme enzyme were measured in 70% glycerol/buffer frozen glasses. After annealing at 210215 K this complex converts to the product complex, alpha-meso hydroxyheme-HO. No heme degradation products were formed in control experiments with ferric HO or other heme proteins. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.