Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to yield biliverdin, CO and iron. It is established that heme degrades via two intermediates: α-hydroxyheme and verdoheme, however, the redox stoichiometry for the overall HO reaction remains unclear. We determined the crystal structure of the rat HO-1 in complex with heme and found that the proximal ligand of the heme iron is His-25. At the distal side, no side chains made direct contact with the heme iron but a hydroxide that can be stabilized by hydrogen bonds formed with backbones of Gly-139 and Gly-143. One of the hydrogen bonds would facilitate the electrophilic addition of the ferric hydroperoxide to the porphyrin ring, resulting in heme α-meso-hydroxylation. Furthermore, we prepared a ferric α-hydroxyheme–HO-1 complex and titrated the complex with O2 under strictly anaerobic conditions. The results obtained indicate that the α-hydroxyheme bound to the enzyme can be converted to a verdoheme by an approximately equimolar amount of O2, without any requirement for exogenous electrons. The redox state of the verdoheme formed proved to be ferrous when CO or potassium ferricyanide was added to the resultant verdoheme–HO-1 complex.
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