Mechanism of porphyrin ring cleavage in the heme oxygenase catalysis

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Heme oxygenase (HO) degrades heme to biliverdin through three successive oxygenations. While the third oxygenation, ring opening of verdoheme, is considered as the rate-determining step to regulate HO enzyme activity in vivo, this step has been the least understood in the Ho catalysis. Specific aim of this research project is to delineate the third oxygenation by HO through detailed reaction analysis, crystallographic and spectroscopic characterization. We also have examined a new HO reaction that affords a novel heme catabolite under physiologically relevant condition.In the classical HO reaction, we have found that HO degrades verdoheme through a dual pathway using either O_2 or H_2O_2. Together with inhibitory and mutational studies, we have proposed a mechanism involving an Fe-OOH verdoheme as a reactive intermediate. This proposal is strongly supported by the fact that the macrocycle cleavage with small alkyl hydroperoxides (MeOOH and EtOOH) incorporated the alkyl group into one end of linear tetrapyrroles produced.The reactive peroxy species is crystallographically characterized for the first time in myoglobin, a model protein of heme enzymes. Crystals of the peroxy myoglobin have been successfully generated by radiolytic reduction of the oxy myoglobin crystals at 100 K by irradiation with 1.0 A synchrotron radiation. Diffraction data is collected by 0.6 A synchrotron radiation without further reaction of the peroxy species.The new heme catabolite, S-biliverdin, has been chemically synthesized and identified by LC-MS and NMR analysis. The verdoheme cleavage in the new HO reaction proceeds through an O2-independent manner and the product ratio of S-biliverdin over biliverdin largely depends on the O2 concentration. These mechanistic changes are expected to affect on the physiological functions, namely an O2 sensing property of HO.