Following Heme Oxygenase Reaction at Atomic Scale

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Heme oxygenase (HO) oxidatively degrades heme to biliverdin. Specific aim of this research project is to delineate the molecular mechanism of the enzyme action through determination of the high resolution crystal structures of all the catalytically significant reaction intermediates. We have chosen HmuO, a heme oxygenase of Corynebacterium diphtheriae, the spectroscopic and enzymatic properties of which have been extensively studied by our research team.In the first year, we determined the crystal structures of ferric and ferrous forms of the heme complex of HmuO, the initial and the second steps of the heme degradation at 1.4 and 1.5 Å resolution. We have found that protons required for HO catalysis is channeled from the solvent water to the oxygen activation site through different conduit between mammalian HO and HmuO despite the high similarity in the overall protein fold between these two HO proteins.In the second year, we have successfully obtained the crystals of the catalytically critical oxy form and determined its structure at 1.85 A resolution. Due to low stability of the oxy form, previous crystallization attempts of oxy HO by other investigators had not been successful, and our publication is the first report of the structure of the catalytically significant oxy form. The structure has resolved long standing important questions on HO, including why oxygen affinity of heme oxygenase is very high, how heme oxygenase prevents product inhibition, and which parts of the active site are relevant to regio-selective hydroxylation of the heme group.The crystal structures of the HmuO complexes with the final product (biliverdin) and its immediate precursor (iron biliverin) have also been solved. These structures provide clues to understand the mechanism of the substrate release from the HO protein, the rate limiting step of the HO catalysis.