The heme oxygenases oxidize heme to biliverdin, carbon monoxide, and ferrous iron. Although this process was once considered simply a mechanism for the catabolic elimination of excess heme, work over the past decade has firmly established that the mammalian heme oxygenases and their three products also have important roles in antiinflammatory and signaling pathways. The recent medical literature indicates that CO and biliverdin are involved in the prevention of allograft rejection, inflammation, cardiovascular disorders, and a variety of other medical pathologies. Over the past four years, we have determined the crystal structure of human heme oxygenase and made considerable progess in characterizing the protein, elucidating its mechanism, and defining its interactions with ancillary reductase proteins and agents such as NO. We propose in the continuation of this project to (a) define the structure of the enzyme throughout its catalytic stages by x-ray crystallography, NMR, and other spectroscopic methods; (b) further clarify at the molecular level the mechanism of the enzyme as it sequentially converts heme to alpha-meso-hydroxyheme, verdoheme, iron-biliverdin, and finally to the free products biliverdin and iron; (c) to isolate and characterize the heme oxygenase from Candida albicans, a potential target for anti-fungal agents, and explore its relationship with the heme binding protein obtained from Saccharomyces cerevisiae that only has heme oxygenase activity in the presence of yeast membranes, (d) to further investigate the protein-protein interactions associated with the heme oxygenase system, and (e) to design and construct probes of the heine oxygenase mechanism and agents that can specifically inhibit the activity of the mammalian and fungal enzymes. The work proposed here will yield a detailed characterization of the human heme oxygenase that can be used to clarify the biology of the enzyme and to design small molecule inhibitors and stimulators with practical applications in heme oxygenase-related medical phenomena. The results wilt also contribute to our ongoing development of a general paradigm for catalytic hemoprotein function, as heme oxygenase catalyzes a highly unusual series of steps that diverge completely from the normal pathway followed by other hemoprotein catalysts.
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