Electronic/Molecular Structure of Enzyme Heme Pockets

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DESCRIPTION: (provided by applicant) This proposal deals with experimental approaches to quantitative characterization of both the molecular and electronic structures of the active sites of moderate-sized (30-80 kDa) heme-containing enzymes in order to understand the mechanism (peroxidase vs. oxygenase) and stereospecificity (alpha vs. beta, gamma, or delta-meso heme cleavage) of the reactions with substrate. Solution NMR spectroscopy of the active site of heme enzymes in paramagnetic states is ideally suited for providing detailed description of such active sites, where advantage is taken of both the increased spectral dispersion that significantly facilitates resolution for active site residues, and the great wealth of novel structural information contained in the hyperfine shifts responsible for the shift dispersion. Methodological approaches will emphasize more quantitative structural studies (internuclear distance) of 30-40 kDa low-spin, ferric enzymes and extension of both assignments and structural analyses to larger 78 kDa) low-spin, ferric, and more strongly paramagnetic, 30-40 kDa high-spin, ferric heme enzymes. The methodological advances are expected to contribute not only to effective structural characterization of our own target enzymes, but to contribute significantly to the NMR investigation of a large variety of other paramagnetic metalloproteins currently pursued in other research laboratories. The optimally developed NMR strategies will be applied to determining in detail the effect of mutation and ligation on the nature of the heme cavity and substrate binding pocket of 44 kDa horseradish peroxidase, and elucidating the heme electronic structure that results from the covalent heme links to the protein matrix in 78 kDa lactoperoxidase. Studies on ligated and substrate-bound human heme oxygenase will focus on reconciling the apparent structural differences between the ligated complex in solution and the unligated complex in the crystal with respect to the position of the distal helix, the nature of the distal hydrogen-bond donor to the bound ligand, the identity of the titrating group that inactivates the protein, the exact origin of steric tilt of the ligand in the direction of the alpha-meso position, and the nature of the distal hydrogen-bonds between two Tyr that are likely involved in "closing" the substrate cavity prior to reaction. The effect of mutation on these structural features will also be examined. The prospects for definitive solution NMR studies of cytochrome P450 will also be explored. It is expected that a considerable improved understanding of the solution structures of the target enzyme and the relationship to their functions will result from these studies.