Our long range research goals are: (a) to develop stereochemical theories for ligand capture and bond formation in hemoglobins (Hb) and myoglobins (Mb); (b) to determine the mechanism of how these proteins and microbial flavohemoglobins (flavoHb) oxidize nitric oxide to nitrate without producing toxic side products; and (c) to develop strategies for inhibiting the NO dioxygenase activity of flavoHbs from pathogenic microorganisms. We have identified the roles of specific amino acids, structural motifs, and stereochemical effects in regulating O2 affinity, ligand discrimination, rates of ligand binding, and NO dioxygenation using mammalian Mb as a model system. Proof of the validity of these mechanisms and their extension to other proteins requires further testing with Mb and detailed comparisons of the functional and structural properties of four unique animal hemoglobins and three microbial flavoHbs. Three new projects are planned to achieve these goals. (1) More rigorous tests of the side path kinetic model for ligand binding in Mb will be carried out using time resolved X-ray crystallography, mutagenesis of amino acids located along the putative pathways, and measurements of the binding of alkyl isocyanides as stereochemical probes of the distal pocket. (2) The generality of the histidine gate for ligand entry and the electrostatic theory for distal regulation of O2 affinity will be tested in four different heme protein systems in which: (a) the distal histidines are exposed to solvent - alpha and beta subunits of tetrameric human HbA; (b) movement of the distal histidine is restricted by a novel dimeric interface between adjacent E-helices - lamprey Hb; (c) the distal histidine gate is completely blocked by a large polar interface - Scapharca inequivalvis Hbl dimers; and (d) a well-defined internal hydrophobic channel appears to be the pathway for ligand entry and exit - Cerebratulus lacteus Hb. (3) The mechanisms and intermediates involved in NO dioxygenation by MbO2, HbO2, and microbial flavoHbO2 will be determined by rapid-mixing absorbance, freezequench EPR, and flow-flash laser photolysis techniques, and the applicability of the results will be tested in two new flavoHbs from fungal pathogens, Candida albicans and Aspergillus fumigatus.
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