CONTROL MECHANISM OF HEMOPROTEIN REACTIVITY

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The objective of the proposed research is to elucidate the molecular mechanism which controls reactivity of hemoproteins. We use myoglobin, the protein which reversibly binds O2, as a functional prototype. In the proposed research, mutations of specific amino acids located in the oxygen binding heme cavity, such as His64, Val68, Leu29, Phe43, Phe46, Leu89 and Ser92, will be attained by site-directed mutagenesis, and mutant myoglobin proteins will be expressed in E. coli for the structural and functional studies. The amino acid replacements in the heme cavity modulate (at least) three important factors: steric crowding, hydrogen bonding, and local polarity. We will prepare myoglobin mutants with these heme pocket amino acids replaced by other residues with similar size but different polarity, or those with different size but similar polarity. Each mutant we prepare will be examined by electron paramagnetic resonance, nuclear magnetic resonance, X-ray crystallography, Raman scattering, electron- nuclear double resonance, X-ray spectroscopy, and kinetics of ligand binding. To do this we are combining resources and skills of the research groups at Case Western Reserve Univ., Rice Univ., Cornell Univ., Univ. of California, Davis, Univ. of Rome, Northwestern Univ., Princeton Univ. and Stanford Synchrotron Radiation Lab. for an efficient utilization of the power of merging protein engineering with advanced spectroscopic techniques. The structure of the heme pocket will be delineated in order to determine the effects of heme pocket amino acid substitutions upon (i) the geometry of the bound ligands, (ii) the dynamics of ligand binding, and (iii) ligand accessibility to the heme iron. The proposed study will further advance the principal investigator's current mutant myoglobin research and will provide structural interpretation of functional alterations induced by mutations. Thus, information about functional roles of heme pocket amino acids in controlling the reactivity of the heme iron in myoglobin will be obtained. The research proposed in this application will help to ascertain the structure-function relationships of hemoproteins in general, and will provide vital information necessary for the eventual preparation of artificial hemoproteins of biomedical importance, such as hemoglobin-based blood substitutes.