This research program continues to explore, develop and exploit the information content of resonance Raman spectra of hemoproteins with with special emphasis on the detection/interpretation of metal-axial ligand vibrations and their utilization for understanding the mechanisms of protein control of heme reactivity in monomeric, dimeric and tetrameric hemoglobins. Dioxygen, carbon monoxide, nitric oxide, cyanide ion and azide ion are employed to probe their bounding interactions and the interactions between bound ligand and distal residues, and to provide direct evidence for the trans effect between metal-His (proximal) and metal-ligand (exogenous) bonds. To provide new insight into the specific distal H-bounding and non-bonding steric hindrance, we propose to investigate a series of E. coli synthesized human mutant hemoglobins with single amino acid replacements at His E7 (gamma Glu, Val and Gln) and at Val E11 (Ala, Leu, Ile and Met) in either alpha or beta-subunits. We also plan to investigate the effects of distal H-bonding on ligand vibrations in a series of synthetic etioporhyrins with covalently attached functional groups (-CH2OH, -COOH,-CONH2, -CONHNG2) near the metal center, capable of formine H-bonds of varying strength. To search for the origin of the extremely high 02 affinity of liver fluke (Dicrocoelium dendriticum) hemoglobin, we will carry out systematic resonance Raman studies to find evidence for the structural factors responsible for the possible elevation of free energy in the deoxy state, or the unusual stabilization (hence lower free energy) of the ligated Hb complexes. Furthermore, the proposed research has the following additional aims: (1) Experimental and theoretical studies of the Raman excitation profile of an unusually intense upilson(Fe-Im) stretching mode (at 214 cm-1) and its overtone in a synthetic adamantane heme system; (2) Development of new resonance Raman probes: vinyl bending mode for monitoring heme rotational disorder components and protein-vinyl interactions, Mn-NO stretching and Mn-N-0 bending as monitors of distal steric effects; Fe-N-O bending/or Fe-N0 strectching as a monitor of the Fe-N-O bond angle; (3) Elucidation of the allosteric control mechanism for ligand binding to dimeric insect hemoglobins CTT VIII and CTT IX; (4) Investigation of the influence of tertiary structural change of ligand bonding within the frozen T- quaternary structure of tetrameric HbM Iwate, and (5) Investigation into the unusual heme active site in ligninase, a peroxidase-like lignin-degrading enzyme from wood-degrading fungus.
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