Protein Engineering and Reactivity Control in Multi-functional Chimeric Metalloproteins

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This research project includes the following two subjects.(1)Structural Characterization of Natural Chimeric Protein, CooA Protein, and Its Molecular MechanismTo investigate structural characterization of CooA protein, which has a myoglobin-like heme binding domain and helix-turn-helix type DNA binding domain, NMR, resonance Raman spectra and ligand binding were utilized. The resonance Raman spectrum for the ferrous CO adduct of CooA protein confirmed the ligation of a histidine residue to the heme iron, while, in the absence of CO, the ligation of an arginine or lysine residue instead of CO were suggested by the NMR spectrum, which is quite unusual in hemoproteins. Although the association rate for the CO rebinding in CooA protein was comparable to that in myoglobin or hemoglobin, it was characterized by its large fraction of the very fast (<ns) rebinding process in the CO recombination. Such a large fraction of the very fast rebinding process can be ascribed to the steric hindrance around the CO binding site and suggest that the ligation of CO kicks or pushes out some amino acid residues near the binding site, inducing the large structural rearrangements for the efficient DNA binding.(2)Protein Engineering for New Chimeric Metalloproteins by Module Substitution.Systematic module substitutions for globins in this research project have shown that the previously proposed module is not a minimum structural unit, but can be further divided into two "sub-modules". Particularly, I focused on one of the "sub-modules", "heme binding module", which consists of the iron-liganded histidine and about 20 amino acid residues in the heme proximal side.On the basis of various spectroscopic data including NMR and resonance Raman spectra, it can be concluded that the "heme binding module" would be a primary determinant for the electronic state of the heme and configuration of the liganded histidine in globins.