H-1-NMR CHARACTERIZATION OF MYOGLOBINS WHERE EXOGENOUS LIGANDS REPLACE THE PROXIMAL HISTIDINE

The role of the proximal ligand in determining the structure and ligand binding properties of sperm whale myoglobin has been investigated using the mutant H93G(L), where the proximal histidine has been replaced with glycine, creating a cavity which can be occupied by a variety of exogenous ligands, L, to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554; DePillis, G. D., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Sec. 116, 6981-6982]. In this report, we present the assignments of selected protons of the heme and heme pocket residues in the metcyano complexes of H93G with Im and a series of methyl-substituted Ims [H93G(Im)CN, H93G(N-MeIm)CN, H93G(2-MeIm)CN, H93G(4-MeIm)CN]. Each complex has a unique H-1 NMR spectrum, providing a fingerprint for documenting the ligand exchange phenomenon. Moreover, the identification of NOEs between the protons of proximal Ligands and protons of proximal pocket amino acid residues confirms that the new ligand occupies the proximal cavity in solution. The pattern of hyperfine-shifted heme methyl resonances in H93G(Im)CN is very different from that of wild-type Mb, consistent with the differences compared to wild-type in orientation of the proximal imidazole observed in the X-ray crystal structure of H93G(Im) [Barrick, D. (1994) Biochemistry 33, 6546-6554]. Addition of deuterated Lm to H93G(Lm)CN permits direct observation of exchange of proximal ligands with Ligands from solution; exchange of Im for deuterated Im in the metcyano complex occurs with half-life of around 10 min. The heme methyl pattern is very similar in the series of H93G(MeIm) complexes, suggesting that the orientation of the imidazole is similar in these proteins. While Im and 4-MeIm have the same affinity for the proximal binding site, N-MeIm and 2-MeIm bind more weakly than Im in the metcyano complex. By characterization of the NMR spectra of these complexes, it is possible to describe interactions between the proximal ligand, the heme, and the protein pocket which play a role in determining the structure and function of the complexes.