1H 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., & Boxer, S.G. (1994) J. Am. Chem. Soc. 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 1H 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 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 Im to H93G(Im)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.(ABSTRACT TRUNCATED AT 250 WORDS)
Decatur
S M
SM
Department of Chemistry, Stanford University, California 94305-5080.
Boxer
S G
SG
D000818:Animals; D006639:Histidine / Q000737:chemistry; D066298:In Vitro Techniques; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014907:Whales
D008024:Ligands; D009211:Myoglobin; D011994:Recombinant Proteins; D006639:Histidine