NMR determination of the orientation of the magnetic susceptibility tensor in cyanometmyoglobin: a new probe of steric tilt of bound ligand.
The experimentally determined paramagnetic dipolar shifts for noncoordinated amino acid side-chain protons in the heme pocket of sperm whale cyanometmyoglobin [Emerson, S. d., & La Mar, G. N. (1990) Biochemistry (preceding paper in this issue]) were used to determine in solution the orientation of the principal axes for the paramagnetic susceptibility tensor relative to the heme iron molecular coordinates. The determination was made by a least-squares search for the unique Euler rotation angles which convert the geometric factors in the molecular (crystal) coordinates to ones that correctly predict each of 41 known dipolar shifts by using the magnetic anisotropies computed previously [Horrocks, W. D., Jr., & Greenberg, E. S. (973) Biochim. Biophys. Acta 322, 38-44]. An excellent fit to experimental shifts was obtained, which also provided predictions that allowed subsequent new assignments to be made. The magnetic axes are oriented so that the z axis is tipped approximately 15 degrees from the heme normal toward the hem delta-meso-H and coincides approximately with the characterized FeCO tilt axis in the isostructural MbCO complex [Kuriyan, J., Wilz, S., Karplus, M., & Petsko, G. A. (1986) J. Mol. Biol. 192, 133-154]. Since the FeCO and FeCN units are isostructural, we propose that the dominant protein constraints that tips the magnetic z axis from the heme normal is the tilt of the FeCN by steric interactions with the distal residues. The rhombic magnetic axes were found to align closely with the projection of the proximal His imidazole plane on the heme, confirming that the His-Fe bonding provides the protein constraints that orients the in-plane anisotrophy. The tipped magnetic z axis is shown to account quantitatively for the previously noted major discrepancy between the hyperfine shift patterns for the bound imidazole side chain in models and protein. Moreover, it is shown that the proximal His ring nolabile proton hyperfine shifts provide direct and exquisitely sensitive indicators of the degree of the z axis tilt that may serve as a valuable probe for characterizing variable steric interactions in the distal pocket of both point mutants and natural genetic variants of myoglobin.
Emerson
S D
SD
Department of Chemistry, University of California, Davis 95616.
La Mar
G N
GN
D000596:Amino Acids; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006418:Heme; D006420:Hemeproteins; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008786:Metmyoglobin / Q000031:analogs & derivatives; D013696:Temperature; D014907:Whales
D000596:Amino Acids; D006420:Hemeproteins; D008024:Ligands; C007177:cyanometmyoglobin; D008786:Metmyoglobin; D006418:Heme