NMR determination of the orientation of the magnetic susceptibility tensor in cyanometmyoglobin: a new probe of steric tilt of bound ligand

Abstract

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.

Characterization of the haem environment in Methylophilus methylotrophus ferricytochrome c″ by 1H‐NMR

“…Furthermore, both the axial histidines experience reasonably large hyperfine shifts, suggesting roughly similar interactions with the haem iron, in contrast to ferricytochrome b5, the only other bis-histidinyl haem protein for which the haem electronic structure has so far been published [33]. It has often been suggested that the orientation of the axial ligands has a major influence on the distribution of the unpaired electron [31,[33][34][35][36], but it seems that this influence is unimportant as far as functional properties of the protein are concerned, such as redox potential and electron-transfer kinetics [23]. The high symmetry of the unpaired-electron distribution observed in cytochrome c" agrees very well with the proposed perpendicular orientation of the imidazole planes [2].…”

Application of the paramagnetic dipole field for solution NMR active site structure determination in low‐spin, cyanide‐inhibited ferric hemoproteins

“…First, a set of iron-centered, reference coordinates, x 0 , y 0 , z 0 , are generated from the crystal coordinates, and the dipolar shift expressed in terms of these (known) coordinates, r, y 0 , O 0 , as follows (1,4,5): Figure 1, with the heme normal serving as the z 0 axis, and x 0 , y 0 in the heme plane. The major magnetic axis, z, is tilted from the heme normal (z 0 ) by the angle b in Figure 1B, and the projection of this tilt on the x 0 , y 0 plane, given by the angle, a, defines the direction of tilt (Fig.…”

Solution Structure of Oxidized Horse Heart Cytochrome c,

“…The different orientation of the histidine ring with respect to the heme plane (the projection of the ring in the two average structures differs of about 10°) may only account for about 20% of this discrepancy. The major contribution is due to the strong dependence of such pseudocontact shift on the deviation of the zz axis from the normal to the heme plane (Emerson & La Mar, 1990). Such deviation is larger in the case of the S. cereVisiae protein.…”