Nuclear Magnetic Resonance Investigation of the Electronic Structure of Deoxymyoglobin
The heme methyl resonances of sperm whale high-spin ferrous or deoxymyoglobin, Mb, have been assigned by a combination of 1H and 2H NMR of the protein reconstituted with specifically deuterated hemes. The complete methyl assignments were carried out on deoxyMb with the native protoheme in both the equilibrium orientation and the reversed orientation differing by 180° rotation about the α, γ-meso axis that is populated transiently upon assembly of apomyoglobin with protoheme. The 1H NMR spectra of deoxyMb reconstituted with a variety of synthetic hemes also lead to specific assignment of pyrrole proton signals. The heme methyl assignments for the reversed protoheme orientation are also provided for the high-spin ferric or metaquo derivative. The pattern of the dominant heme methyl contact shift in the two heme orientations of low-spin and high-spin metMb are shown to be interpretable solely on the basis of the imposition of a fixed protein-based rhombic influence on the heme in-plane asymmetry, with the net equatorial bonding to the heme conserved for the altered orientations. It is shown, moreover, that the equilibrium, as well as rates of ligation, in the conversion of metaquoMb to metcyanoMb, are independent of heme orientation. The much larger magnitude as well as the distinctive pattern of the protein-imposed rhombic influence in low-spin as compared to high-spin metMb is shown to be characteristic of an orbitally degenerate ground state of the low-spin d5 system. Analysis of the heme methyl contact shifts for the two heme orientations of deoxyMb reveals a magnitude and pattern of the protein-induced rhombic influence that strongly favor an orbital ground state derived from 5E rather than 5B2 in 4-fold symmetry. However, significant and systematic differences in the mean heme methyl contact shift and proposed E7 Val dipolar shifts in the two heme orientations provide evidence that there are likely contributions from two electronic states to the hyperfine shift pattern and that the relative contributions of the two states differ with heme orientation. Since these significant differences in the deoxyMb NMR characteristics for the two heme orientations do not manifest themselves in any ligation properties of the reduced protein, considerable caution should be exercised in interpreting heme hyperfine shifts in deoxyMb in terms of structural or functional properties. © 1993, American Chemical Society. All rights reserved.
Publication Source (Journal or Book title)
Journal of the American Chemical Society
La Mar, G., Davis, N., Johnson, R., Smith, W., Hauksson, J., Budd, D., Dalichow, F., Langry, K., Morris, I., & Smith, K. (1993). Nuclear Magnetic Resonance Investigation of the Electronic Structure of Deoxymyoglobin. Journal of the American Chemical Society, 115 (10), 3869-3876. https://doi.org/10.1021/ja00063a003