SOLUTION H-1-NMR STUDY OF THE ELECTRONIC-STRUCTURE AND MAGNETIC-PROPERTIES OF HIGH-SPIN FERROUS OR DEOXY MYOGLOBINS

Solution 1D and 2D NMR, together with limited isotope labeling, have l ed to the assignment of the heme, axial His, and numerous heme contact residues in sperm whale, horse, and human deoxy myoglobin. The parama gnetic relaxivity leads to increased line widths and shorter T(1)s wit h little compensation in increased dispersion due to dipolar shifts. H ence only limited, but crucial F helix standard backbone sequence spec ific assignment could be made for heme cavity residues. Numerous other residues with significant dipolar shifts could be assigned from the c haracteristic scalar connectivities and dipolar contacts to the heme p redicted by the crystal structure. It is concluded that the complete a nd unambiguous assignment of the heme pyrrole substituent signals is n ot attainable by 2D NMR alone without either partial deuterium labelin g of the heme or parallel assignment of key residues in dipolar contac t with the heme; hence the present study revises some earlier assignme nts. The resulting dipolar shifts for nonligated residues, together wi th the crystal coordinates of deoxy myoglobin, were used to determine the orientation relative to the heme and the anisotropy of the paramag netic susceptibility tenser. The significant anisotropy, \Delta chi\ s imilar to 1 x 10(-9) m(3)/mol, however, is shown to result in dipolar shift with reciprocal square, rather than just reciprocal, absolute te mperature dependence, which is indicative of large zero field splittin g rather than g-tensor anisotropy. The appropriate equation for a B-5( 2) ground state allows an estimate of the zero-field splitting, D simi lar to-10 cm(-1), which is in good agreement with earlier results. The present NMR data favor a spin-only magnetic moment with S = 2 and D s imilar to-10 cm(-1) over a ground state with S < 2 and significant orb ital contribution (Hendrich and Debrunner, 1989).