DETERMINATION OF THE ZERO-FIELD SPLITTING CONSTANT FOR PROTON NMR CHEMICAL-SHIFT ANALYSIS IN METAQUOMYOGLOBIN - THE DIPOLAR SHIFT AS A STRUCTURAL PROBE

Two-dimensional H-1 NMR experiments were carried out on sperm whale me taquomyoglobin (paramagnetic complex, S = 5/2) and carbonmonoxymyoglob in (diamagnetic complex) in order to determine the zero-field splittin g constant (D) of the Fe(III) atom in metaquomyoglobin. The paramagnet ic shift contains contact and dipolar contributions. The former is neg ligible for the protons included in the study while the latter is dire ctly proportional to the geometric factor and D, and can be estimated by taking the difference of the chemical shifts in the two myoglobin c omplexes. The value so obtained is accurate if the carbonmonoxymyoglob in shift is an adequate diamagnetic reference for metaquomyoglobin. In order to determine D, 37 backbone resonances were selected for which this assumption was likely to be closely satisfied. The selection was achieved by identifying protons with a constant ring current shift as calculated from the solid-state structures of metaquomyoglobin and car bonmonoxymyoglobin. With this restricted set of dipolar shifts and the geometric factors calculated from the solid state coordinates, D was found to be 9.66 4 +/- 0.08 cm-1. This constant was then used to evalu ate the dipolar contribution to the chemical shift of the other 104 as signed protons. Comparison of the predicted and the observed values pr ovided a description of the structural alterations occurring upon chan ge in complexation and dissolution. In nearly 80% of the cases, good a greement was observed, which indicated that the X-ray structure is the same as the solution structure and that the dipolar shift can be used to confirm spectral assignments. However, discrepancies were noted in the A-G-H and G-H interfaces and in turns. For several regions, the s tructural perturbations could be rationalized with features specific t o the metaquomyoglobin solid-state structure, such as proximity to an ion binding site and unallowed steric interactions. The dipolar shift was demonstrated to be a sensitive probe for the elucidation of the co nformational properties of metaquomyoglobin in solution and for a comp arison to the solid-state structure.