H-1 and C-13 NMR investigation of the influence of nonligated residue contacts on the heme electronic structure in cyanometmyoglobin complexes reconstituted with centro- and pseudocentrosymmetric hemins

The H-1 and C-13 chemical shifts for the heme methyls of low-spin, ferric s perm whale cyanometmyoglobin reconstituted with a variety of centrosymmetri c and pseudocentrosymmetric hemins have been recorded and analyzed to shed light on the nature of heme-protein contacts, other than that of the axial His, that modulate the rhombic perturbation to the heme's in-plane electron ic asymmetry. The very similar H-1 dipolar shifts for heme pocket residues in all complexes yield essentially the same magnetic axes as in wild type, and the resultant dipolar shifts allow the direct determination of the heme methyl proton and C-13 contact shifts in all complexes. It is demonstrated that, even when the magnetic axes and anisotropies are known, the intrinsi c uncertainties in the orientational parameters lead to a sufficiently larg e uncertainty in dipolar shift that the methyl proton contact shifts are in herently significantly less reliable indicators of the unpaired electron sp in distribution than the methyl C-13 contact shifts. The pattern of the non inversion symmetry in C-13 contact shifts in the centro- or pseudocentrosym metric hemes is shown to correlate with the positions of aromatic rings of Phe43(CD1) and His97(FG3) parallel to, and in contact with, the heme. These results indicate that such pi-pi -interactions significantly perturb the i n-plane asymmetry of the heme a spin distribution and cannot be ignored in a quantitative interpretation of the heme methyl C-13 contact shifts in ter ms of the axial His orientation in b-type hemoproteins.