H-1 NMR investigation of the role of intrinsic heme versus protein-inducedrhombic perturbations on the electronic structure of low-spin ferrihemoproteins: Effect of heme substituents on heme orientation in myoglobin

Solution H-1 NMR spectroscopy has been used to characterize the cyanomet my oglobin complexes of a variety of chemically modified hemins in order to el ucidate the importance of hemin peripheral electronic, relative to axial Hi s imidazole-induced, rhombic perturbations in raising the orbital degenerac y of the pi-bonding d(xz),d(yz) orbitals. Variation of the hemin 2- and/or 4-position substituents among hydrogen, ethyl, vinyl, acetyl, and formyl gr oups leads to conserved molecular structure of the heme pocket and orientat ion of the major magnetic axis for the heme iron, but systematically pertur bed heme methyl contact shift patterns. Two strongly rhombically perturbed hemins with single acetyl groups on either pyrrole I or II exhibit heme met hyl contact shift patterns and characteristic deviations from Curie law tha t are very similar to that induced in pseudosymmetric hemins upon incorpora tion into metMbCN in the alternate orientations about the alpha,gamma-meso axis. The perturbation due to the 4-acetyl group and the axial His bond lea ds to increased contact shift spread and stronger deviations from Curie beh avior compared to WT, indicative of an increased d(xz)/d(yz) spacing relati ve to WT. In contrast, the perturbation due to the 2-acetyl group and axial His nearly cancel, leading to a highly compressed methyl contact shift spr ead and weaker deviations from Curie behavior than WT. It is shown, moreove r, that the larger d(xz)/d(yz) splitting with 4-acetylhemin, and the smalle r splitting with 2-acetylhemin, relative to WT, result in the expected incr ease and decrease, respectively, for the axial His contact shift relative t o WT. Comparison of the methyl shifts for 16 peripherally modified hemins a s model compounds and incorporated into metMbCN shows that the rhombic infl uences are additive in each of the complexes. Thus, the present results sho w that chemical functionality of the heme periphery contributes to raising the orbital degeneracy of the heme iron and that such influences can accoun t for orbital ground states that are not necessarily aligned with the axial His orientation. The range of variant 2- and/or 4-substitutions have led t o equilibrium heme orientations that are largely the same as found in WT Mb , except for a 4-ethyl group, which favors the reversed heme orientation by 2:1.