¹H NMR Investigation of the Role of Intrinsic Heme versus Protein-Induced Rhombic Perturbations on the Electronic Structure of Low-Spin Ferrihemoproteins:  Effect of Heme Substituents on Heme Orientation in Myoglobin

Abstract

Solution 1H NMR spectroscopy has been used to characterize the cyanomet myoglobin complexes of a variety of chemically modified hemins in order to elucidate the importance of hemin peripheral electronic, relative to axial His imidazole-induced, rhombic perturbations in raising the orbital degeneracy of the π-bonding d xz ,d yz orbitals. Variation of the hemin 2- and/or 4-position substituents among hydrogen, ethyl, vinyl, acetyl, and formyl groups leads to conserved molecular structure of the heme pocket and orientation of the major magnetic axis for the heme iron, but systematically perturbed heme methyl contact shift patterns. Two strongly rhombically perturbed hemins with single acetyl groups on either pyrrole I or II exhibit heme methyl contact shift patterns and characteristic deviations from Curie law that are very similar to that induced in pseudosymmetric hemins upon incorporation into metMbCN in the alternate orientations about the α,γ-meso axis. The perturbation due to the 4-acetyl group and the axial His bond leads to increased contact shift spread and stronger deviations from Curie behavior compared to WT, indicative of an increased d xz /d yz spacing relative 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 spread and weaker deviations from Curie behavior than WT. It is shown, moreover, that the larger d xz /d yz splitting with 4-acetylhemin, and the smaller splitting with 2-acetylhemin, relative to WT, result in the expected increase and decrease, respectively, for the axial His contact shift relative to WT. Comparison of the methyl shifts for 16 peripherally modified hemins as model compounds and incorporated into metMbCN shows that the rhombic influences are additive in each of the complexes. Thus, the present results show that chemical functionality of the heme periphery contributes to raising the orbital degeneracy of the heme iron and that such influences can account 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 to 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.

Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

“…2B) orientation of the etioheme likely results from the ethyl group selecting the position occupied by the 2- [53,54] g Taken from Refs. [49,51] and 4-vinyls of protoheme [57], and the demonstrated [19] lack of space for accommodating groups larger than methyls at position e in Fig. 2.…”

Solution NMR study of environmental effects on substrate seating in human heme oxygenase: Influence of polypeptide truncation, substrate modification and axial ligand

“…The complete heme assignment of the latter unique complex [13], and the major isomer of the former complex [12,31,43], have been presented. The significant differences in the heme methyl hyperfine [27] (largely contact) shifts for PH and DMDH (for all but the propionate C b Hs) arise from variable in-plane electronic influences of 2,4-vinyl versus 2,4-methyl substituents [27,45]. Similar differences in the heme hyperfine shifts are observed in bis-cyano complexes outside of a protein matrix [46,47], and hence the observed differences in the heme hyperfine shifts do not reflect any significant differences between the interaction of the two substrates with the folded protein.…”

Functional aspects of the heme bound hemophore HasA by structural analysis of various crystal forms

“…29 It was found that one of the orientations was accompanied by a 0.9Å displacement of the heme towards the solvent. The heme insertion in myoglobin has been studied by Circular Dichroism 30 and NMR 31 methods and the insertion mode in hemoglobin by NMR studies. 32 The results of all these studies indicate that there is a "correct" and a "reversed" heme orientation in the heme crevice and, in the case of the hemoglobin, an equilibrium ratio of 20:1 was determined.…”