¹H and ¹³C NMR Investigation of the Influence of Nonligated Residue Contacts on the Heme Electronic Structure in Cyanometmyoglobin Complexes Reconstituted with Centro- and Pseudocentrosymmetric Hemins

Abstract

The 1H and 13C chemical shifts for the heme methyls of low-spin, ferric sperm whale cyanometmyoglobin reconstituted with a variety of centrosymmetric 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 electronic asymmetry. The very similar 1H 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 13C contact shifts in all complexes. It is demonstrated that, even when the magnetic axes and anisotropies are known, the intrinsic uncertainties in the orientational parameters lead to a sufficiently large uncertainty in dipolar shift that the methyl proton contact shifts are inherently significantly less reliable indicators of the unpaired electron spin distribution than the methyl 13C contact shifts. The pattern of the noninversion symmetry in 13C contact shifts in the centro- or pseudocentrosymmetric 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 in-plane asymmetry of the heme pi spin distribution and cannot be ignored in a quantitative interpretation of the heme methyl 13C contact shifts in terms of the axial His orientation in b-type hemoproteins.

Recent developments in the 13 C NMR spectroscopic analysis of paramagnetic hemes and heme proteins

“…These authors demonstrated that even if the magnetic axes and anisotropies are known, the intrinsic uncertainties in the orientational parameters lead to a relatively large uncertainty in the determination of the dipolar contribution to the methyl proton isotropic shifts. By comparison, the relatively small contribution of the methyl carbon dipolar shift to the isotropic shift makes the methyl carbon contact shifts more reliable indicators of the unpaired electron distribution on the heme macrocycle [114,131]. Thus, by utilizing the 13 CH 3 pattern of non-inversion symmetry in centro-and pseudocentro-symmetric hemes reconstituted into myoglobin, it was shown that π-1480 Fig.…”

“…Other factors such as the nature of heme substituents (i.e., vinyl, methyl, and propionate), van der Waals interactions between the heme and side chains lining the heme pocket, and heme conformational distortions from planarity can provide secondary modifications of the in-plane asymmetry. A recent study pointed out that 13 C NMR spectroscopy is well suited to elucidate the nature and extent of these secondary regulatory mechanisms [131]. These authors demonstrated that even if the magnetic axes and anisotropies are known, the intrinsic uncertainties in the orientational parameters lead to a relatively large uncertainty in the determination of the dipolar contribution to the methyl proton isotropic shifts.…”

“…This figure was reproduced from reference [69] [69] π interactions between the heme and aromatic residues in close contact perturb the in-plane asymmetry of unpaired electron distribution. Hence, it is likely that in the quantitative interpretation of heme methyl 13 C contact shifts in the context of axial ligand plane orientation in b hemes, the regulatory effect of π-π interactions will have to be taken into account [131]. Furthermore, it is important to investigate whether these secondary modulations of inplane asymmetry have important functional consequences.…”

1H-NMR Study of the Effect of Temperature through Reversible Unfolding on the Heme Pocket Molecular Structure and Magnetic Properties of Aplysia limacina Cyano-Metmyoglobin

“…Lastly, it is observed that each of the meso-Hs exhibit 2-4 ppm low-field d con , as previously observed for sperm whale metMbCN (29), which is contrary to the difference in the a-/ d-meso versus b-/d-meso-H d con as predicted ( 18) by the prevailing model for hyperfine shifts in low-spin ferric hemins. The failure to account for meso-H d con patterns in other metMbCN complexes has been noted previously (52). Detailed calculations of the unpaired spin distribution in low-spin ferric hemes have shown that correlation leads to both positive and negative spin density on the heme periphery that cannot be interpreted simply based on MO theory of p-spin density in a single molecular orbital (53) .…”

Linear Correlation between 1H and 13C Chemical Shifts of Ferriheme Proteins and Model Ferrihemes

“…$${\delta }^{\text{H}}={{\delta }^{\text{H}}}_{\text{dia}}+{{\delta }^{\text{H}}}_{\text{con}}+{{\delta }^{\text{H}}}_{\text{pcM}}$$ $${\delta }^{\text{C}}={{\delta }^{\text{C}}}_{\text{dia}}+{{\delta }^{\text{C}}}_{\text{con}\pi }+{{\delta }^{\text{C}}}_{\text{pcM}}+{{\delta }^{\text{C}}}_{\text{pcL}\pi }$$ where δ H dia and δ C dia are the shifts of the same nuclei in a diamagnetic complex (usually Zn(II)-substituted complexes are used to evaluate these27,39–41), δ H con is the proton contact shift contribution, and δ C conπ is the corresponding carbon contact shift for delocalization of the unpaired electron from the metal to the ligand through π bonds. The pseudocontact (formerly called dipolar39,40) shifts, δ H pcM and δ C pcM , are the result of the through-space dipolar coupling between the nucleus of interest (H or C) and the electron, which is centered on the metal, because of the magnetic anisotropy of d-orbitals, which produce an angular and distance dependence on the size of the pseudocontact shift and can be calculated if one knows the distance and angular relationship between each nucleus and the metal center from the X-ray crystal structure 24,39–41.…”