Porphyrin and Ligand Protons as Internal Labels for Determination of Ligand Orientation in ESEEMS of Low-Spin d⁵ Complexes in Glassy Media:  ESEEM Studies of the Orientation of the g Tensor with Respect to the Planes of Axial Ligands and Porphyrin Nitrogens of Low-Spin Ferriheme Systems

Abstract

The proton sum frequency peak(s), I(ν + ), in the ESEEM spectra of low-spin ferriheme complexes provide single-crystal-like information concerning the orientation of the g tensor in samples in frozen glassy media. In this work we have investigated two model heme complexes, [OEPFe(imidazole) 2 ] + and [OEPFe-(4-(dimethylamino)pyridine) 2 ] + (OEP ) octaethylporphyrinate). Both experimental intensities and frequency shifts from twice the 1 H Larmor frequency of the observed signals were measured at various points across the EPR spectrum and compared to the expected spectra, simulated using the known crystal structure data, isotropic hyperfine coupling constants, and g strain. In each case the z magnetic axis direction was defined as perpendicular to the mean plane of the porphyrinate, and it was found that g zz is the largest g value in both cases. The in-plane magnetic axis directions could also be determined from the ESEEM data, and it was found that the orientations of g xx and g yy differ, depending on the orientation of the (parallel) axial ligands with respect to the porphyrinate nitrogens: For the bis(imidazole) complex, for which the axial ligands nearly eclipse opposite porphyrinate nitrogens (φ ) 7°) in the crystalline state, g xx and g yy are aligned at (45°to the normal to the plane of the axial ligands, while for the bis(4-(dimethylamino)pyridine) complex, for which the axial ligands lie in parallel planes nearly bisecting the porphyrinate nitrogens (φ ) 41°) in the crystalline state, g yy is aligned along the plane of the axial ligands. The significance of these results with respect to the concept of counterrotation of the g tensor with rotation of axial ligands and the interpretation of the in-plane magnetic anisotropy of heme proteins measured by NMR techniques is discussed.

1H NMR Investigation of the Distal Hydrogen Bonding Network and Ligand Tilt in the Cyanomet Complex of Oxygen-avidAscaris suum Hemoglobin

“…The re-evaluation of the x-ray diffraction data to reconcile the alternate heme orientation in the crystal and solution has shown that the heme in the crystal is, in fact, rotated by 180°about the ␣,␥-meso axis from that originally reported (8) 4 and the same as found by 1 H NMR in solution. Theoretical considerations (61,64) confirmed in model compounds (65,66) dictate that if the orbital ground state is determined by the axial His(F8) bonding, the rhombic axes, , and the angle between the heme N-Fe-N and imidazole plane, (Fig. 1C), obey the counter-rotation rule where ϭ Ϫ.…”

Structure and spin density of ferric low-spin heme complexes determined with high-resolution ESEEM experiments at 35 GHz

“…The distribution of the unpaired electron within the 3d orbitals of Fe(III) can be obtained from the g values measured in a continuous-wave (CW) EPR spectrum [4][5][6], but determining the orientation of these orbitals in disordered samples requires more effort. Dipolar interactions between electron and proton nuclear spins depend on the position of the proton relative to the iron; therefore, one can use the proton hyperfine couplings obtained by ENDOR [7][8][9][10][11][12] or ESEEM [8,[13][14][15][16][17] to determine the relative orientations between the g matrix, heme molecular frame, and axial ligands. The main difficulty with this approach is that typically there are many protons in the active site and proton signals often cannot be unambiguously assigned.…”

CW-EPR and ENDOR Study of Cytochrome c6 from Anabaena PCC 7119

“…Alternatively, EPR-related techniques such as ENDOR or ESEEM may reveal weak hyperfine interactions with adjacent ( 1 H, 14 N) nuclei. Nevertheless, relatively few works using these methods to study cytochromes or related heme systems have been reported (11)(12)(13)(14)(15)(16)(17).…”