Magnetic Susceptibility Tensor and Heme Contact Shifts Determinations in the Rhodobacter capsulatus Ferricytochrome c‘: NMR and Magnetic Susceptibility Studies

Abstract

The 1H and 15N resonances of the carbon monoxide complex of ferrocytochrome c' of Rhodobacter capsulatus, a ferrous diamagnetic heme protein, have been extensively assigned by TOCSY-HSQC, NOESY-HSQC, and HSQC-NOESY-HSQC 3D heteronuclear experiments performed on a 7 mM sample labeled with 15N. Based on short-range and medium-range NOEs and H(N) exchange rates, the secondary structure consists of four helices: helix 1 (3-29), helix 2 (33-48), helix 3 (78-101), and helix 4 (103-125). The 15N, 1HN, and 1H(alpha) chemical shifts of the CO complex form are compared to those of the previously assigned oxidized (or ferric) state. From the chemical shift differences between these redox states, the orientation and the anisotropy of the paramagnetic susceptibility tensor have been determined using the crystallographic coordinates of the ferric state. The chi-tensor is axial, and the orientation of the z-axis is approximately perpendicular to the heme plane. The paramagnetic chemical shifts of the protons of the heme ligand have been determined and decomposed into the Fermi shift and dipolar shift contributions. Magnetic susceptibility studies in frozen solutions have been performed. Fits of the susceptibility data using the model of Maltempo (Maltempo, M. M. J. Chem. Phys. 1974, 61, 2540-2547) are consistent with a rather low contribution of the S = 3/2 spin state over the range of temperatures and confirm the value of the axial anisotropy. Values in the range 10.4-12.5 cm(-1) have been inferred for the axial zero-field splitting parameter (D). Analysis of the contact shift and the susceptibility data suggests that cytochrome c' of Rb. capsulatus exhibits a predominant high-spin character of the iron in the oxidized state at room temperature.

Cytochromesc‘: Biological Models for theS=3/2,5/2Spin-State Admixture?

“…capsulatus Bath), and 1 H NMR studies of the ferricytochromes c ‘ conclude that, with the possible exception of Ch . vinosum , the ground state of these proteins is essentially high-spin. − …”

The Magnetic Properties of Myoglobin as Studied by NMR Spectroscopy

“…15 N chemical shift differences between Mb and Mb–CO are also consistent with Equation (1), but with a much larger RMSD (0.45 ppm). As found in the analogous reduced cytochrome c′26 and in other redox proteins26, 26–29 the 15 N shifts of the diamagnetic protein do not correspond exactly to the diamagnetic contribution of the paramagnetic species. The average magnetic anisotropy values are similar to those of low‐spin iron( III ) heme proteins and larger than previously guessed,15 although smaller than those of reduced cytochrome c′ 16.…”

29 Bioinorganic chemistry

“…The results together with magnetic susceptibility data indicate a predominant high-spin character for the Fe centre in the oxidised form of this enzyme at room temperature. 49 The effect of axial ligand ligation on the electronic structure of cytochrome c 50, 51 and small molecule analogues 52 has been probed by NMR spectroscopy.…”