Magnetic susceptibility tensor and heme contact shifts determinations in the Rhodobacter capsulatus ferricytochrome c ': NMR and magnetic susceptibility studies

The H-1 and N-15 resonances of the carbon monoxide complex of ferrocytochro me c' of Rhodobacter capsulatus, a ferrous diamagnetic heme protein, have b een extensively assigned by TOCSY-HSQC, NOESY-HSQC, and HSQC-NOESY-HSQC 3D heteronuclear experiments performed on a 7 mM sample labeled with N-15. Bas ed on short-range and medium-range NOEs and H-N exchange rates, the seconda ry structure consists of four helices: helix 1 (3-29), helix 2 (33-48), hel ix 3 (78-101), and helix 4 (103-125). The N-15, H-1(N), and H-1(alpha) chem ical shifts of the CO complex form are compared to those of the previously assigned oxidized (Or ferric) state. From the chemical shift differences be tween these redox states, the orientation and the anisotropy of the paramag netic susceptibility tensor have been determined using the crystallographic coordinates of the ferric state. The X-tensor is axial, and the orientatio n of the z-axis is approximately perpendicular to the heme plane. The param agnetic chemical shifts of the protons of the heme ligand have been determi ned and decomposed into the Fermi shift and dipolar shift contributions. Ma gnetic susceptibility studies in frozen solutions have been performed. Fits of the susceptibility data using the model of Maltempo (Maltempo, M. M. J. Chern. Phys. 1974, 61, 2540-2547) are consistent with a rather low contrib ution 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) hav e 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.