Magnetic susceptibility tensor and heme contact shifts determinations in the Rhodobacter capsulatus ferricytochrome c ': NMR and magnetic susceptibility studies
P. Tsan et al., Magnetic susceptibility tensor and heme contact shifts determinations in the Rhodobacter capsulatus ferricytochrome c ': NMR and magnetic susceptibility studies, J AM CHEM S, 123(10), 2001, pp. 2231-2242
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.