Unusual contact shifts and magnetic tensor orientation in Rhodobacter capsulatus ferrocytochrome c′:: NMR, magnetic susceptibility, and EPR studies

In contrast to high-spin ferrous paramagnetic heme proteins, the chemical shifts of the heme protons are very unusual in the ferrocytochromes c'. Magnetic susceptibility studies of Rhodobacter capsulatus ferrocytochrome c' in frozen solutions have been performed and indicate an S = 2 spin state and a large negative axial (D) zero-field splitting parameter (-18.3 cm(-1)) as well as a significant rhombic (E) value (-4.9 cm(-1)). The H-1 and N-15 resonances have been extensively assigned by TOCSY-HSQC, NOESY-HSQC, and HSQC-NOESY-HSQC 3-D heteronuclear experiments performed on a 8 mM sample labeled with N-15. Based on short-range and medium-range NOEs and HN exchange rates, the secondary structure consists of four helices: helix-1 (3-30), helix-2 (34-49), helix-3 (78-97), and helix-4 (103-117). The N-15, HN, and Ha chemical shifts of the reduced (or ferro) state are compared to those previously assigned for the diamagnetic carbon monoxide complex form. 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. Values of -23 and -3 cm(-1) have been inferred for D and E, and the z-axis of the tensor is tilted approximately 30 degrees from the normal to the heme. The paramagnetic chemical shifts of the heme protons have been determined and split up into Fermi shift and the dipolar shift contributions. The pattern of the contact shifts is very unusual, exhibiting a 2-fold symmetry, and is discussed in terms of molecular orbital interactions between the porphyrin macrocycle and the imidazole ring.