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 s hifts of the heme protons are very unusual in the ferrocytochromes c'. Magn etic 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 re sonances have been extensively assigned by TOCSY-HSQC, NOESY-HSQC, and HSQC -NOESY-HSQC 3-D heteronuclear experiments performed on a 8 mM sample labele d with N-15. Based on short-range and medium-range NOEs and HN exchange rat es, 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 ch emical shifts of the reduced (or ferro) state are compared to those previou sly assigned for the diamagnetic carbon monoxide complex form. From the che mical 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 a nd -3 cm(-1) have been inferred for D and E, and the z-axis of the tensor i s tilted approximately 30 degrees from the normal to the heme. The paramagn etic chemical shifts of the heme protons have been determined and split up into Fermi shift and the dipolar shift contributions. The pattern of the co ntact shifts is very unusual, exhibiting a 2-fold symmetry, and is discusse d in terms of molecular orbital interactions between the porphyrin macrocyc le and the imidazole ring.