Unusual Contact Shifts and Magnetic Tensor Orientation in Rhodobacter capsulatus Ferrocytochrome c‘:  NMR, Magnetic Susceptibility, and EPR Studies

Abstract

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 1 H and 15 N 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 15 N. Based on short-range and medium-range NOEs and H N 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 15 N, H N , and H R 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°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.

De novo determination of protein structure by NMR using orientational and long-range order restraints

“…The well-ordered backbone region of the NMR structure (5-55,75-125) superposes to 1.7 A Ê on the equivalent regions of the crystal structure for the closest structure, and 2.05(AE0.26) A Ê for the ensemble (Figure 5), while the most signi®cant differences exist in regions where the disorder of the NMR ensemble is very high in any case. Note that the tensor component values and orientations determined in this calculation are also similar to those recently estimated (Boisbouvier et al, 1999;Tsan et al, 1999;Tsan 1999) with respect to the crystallographic coordinates.…”

“…To determine the capacity of long-range paramagnetic and orientational information to unambiguously de®ne protein structure we have used only experimental data from the diamagnetic form and one of the two available paramagnetic states (S 2). Recent studies have shown that this molecule is monomeric in solution in all three forms (De Âme Âne  et al, 2000;Tsan et al, 1999), and in this study we assume that the overall fold of the two forms of the protein is independent of the spin-state of the electron.…”

“…Cytochrome c H : a model for spin-labelled proteins As an experimental model system we have chosen the cytochrome c H (Cytc H ) from Rhodobacter capsulatus, for which experimental data from the above phenomena have been measured in our laboratory (Boisbouvier et al, 1999;Tsan et al, 1999;Tsan 1998) in addition to diamagnetic 15 N relaxation data (Tsan, 1998). Cytc H is a ®ve-ligand heam protein possessing two paramagnetic states for the reduced (S 2) and oxidised (S 5/2) Fe, and which is diamagnetic when bound to CO as a sixth ligand.…”

“…Details of sample preparation and spectroscopic measurements have been published for the heteronuclear auto-relaxation, assignment of 13 C chemical shifts (Caffrey et al 1995), assignment of diamagnetic and paramagnetic H N and H a chemical shifts of the S 2 form of the molecule (Tsan et al, 1999), measurement of J HN residual dipolar coupling constants for the S 2 and diamagnetic forms at 600 MHz 1 H frequency and measurement of the Curie-dipolar cross correlation effect in the S 2 form (Boisbouvier et al 1999).…”

“…Similar methods, based on the measured coupling constants (Clore et al 1998b), allied to magnetic susceptibility measurements were used to estimate approximate initial values for Áw ax and Áw rh (Tsan et al, 1999). " w was estimated from the theoretical calculation of the interaction strength from physically known parameters, which gave similar values to estimates based on the range of measured values.…”