Use of paramagnetic NMR probes for structural analysis in cytochrome c3 from Desulfovibrio vulgaris.

The dipolar field generated by each of the four haems in the tetrahaem ferricytochrome c3 from Desulfovibrio vulgaris (Hildenborough) (c3DvH) is determined by means of a novel procedure. In this method the 13C chemical shifts of the nuclei directly bound to the haems are used to determine the in-plane orientations of the rhombic perturbation in each of the four haems with respect to a model of molecular orbitals of e(g) symmetry which are subject to a rhombic perturbation [Turner, D. L., Salgueiro, C. A., Schenkels, P., LeGall, J. & Xavier, A. V. (1995) Biochim. Biophys. Acta 1246, 24-281. These orientations, together with the components of the magnetic susceptibility tensors obtained from the EPR g values and the crystal structure of c3DvH, can be used to calculate the dipolar shifts induced by each haem throughout the protein. Thus the observed 13C paramagnetic shifts of the c3DvH haem substituents were fitted considering both the pseudocontact and contact shifts of each haem simultaneously. The dipolar shifts calculated by this method were tested against the observed dipolar shifts for some amino acid residues strategically placed in the protein and also for the haem propionate groups. The effect of considering the calculated dipolar extrinsic shifts on the behaviour of the chemical shifts of the haem methyl groups in the intermediate stages of oxidation at different pH values was also analysed. The several tests applied to the calculated dipolar shifts have shown that the method is extremely useful for predicting chemical shifts as an aid to complete proton assignment, and to add further constraints in the refinement of solution structures of paramagnetic proteins and hence to probe subtle structural rearrangements around the haem pocket.