USE OF PARAMAGNETIC NMR PROBES FOR STRUCTURAL-ANALYSIS IN CYTOCHROME C(3) FROM DESULFOVIBRIO-VULGARIS

The dipolar field generated by each of the four haems in the tetrahaem ferricytochrome c(3) from Desulfovibrio vulgaris (Hildenborough) (c(3 )DvH) is determined by means of a novel procedure. In this method the C-13 chemical shifts of the nuclei directly bound to the haems are use d to determine the in-plane orientations of the rhombic perturbation i n 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. (1 995) Biochim. Biophys. Acta 1246, 24-28]. These orientations, together with the components of the magnetic susceptibility tensors obtained f rom the EPR g values and the crystal structure of c(3)DvH, can be used to calculate the dipolar shifts induced by each haem throughout the p rotein. Thus the observed C-13 paramagnetic shifts of the c(3)DvH haem substituents were fitted considering both the pseudocontact and conta ct shifts of each haem simultaneously. The dipolar shifts calculated b y 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 d ipolar extrinsic shifts on the behaviour of the chemical shifts of the haem methyl groups in the intermediate stages of oxidation at differe nt pH values was also analysed. The several tests applied to the calcu lated dipolar shifts have shown that the method is extremely useful fo r predicting chemical shifts as an aid to complete proton assignment, and to add further constraints in the refinement of solution structure s of paramagnetic proteins and hence to probe subtle structural rearra ngements around the haem pocket.