Use of Paramagnetic NMR Probes for Structural Analysis in Cytochrome c₃ from Desulfovibrio Vulgaris

Abstract

The dipolar field generated by each of the four haems in the tetrahaem ferricytochrome c, from Desulfovibrio vulgaris (Hildenborough) (c,DvH) is determined by means of a novel procedure. In this method the I3C chemical shifts of the nuclei directly bound to the haems are used to determine the inplane orientations of the rhombic perturbation in each of the four haems with respect to a model of molecular orbitals of e, symmetry which are subject to a rhombic perturbation [Turner, D. L., Salgueiro, C. A., Schenkels, P., LeGall, J. & Xavier, A. ) Biochim. Biophys. Acta 1246. These orientations, together with the components of the magnetic susceptibility tensors obtained from the EPR g values and the crystal structure of c,DvH, can be used to calculate the dipolar shifts induced by each haem throughout the protein. Thus the observed 13C paramagnetic shifts of the c,DvH 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.

Proton-assisted Two-electron Transfer in Natural Variants of Tetraheme Cytochromes from Desulfomicrobium Sp.

“…In this context, heme I has the strongest of all the significant interactions with the protonic center in both proteins. This makes propionate 13 of heme I a likely candidate for the redox-Bohr group, as proposed previously for other cytochromes c 3 (4,13,17,40,59). In Dsmb the hydroxyl group of Tyr-88 is pointing to propionate 13 of heme I and is at a distance of 2.56 Å.…”

“…Conclusions-The thermodynamic parameters obtained for these two Dsm cytochromes suggest that they are capable of performing e Ϫ /H ϩ energy transduction using a different mechanism from that described previously (4,17,40,43,65) for D. vulgaris and D. gigas type I cytochromes c 3 . The variations in the microscopic thermodynamic parameters of these two phylogenetically related strains are caused by a small difference in their amino acid sequences and may have resulted from evolutionary advantages associated with the different habitats from which these two organisms were isolated.…”

Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR

“…In the case of PpcA, only four heme methyl signals could be assigned with confidence to their final position in the paramagnetic protein: 2 1 H-13 C-HSQC (Fig. 1) NMR experiments, following the strategy first described for Desulfovibrio vulgaris (Hildenborough) cytochrome c 3 [21,28]. All assignments are listed in Table 1.…”

Effect of Hydrogen-Bond Networks in Controlling Reduction Potentials in Desulfovibrio vulgaris (Hildenborough) Cytochrome c3 Probed by Site-Specific Mutagenesis

“…Therefore, the paramagnetic shift observed for a heme substituent is directly proportional to its fractional oxidation only in the absence of an extrinsic contribution. As shown previously for DVHc 3 (38), the heme methyl groups 18 1 CH 3 I , 18 1 CH 3 II , 12 1 CH 3 III , and 18 1 CH 3 IV (Roman numbers designate the heme order in the polypeptide chain) have large paramagnetic shifts and negligible extrinsic dipolar contributions; hence they are suited to monitor the thermodynamic properties of this cytochrome.…”

“…Thus, the paramagnetic shifts of heme substituents can be used to obtain the relative microscopic reduction potentials of the heme groups in each stage of oxidation (10,11,36). However, the paramagnetic shift observed for a particular heme substituent depends not only on the Fermi contact and the dipolar contributions of its own heme (intrinsic shift) but also on the dipolar shift due to the other hemes (extrinsic shift) (36)(37)(38). Therefore, the paramagnetic shift observed for a heme substituent is directly proportional to its fractional oxidation only in the absence of an extrinsic contribution.…”