DETERMINATION OF THE STRUCTURE OF OXIDIZED DESULFOVIBRIO-AFRICANUS FERREDOXIN-I BY H-1-NMR SPECTROSCOPY AND COMPARISON OF ITS SOLUTION STRUCTURE WITH ITS CRYSTAL-STRUCTURE

The solution structure of the 64 amino acid Fe4S4 ferredoxin I from De sulfovibrio africanus has been determined using two-dimensional H-1 NM R spectroscopy. Sequence-specific assignments were obtained for 59 ami no acid residues and the structure determined with the program DIANA o n the basis of 549 nuclear Overhauser enhancement (NOE) upper distance limits, and four dihedral angle and 52 distance constraints for the F e4S4 cluster. The NMR structure was refined using the simulated anneal ing and energy minimisation protocols of the program X-PLOR to yield a final family of 19 structures selected on the basis of good covalent geometry and minimal restraint violations. The r.m.s.d. values to the average structure for this family are 0.49(+/-0.07) Angstrom and 0.94( +/-0.09) Angstrom for the backbone and heavy-atoms of residues 3 to 62 , respectively. The NMR structure has been compared to the previously reported X-ray structures for the two molecules within the asymmetric unit of the crystal, which have a network of seven hydrogen bonds betw een them. This intermolecular interface, involving residues 38, 40 to 43 and 46, has the same conformation in the solution structures showin g that the crystal packing does not perturb the structure. There are t hree regions in which the NMR and X-ray structures differ: around the cluster, a turn involving residues 8 to 10, and a loop involving resid ues 29 to 32. In the family of solution structures the backbone of the loop region incorporating residues 29 to 32 is well-defined whilst in both of the X-ray molecules it is ill-defined. The small differences between the X-ray and NMR structures for the cluster environment and t he turn between residues 8 to 10 probably reflects a lack of NMR const raints. The observation of relatively rapid amide NH hydrogen exchange of NH groups close to the cluster, together with rapid flipping for P he25, which is also close to the cluster, indicates that the cluster e nvironment is more dynamic than the corresponding regions of related F e/S proteins. (C) 1998 Academic Press Limited.