NMR of Chromatium vinosum ferredoxin: evidence for structural inequivalence and impeded electron transfer between the two [4Fe-4S] clusters.

The 2[4Fe-4S] ferredoxin from Chromatium vinosum has been investigated by 1H and 13C nuclear magnetic resonance. 1H NMR sequence-specific assignments have been obtained for a large majority of the residues. They indicate that the protein folds along a pattern similar to that previously evidenced for shorter 2[4Fe-4S] ferredoxins. However, C. vinosum ferredoxin differs from other ferredoxins by the occurrence of a turn in an eight amino acid region separating two successive cysteines, Cys-40 and Cys-49, liganding one cluster. Also, the unique C-terminal end of C. vinosum ferredoxin contains a 10 amino acid alpha-helix which interacts with one side of the above turn. The only cysteine of the sequence not involved in the ligation of the [4Fe-4S] clusters is Cys-57. Specific NMR experiments helped characterizing the signals arising from the ligands of these clusters: most of them display properties reminiscent of those of homologous ferredoxins, except for the signals associated with Cys-40. Despite the general similarity between C. vinosum ferredoxin and other 2[4Fe-4S] ferredoxins, the electron paramagnetic resonance and NMR spectra of the former reduced protein are significantly different from those previously observed for S = 1/2 [4Fe-4S]+ clusters. In addition, the intramolecular electron transfer rate in C. vinosum is far slower than in other similar cases. This is the first report of impeded electron exchange between two [4Fe-4S] clusters expected to be less than 12 A apart.