The 3Fe containing ferredoxin from desulfovibrio gigas: an NMR characterization of the oxidised and intermediate states

Ferredoxin II (FdII), isolated from Desulfo6ibrio gigas, is a small electron transfer protein that contains one [3Fe–4S] cluster per monomer (6 kDa). The characterization of the oxidized and intermediate forms of FdII was carried out by NMR spectroscopy, in conjunction with other spectroscopic techniques, such as EPR, UV–vis and Mo¨ssbauer spectroscopy, in order to fully understand the redox and electronic properties of the protein. The native oxidized state of FdII has been studied from the point of view of the spin coupling between the iron atoms, via the 1H-NMR temperature dependence of the b-CH2 proton resonances of the cysteinyl cluster ligands (Cys8, Cys14 and Cys50). The assignment of the 2D-NOESY spectrum has also been carried out: distances have been obtained for the diamagnetic region and 1D-NOE experiments were performed in order to detect NOEs for protons in the vicinity of the cluster, allowing the determination of the protein structure in solution. An intermediate state, FdIIint, detected by NMR spectroscopy, was attributed to the opening of the S–S bridge between Cys18 and Cys42, in the potential range of cluster reduction. This state has been characterized by several spectroscopic techniques, showing that the protein can transfer three electrons in one redox step, that involves the [3Fe–4S] cluster and the disulfide bridge. The temperature behavior of the hyperfine shifts, when compared with FdIIox, lead to the conclusion that the spin coupling of the cluster changes upon reduction of the S–S bond. 2D-NOESY spectra of the diamagnetic region were also collected for FdIIint, and compared with the native state, in order to clarify the structural changes occurring in the protein on cleavage of the S–S bridge