Browsing by Author "Goodfellow, B. J."
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- Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe-4S] clusterPublication . Rodrigues, Pedro; Macedo, Anjos L.; Goodfellow, B. J.; Moura, Isabel; Moura, José J. G.Abstract Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe3S4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdIIint) with four 1H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdIIox and FdIIint were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdIIox) was used here for the assignment of the same three ligands in the intermediate state (DgFdIIint). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm 1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdIIint. This study, together with the spin delocalization mechanism proposed here for DgFdIIint, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdIIox and DgFdIIint.
- The 3Fe containing ferredoxin from desulfovibrio gigas: an NMR characterization of the oxidised and intermediate statesPublication . Macedo, Anjos L.; Rodrigues, Pedro; Goodfellow, B. J.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.
- The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from desulfovibrio gigasPublication . Goodfellow, B. J.; Macedo, Anjos L.; Rodrigues, Pedro; Moura, Isabel; Wray, Victor; Moura, José J. G.The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe-4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80 Å), with the majority of f/c angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdIIox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdIIint), for which no X-ray structure is available.