Browsing by Author "Moura, Isabel"
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- Characterization of recombinant desulfovibrio gigas ferredoxinPublication . Rodrigues, Pedro; Graça, Fernando; Macedo, Anjos L.; Moura, Isabel; Moura, José J. G.Dg ferredoxin gene was cloned using the polymerase chain reaction (PCR), inserted into vector pT7-7, and overexpressed in Escherichia coli (E. coli) grown in aerobic media. The recombinant protein is a dimer and contains a [3Fe–4S] cluster per monomer. EPR and 1H NMR data of recombinant and wild-type protein are compared.
- 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.
- Spectroscopic characterization of a novel 2×[4Fe–4S] ferredoxin isolated from Desulfovibrio desulfuricans ATCC 27774Publication . Rodrigues, Pedro; Moura, Isabel; Macedo, Anjos L.; Moura, José J. G.A novel iron /sulfur containing protein, a ferredoxin (Fd), was purified to homogeneity from the extract of Desulfovibrio desulfuricans American type culture collection (ATCC) 27774. The purified protein is a 13.4 kDa homodimer with a polypeptide chain of 60 amino acids residues, containing eight cysteines that coordinate two [4Fe /4S] clusters. The protein is shown to be air sensitive and cluster conversions take place. We structurally characterize a redox state that contains two [4Fe /4S] cores. 1D and 2D 1H NMR studies are reported on form containing the clusters in the oxidized state. Based on the nuclear Overhauser effect (NOE), relaxation measurements and comparison of the present data with the available spectra of the analogous 8Fe Fds, the cluster ligands were specifically assigned to the eight-cysteinyl residues.
- 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.