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C Luchinat

Publications and source records attributed to C Luchinat.

At least 73 records · Page 4Linked to original sources
PubMed1995

Evidence of histidine coordination to the catalytic ferrous ion in the ring-cleaving 2,2',3-trihydroxybiphenyl dioxygenase from the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1.

The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytically active reduced form of the monomeric enzyme (MW = 32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D2O solution. Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines. These signals show strong sensitivity to the presence of the substrate. The oxidized form of the enzyme shows no hyperfine shifted signals. It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

Benzofurans↗
PubMed1995

Solution structure of the oxidized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two [4Fe-4S] clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non-selective T1 smaller than 25 ms. The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN-H alpha, H-bond constraints and dihedral angle constraints on the Cys chi 2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine beta CH2 protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.-M. (1994) J. Mol. Biol. 243, 683-695]. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The C alpha root-mean-square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region.

Amino Acid Sequence↗
PubMed1995

Three-dimensional solution structure of the oxidized high potential iron-sulfur protein from Chromatium vinosum through NMR. Comparative analysis with the solution structure of the reduced species.

The NMR solution structure of the oxidized HiPIP from Chromatium vinosum has been solved. Despite the fact that the protein is paramagnetic, 85% of the 1H and 80% of the 15N signals have been assigned. Through 1537 NOEs, out of which 1142 were found to be relevant for the structure determination, a family of structures has been obtained by distance geometry calculations. These structures have then been subjected to restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations in vacuum. Finally, the mean structure of the RMD family has been treated through RMD in water. The RMSD values for the backbone and heavy atoms within the RMD family are 0.57 +/- 0.14 and 1.08 +/- 0.16 A, respectively. These values together with other parameters indicate that the structure is of good quality and as good as the structure of the reduced protein. The RMDw structures of the reduced and oxidized proteins are different beyond the experimental indetermination. The set of constraints for the reduced and oxidized forms have been used to treat the available X-ray structure by RMD in water. The two structures generated in this way are quite similar to their respective solution structures, thus confirming that the experimental constraints are capable of yielding two different structures from the same starting structural model. This is the first time that independently determined solution structures of two redox states of a paramagnetic protein are available. Differences between them and the X-ray structure are discussed.

Amino Acid Sequence↗
PubMed1995

Magnetic resonance of Fe-S clusters: isolation and characterization of a 7Fe ferredoxin from Rhodopseudomonas palustris.

A novel iron-sulfur protein from the photosynthetic purple bacterium Rhodopseudomonas palustris was purified to homogeneity and identified as a ferredoxin on the basis of its physicochemical properties. Based on the uv/vis spectrum, iron quantitation, cyclic voltammetry, EPR, and 1H NMR data, the ferredoxin is found to contain two iron-sulfur clusters, one [3Fe-4S] and one [4Fe-4S], which places this protein in the class of 7Fe ferredoxins. The voltammetric peak potentials of the two clusters are -0.260 and -0.560 V at pH 8.0. The molecular mass around 19 kDa makes this protein the heaviest known in this class. This paper further demonstrates the diagnostic power of magnetic resonance spectroscopies in recognition of the two types of clusters in iron-sulfur proteins.

Electrochemistry↗
PubMed1995

Determination of the [Fe4S4]Cys4 cluster geometry of Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy.

1D and 2D 1H NMR studies of the Fe4S4 cluster containing ferredoxin I from Desulfovibrio africanus have been carried out with the aim of determining the geometry of the cluster linkages with the 4 Cys side chains that bind the cluster. This required the Cys beta CH resonances of the oxidised protein to be sequence-specifically and stereo-specifically assigned, and this was accomplished by a combination of TOCSY and NOE measurements, allied to model building based on X-ray structures of related ferredoxins. An analysis of the estimated hyperfine shifts of the Cys beta CH resonances with a Karplus-type equation relating the shifts to iron-sulfur-beta carbon-beta proton dihedral angles, taken together with the relative relaxation rates of the two beta CH2 resonances, estimated from their linewidths, then allowed the iron-sulfur-beta-carbon-alpha-carbon dihedral angles to be determined. A novel representation of the NMR data is presented which shows that the cluster dihedral angles are uniquely determined by the NMR data. The analysis reveals that the dihedral angles for D. africanus ferredoxin I are similar to the corresponding angles of other ferredoxins even though there are differences in their 1H NMR spectra. The sequence-specific and stereospecific assignments have been extended by analogy to the related Fe4S4-containing D. gigas ferredoxin I, and the stereospecific assignments to the Fe4S4-containing Thermococcus litoralis ferredoxin.

Chemical Phenomena↗
PubMed1995

The three-dimensional solution structure of the reduced high-potential iron-sulfur protein from Chromatium vinosum through NMR.

The 1H NMR assignment of the reduced HiPIP from Chromatium vinosum available in the literature [Gaillard, J., Albrand, J.-P., Moulis, J.-M., & Wemmer, D. E. (1992) Biochemistry 31, 5632-5639] has been extended up to 85% of the total protein protons. Ninety percent of the nitrogens have been assigned. Then the solution structure has been obtained using as many as 1147 meaningful NOE connectivities. The protein is sizably paramagnetic even though the ground state is a singlet. Nevertheless, the final RMSD values are 0.62 and 1.19 A for the backbone and the heavy atoms, respectively. These values compare well with those for diamagnetic proteins of the same size. The solution structure is discussed in the light of the available structural information from X-ray data.

Amino Acid Sequence↗
PubMed1994

Sequence-specific assignment of the 1H and 15N nuclear magnetic resonance spectra of the reduced recombinant high-potential iron-sulfur protein I from Ectothiorhodospira halophila.

A 1H and 15N NMR investigation through two-dimensional and three-dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are paramagnetic with a relatively low mu eff of about 0.8 mu B/iron ion, but the paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closet to the paramagnetic center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X-ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a paramagnetic protein to date. Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter-residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein. These data are meaningful for the detection of structural differences among mutants in future studies.

Amino Acid Sequence↗
PubMed1994

The three-dimensional structure in solution of the paramagnetic high-potential iron-sulfur protein I from Ectothiorhodospira halophila through nuclear magnetic resonance.

The three-dimensional structure in solution of reduced recombinant high-potential iron-sulfur protein iso-I from Ectothiorhodospira halophila was determined using 948 relevant interproton NOEs out of the 1246 observed NOEs. The determination was accomplished using the XEASY program for spectral analysis and the distance geometry (DG) program DIANA for generation of the structure as described by Wüthrich [Wüthrich, K. (1989) Acc. Chem. Res. 22, 36-44]. The FeS cluster was simulated using an amino acid residue constructed for the present work from a cysteinyl residue with an iron and a sulfur atom attached to the terminal thiol. The family of structures obtained from distance geometry were subjected to energy minimization and molecular dynamics simulations using previously defined force field parameters. The quality of these structures at each stage of the refinement process is discussed with respect to the dihedral angle order parameter and the root-mean-square deviation of the atomic coordinates. The latter values for the backbone atoms vary from 67 pm for the distance-geometry structures to 60 pm for the energy-minimized structures to 51 pm for the structures subjected to restrained molecular dynamics. Finally, the structure in best agreement with the NOE constraints has been further treated with extensive restrained molecular dynamics in water. The solution structure is well defined and is very similar to the available X-ray structure. We do not know of any previous determination of the structure of a paramagnetic protein in solution by NMR. The effect of paramagnetism on the quality of the structure determination is discussed.

Bacteria↗
PubMed1994

Influence of surface charges on redox properties in high potential iron-sulfur proteins.

The pH-dependence of the reduction potential determined through differential pulse voltammetry for the high potential iron sulfur proteins (HiPIP) from R. globiformis, C. vinosum, R. gelatinosus, E. vacuolata (I and II), E. halophila (I and II) is reported. A decrease in reduction potential with pH is invariably observed in the pH range where deprotonation of the imidazolium nitrogen of histidine residue(s) occurs. No pH dependence is observed for the only protein lacking histidines. It appears that surface charges like the His imidazolium groups are capable of influencing the reduction potential despite the known quencing of the electrostatic interactions due to solvent effects.

Bacterial Proteins↗
PubMed1994

X-ray, NMR and molecular dynamics studies on reduced bovine superoxide dismutase: implications for the mechanism.

Single crystals of the reduced form of Cu, Zn superoxide dismutase (space group P2(1)2(1)2(1), one dimer per asymmetric unit) have been obtained and their X-ray structure refined at 1.9 A resolution. The structure shows that the imidazolate bridge is maintained in the present crystalline form. It is confirmed that in solution the bridge is broken and the involved histidine is protonated on the side of copper. Based on the NOE constraints, and with the aid of molecular dynamics calculations, a structural model is proposed for the molecule in solution. Both structures are considered significant as far as the enzymatic mechanism is concerned.

Animals↗
PubMed1994

Polymetallic macromolecules are potential contrast agents of improved efficiency.

The commercial synthetic homopolypeptide polyaspartate (average MW = 30,000, approximately 220 monomers) in solution quantitatively binds up to 40 mol Gd3+ ions per mole of polyaspartate. The water proton relaxivity of the solutions is far higher than that of clinically used or commonly investigated gadolinium(III) complexes. It is shown that polymetallic macromolecular complexes combine the high relaxing efficiency of monometallic macromolecular complexes with the favorable metal/ligand mass ratio of small monometallic complexes.

Contrast Media↗
PubMed1994

Strategies of signal assignments in paramagnetic metalloproteins. An NMR investigation of the thiocyanate adduct of the cobalt (II)-substituted human carbonic anhydrase II.

The title protein with MW 30,000 containing high-spin cobalt (II) has been thoroughly investigated through 1H NMR spectroscopy with the aid of selectively deuterated amino acids and 15N enrichment. The aim is that of showing the potentiality of the approach when local information by NMR is needed and the X-ray structure is available. The potential use of the pseudocontact shift is discussed; 90, 200, and 600 MHz spectrometers are used to investigate a spherical region at various distances from the metal ion. More than 35 signals of protons around cobalt (II) have been assigned.

Carbonic Anhydrases↗
PubMed1993

Electron self-exchange in high-potential iron-sulfur proteins. Characterization of protein I from Ectothiorhodospira vacuolata.

During previous research on oxidized and reduced high-potential iron-sulfur proteins (HiPIP hereafter), qualitative different electron self-exchange rates were noticed. We have now investigated this phenomenon in detail for HiPIP I and II from Ectothiorhodospira vacuolata, which differ significantly in total charge and in which the sequence homology is the largest among all known HiPIPs. We have also characterized the electronic structure of HiPIP I through 1H NMR and EPR spectroscopies to parallel the existing characterization of HiPIP II and other HiPIPs. This investigation has allowed us to propose a model, according to which the productive collisions for electron transfer occur through a hydrophobic patch near the cluster. The effects of total charge and redox potential are considered. The possible formation of dimers through the hydrophobic patch at liquid helium temperature is discussed in light of the EPR spectra.

Amino Acid Sequence↗
PubMed1993

The electronic structure of [Fe4S4]3+ clusters in proteins. An investigation of the oxidized high-potential iron-sulfur protein II from Ectothiorhodospira vacuolata.

Within the framework of an investigation of the electronic structure of oxidized high-potential iron-sulfur proteins (HiPIP), we have studied the HiPIP II from Ectothiorhodospira vacuolata, which was known to have a peculiar temperature dependence of the 1H NMR isotropic hyperfine shifts. The signals of the cysteine ligand protons have been sequence specifically assigned through NOE, NOESY, and TOCSY experiments. Nine hyperfine-shifted signals are observed: seven in the downfield and two in the upfield region. They have been assigned to the eight beta-CH2 protons of the four coordinated cysteines and to one alpha-CH cysteine proton. The two most downfield-shifted signals belong to the beta-CH2 protons of Cys 63 (Chromatium vinosum numbering) and the two upfield protons to those of Cys 43. These two pairs of protons show a Curie-type temperature dependence of the hyperfine shifts. Among the remaining five downfield-shifted signals, three show a Curie-type temperature dependence and two have an anti-Curie temperature dependence. The former are assigned to the beta-CH2 and alpha-CH protons of Cys 77 and the latter to the beta-CH2 protons of Cys 46. The shift patterns are thus similar, in a sequence-specific sense, to those of the analogous proteins from C. vinosum and Rhodocyclus gelatinosus, whereas they differ from those of Rhodocyclus globiformis HiPIP and even more from those of Ectothiorhodospira halophila HiPIP II. Oxidized HiPIPs can be formally viewed as containing a cluster of four ferric ions plus one extra electron. We present here a model based on a chemical equilibrium, fast on the NMR time scale, between two species, both of which contain a pair of iron(III) ions and a mixed-valence pair but are differently oriented within the protein frame. The EPR data are also discussed in the light of the debate on the nature of the different species detected at low temperature. The interpretation of the whole set of data on HiPIPs in the light of the present model is compared with that based on previous models.

Bacteria↗
PubMed1993

1H-NMR investigation of oxidized and reduced high-potential iron-sulfur protein from Rhodopseudomonas globiformis.

1H one-dimensional and two-dimensional NMR spectra have been recorded for the oxidized and reduced forms of the high-potential iron-sulfur protein (HiPIP) from Rhodopseudomonas globiformis which has the highest known reduction potential. The spectrum of the oxidized protein is similar to that of Chromatium vinosum and Rhodocyclus gelatinosus HiPIP but different from that of the HiPIP II from Ectothiorhodospira halophila. Surprisingly, site-specific assignment has shown that in the oxidized protein the distribution of oxidation numbers within the cluster is very similar to that found for E. halophila HiPIP II and different from that of the other two proteins. The spectrum of the reduced species is very similar to that of all other HiPIPs known to date, indicating very similar electronic and geometric structures for the reduced forms. These findings are discussed in terms of cluster structure in HiPIPs and of redox potentials.

Amino Acid Sequence↗
PubMed1993

One- and two-dimensional NMR characterization of oxidized and reduced cytochrome c' from Rhodocyclus gelatinosus.

1D and 2D NMR spectra of both the reduced and oxidized forms of cytochrome c' from Rhodocyclus gelatinosus have been recorded. The analysis of the pH dependence of the 1H NMR spectrum of the ferric form has been performed, and two main ionizing groups have been identified. By comparison of the pH dependence of the available spectra of cytochromes c', an ambiguity remaining from previous studies on related cytochromes c' has been solved. By means of 2D spectra, an assignment of all the paramagnetically shifted signals is proposed both for the ferrous and for the ferric forms.

Bacteria↗