Abstract: Low energy modes have been calculated for the largest possible number of available representatives (>150) of EF-hand domains belonging to different members of the calcium-binding EF-hand protein superfamily. These proteins are the major actors in signal transduction. The latter, in turn, relies on the dynamical properties of the systems, in particular on the relative movements of the four helices characterizing each EF-hand domain upon calcium binding. The peculiar structural and dynamical features of this protein superfamily are systematically investigated by a novel approach, where the lowest energy (essential) modes are described in the space of the six interhelical angles among the four helices constituting the EF-hand domain. The modes, obtained through a general and transferable coarse-graining scheme, identify the easy directions of helical motions. It is found that, for most proteins, the two lowest energy modes are sufficient to capture most of the helices' fluctuation dynamics. Strikingly, the comparison of such modes for all possible pairs of EF-hand domain representatives reveals that only few easy directions are preferred within this large protein superfamily. This enables us to introduce a novel dynamics-based classification of EF-hand domains that complements existing structure-based characterizations from an unexplored biological perspective.
Abstract: In this review, the contribution of the Magnetic Resonance Center of the University of Florence (CERM) to structural genomics is described. This contribution focuses on metalloproteins. Particular emphasis is given to those metalloproteins that belong to the same biochemical pathway, such as the proteins involved in copper trafficking, in metal detoxification, in the assembly of cytochrome c oxidase, and in the assembly of the urease nickel cofactor. Calcium-dependent signalling proteins studied at CERM are also reviewed, for their peculiar conformational features that are at the basis of the signalling process as well as for the importance of the methodological NMR approach based on the substitution of calcium with lanthanide ions. The structural determination in solution of iron-sulfur proteins and cytochromes was pioneered by us in a pre-genomic era and constitutes the methodological basis of the subsequent studies. Our methodological approach is indeed largely based on NMR, even if not exclusively, with important contributions deriving also from X-ray crystallography and X-ray absorption spectroscopy. The use of NMR is particularly useful for the characterization of proteins that occur in vivo as largely of completely unfolded, or that can become unfolded under extreme solution conditions or upon chemical manipulations. Examples in this sense are provided for the different classes of metal binding proteins and in particular for cytochromes and CuZn superoxide dismutase. Within the worldwide frame of structural genomics, we are pursuing also the characterization of known naturally occurring pathogenic mutations. As the three-dimensional structures provided by structural genomics projects constitute a starting database for post-genomic drug design, mention is also made to the perspectives in this field by reviewing our activity. (c) 2006 Elsevier B.V. All rights reserved.
Abstract: The structure and dynamics of human calmodulin-like skin protein (CLSP) have been characterized by NMR spectroscopy. The mobility of CLSP has been found to be different for the N-terminal and C-terminal domains. The isolated domains were also expressed and analyzed. The structure of the isolated C-terminal domain is presented. The N-terminal domain is characterized by four stable helices, which experience large fluctuations. This is shown to be due to mutations in the hydrophobic core. The overall N-terminal domain behavior is similar both in the full-length protein and in the isolated domain. By exploiting the capability of Tb3+ bound to CLSP to induce partial orientation of the molecule in a magnetic field, restricted motion of one domain with respect to the other was proved. By using NMR, ITC, and ESI-MS, the calcium and magnesium binding properties were investigated. Finally, CLSP is framed into the evolutionary scheme of the calmodulin-like family.
Abstract: Calcium signaling, one of the most widespread signaling mechanisms in cells, is generally carried out by EF-hand proteins, characterized by a helix-loop-helix motif paired in functional domains. EF-hand proteins may be viewed as molecular switches activated by calcium concentration transients. The EF-hand structural database has grown to a point where meaningful inferences on the functional conformational rearrangements upon calcium binding can be made by comparing a fair number of pairs of end points, i.e., the structures of the apo and calcium-bound forms. More compact descriptors of the movement associated with calcium binding, in terms of principal component analysis of the six interhelical angles, have also become available. Dynamic information obtained by NMR, also with the aid of calcium substitution with paramagnetic lanthanides, is shedding light on the intrinsic amplitude of the conformational degrees of freedom sampled by the various members of the EF-hand superfamily, as well as on the time scales of the motions. Particularly, NMR of lanthanide derivatives helps in capturing long time scale motions. Both static and dynamic pictures reveal a large variety of behaviors. It is increasingly recognized that the EF-hand machinery has differentiated its behavior during evolution in several ways, e.g., by modifying one of the loops, by undergoing a further duplication after the initial motif duplication that originated the functional domain, or by acquiring the ability to dimerize.
Abstract: A database of nonredundant structures of EF-hand domains--i.e., pairs of helix-loop-helix motifs--has been assembled, and the six angles among the four helices re-determined. A principal component analysis of these angles allows us to use two such components (PC1 and PC2) to describe the system retaining 80% of the total variance. A PC2 against PC1 plot representation allows us to represent in a compact way the full range of structural diversity of EF-hand domains, their grouping into protein families, and the variation for each family upon calcium and peptide binding.
Abstract: The electron paramagnetic resonance (EPR) spectrum from the [4Fe-4S](3+) cluster in several high-potential iron-sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe-4S](3+) structure the (Fe-Fe)(5+) mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe-4S](3+) paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe-4S](3+) model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.
Abstract: In the last few years the poultry industry has seen a significant deterioration in meat quality properties during the summer season. The objective of this study was to evaluate the seasonal effect (summer and winter) on turkey meat quality assessed by both conventional and low-resolution nuclear magnetic resonance (LR-NMR) analysis. Eighty-eight breast muscle samples (35 winter and 53 summer) from BUT-Big 6 turkeys belonging to 16 different flocks, were randomly collected from a commercial processing plant. The samples were analysed for transverse relaxation times (T-2) by LR-NMR and for initial pH (15 min post mortem), ultimate pH (24 h post mortem) and pH after cooking, temperature at 15 min post mortem, water-holding capacity (WHC, drip loss, filter paper press wetness and cooking loss) at 24h post mortem, colour of raw and cooked meat and chemical composition (moisture, lipids and proteins). The results indicate that, during the summer season, turkey breast meat undergoes a relevant WHC decrease. Cluster analysis of the raw LR-NMR data evidenced the presence of two groups corresponding to samples harvested in each different season. Correlations between the LR-NMR signal and the conventional parameters measuring WHC were obtained by a recently proposed type of principal component regression (PCR) termed relative standard deviation PCR. (C) 2004 Society of Chemical Industry.
Abstract: Direct detection heteronuclear NMR allows us to drastically reduce paramagnetic contributions to the line width as compared to 1H detection. As an example, a calcium binding protein (human oncomodulin), in which one of the calcium ions was selectively substituted with Tb3+, is used. Through a variety of 13C direct detection NMR experiments, resonances were detected as close as 5.5 A from the metal ion. Pseudocontact shifts measured through 13C direct detection experiments provide structural constraints in regions of the protein where 1H resonances are broadened beyond detection through Curie relaxation (up to 16 A from the paramagnetic center).
Abstract: The aim of this research was to determine the structure of human beta-parvalbumin (109 amino acids) and to compare it with its paralog and ortholog proteins. The structure was determined in solution using multinuclear and multidimensional NMR methods and refined using substitution of the EF-hand Ca(2+) ion with a paramagnetic lanthanide. The resulting family of structures had a backbone rmsd of 0.50 A. Comparison with rat oncomodulin (X-ray, 1.3 A resolution) as well as with human (NMR, backbone rmsd of 0.49 A) and rat (X-ray, 2.0 A resolution) parvalbumins reveals small but reliable local differences, often but not always related to amino acid variability. The analysis of these structures has led us to propose an explanation for the different affinity for Ca(2+) between alpha- and beta-parvalbumins and between parvalbumins and calmodulins.
Abstract: The pair-of-pairs model that successfully explained the Mossbauer and NMR data on oxidized High Potential Iron-sulfur Proteins (HiPIP) in terms of equilibria between two electronic isomers is reviewed. A bridging of the high temperature NMR data with the low temperature EPR data is attempted through extrapolation to low temperature of the species distribution detected at room temperature by NMR. This extrapolation is validated by recent H-1 ENDOR data. With one exception, the more abundant isomer is the one with the mixed-valence iron pair localized either on irons II-III or on irons III-IV. It is suggested that one or two more isomers besides these two, such as I-III or II-IV, may be present in smaller amount.
Abstract: The temperature dependence of the mean square displacement of the (57)Fe nuclei due to motion faster than 100 ns are measured by temperature-dependent Mössbauer spectroscopy for oxidized and reduced HiPIPs from Ectothiorhodospira halophila, Chromatium vinosum WT and a Cys77Ser mutant. The behaviour is interpretable in the frame of the general model of protein dynamics distinguishing two temperature intervals. The character of harmonic and quasi-diffusional modes in HiPIPs is discussed. Dynamic information obtained from Mössbauer spectroscopy and Fe K-edge EXAFS are compared. Structure dynamics of the iron-sulfur cluster in the partially unfolded reduced HiPIP from C. vinosum was investigated by Mössbauer spectroscopy and EXAFS, indicating an intact metal centre and a protein backbone with a largely collapsed secondary structure. The role of the cofactor during protein folding is discussed. Differences in the dynamics between the native protein and the molten globule are found at physiological temperatures only. The structure and dynamic behaviour of the [Fe(4)S(4)]Cys(3)Ser cluster in the Cys77Ser mutant of the HiPIP from C. vinosum are analysed. The temperature dependence of electron relaxation in oxidized HiPIPs is investigated by Mössbauer spectroscopy and analysed theoretically, considering spin-spin and spin-lattice relaxation. The latter consists of contributions from direct phonon bottleneck and Orbach mechanisms. The data agree with former pulsed EPR results. Orbach relaxation is interpreted as due to transitions between electronic isomers of oxidized HiPIPs. With this interpretation, the energetic difference between both isomers equals the energy gap estimated from the temperature dependence of the Orbach relaxation.
Abstract: Mossbauer. Fe-57 ENDOR, CW and pulsed EPR experiments were performed on the reduced and the oxidized high-potential iron proteins (HiPIPs) of the wild type (WT) and the C77S mutant from Chromatium vinosum. The EPR spectra of the oxidized WT and mutant show three species respectively having nearly the same g-values but strongly changed spectral contributions. Relaxation times were estimated for oxidized WT and mutant at T=5 K with pulsed EPR. A-tensor components of both iron pairs were obtained by Fe-57 ENDOR, proving a similar magnetic structure for the WT and the mutant. Electronic relaxation has to be taken into account at T=5 K in native and mutated oxidized HiPIPs to achieve agreement between Mossbauer and Fe-57 ENDOR spectroscopies. The Mossbauer spectroscopy shows that the oxidized cluster contains a pure ferric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the Mossbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The Mossbauer parameters confirm a loss of the covalent character of the iron bond when S is replaced by O and indicate a shift of the cluster's electron cloud towards the serine. Mossbauer spectra of the oxidized mutant can be simulated with two models: model I introduces a single electronic isomer with the serine always ligated to a ferric iron. Model II assumes two equally populated electronic isomers with the serine ligated to a ferric iron and a mixed-valence iron, respectively. The latter model is in better agreement with EPR and NMR.
Abstract: In this paper chemometrics (ANOVA and PCR) is used to measure unbiased correlations between NMR spin-echo decays of pork M. Longissimus dorsi obtained through Carr-Purcell-Meiboom-Gill (CPMG) experiments at low frequency (20 MHz) and the values of 14 technological parameters commonly used to assess pork meat quality. On the basis of the ANOVA results, it is also found that the CPMG decays of meat cannot be best interpreted with a "discrete" model (i.e., by expanding the decays in a series of a discrete number of exponential components, each with a different transverse relaxation time), but rather with a "continuous" model, by which a continuous distribution of T-2's is allowed. The latter model also agrees with literature histological results. (C) 2000 Academic Press.
Abstract: Technomimetic NMR experiments were performed in accordance with the lye treatment adopted during table olive industrial procedures for the debittering process causing oleuropein degradation. The site selective hydrolysis of the two ester groups, characterizing the biophenolic secoiridoid molecule, was shown to be dependent on the different reactivities of these functionalities. The process is controlled by the experimental conditions exerted on the olive pulp and determined by the buffering capacity of the olive mesocarp and by the epicarp molecular components influencing the reactant penetration into the fruit pulp. The overall hydrolytic process of oleuropein, the bitter principle of olives, using the technomimetic experimental mode, gave rise to its catabolic derivatives, hydroxytyrosol, 11-methyloleoside, and the monoterpene glucoside, technomimetically produced, isolated, and structurally characterized by H-1, C-13, and COSY spectroscopy as the oleoside.
Abstract: An inexpensive external unit that allows the use of a commercial high-resolution NMR spectrometer as a very low frequency instrument is described. The external unit is phase coherent, the pulse timing being given by the parent spectrometer, With the exception of the probe, the external unit does not contain any tuned elements. This permits easy change of frequency in the range 100 kHz-1 MHz, The external unit may be appropriately employed in food science where, in several cases, low frequency is desirable. An application to hen shell eggs at the frequency of 700 kHz is described, (C) 1999 Academic Press.
Abstract: The crystal structure of the reduced high-potential iron protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure-of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0.93 Angstrom, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the protein atoms. Restrained anisotropic refinement for re-WT and H42Q-1 converged to R-1 = Sigma \\F-0\ - \ F-c\/Sigma \ F-0\ of 12.0 and 13.6%, respectively [data with I > 2 sigma(I)], and 12.8 and 15.5% tall data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 Angstrom. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe4S4](2+) cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe-S bonds along four approximately parallel cube edges, and eight long Fe-S bonds. The unique protein molecules in H42Q-2 and re-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the protein rather than by structural differences in the cluster region.
Abstract: The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with S gamma of Cys77, one of the cluster ligands, The reduction potential of the S79P variant was 104+/-3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins, These results suggest that the influence of amide group 79 on the reduction potential of C, vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins.
Abstract: The thermodynamic parameters for the reduction reaction of the high-potential iron-sulfur protein from Chromatium vinosum, and its H42Q mutant, have been measured by direct cyclic voltammetry using a 'non-isothermal' reference electrode. Histidine 42 is mainly responsible for the pH dependence of the reduction potential (E degrees) with pK(a), values equal to 5.9 and 6.3 for the oxidized and reduced proteins, respectively. The H42Q mutant does not present this pH dependence, although an increasing E degrees at the lowest pH values is still present. The main contribution to the difference in free energy between the protonated and the neutral His42 forms is given by the enthalpic term (Delta Delta H degrees = 7.4 kJ mol(-1)), although the entropic term is not negligible. (C) 1998 Elsevier Science S.A. All rights reserved.
Abstract: The aim of this article is to critically discuss the information available on the influence of the protein environment on the electron transfer properties of Fe-S proteins. First, the intrinsic redox and structural features of synthetic Fe-S clusters are illustrated, in order to provide a background on which the effects of the protein matrix can be subsequently highlighted. The parameters found to influence the reduction potential in metalloproteins are then discussed, and a detailed analysis of structure-function-relationships among the different classes of Fe-S proteins is carried out. This analysis reveals that, within each class, one of the parameters is often more important than the others in determining the reduction potential of the Fe-S center; the parameter changes upon passing from one class to the other. Within the class of high potential Fe-S proteins the major determinant for the variation of reduction potential is the difference in charged residues, whereas innrubredoxins and [2Fe-2S], [3Fe-4S], and [4Fe-4S] ferredoxins, this effect is attenuated and overwhelmed by solvent accessibility to the cluster core and/or inner-fractional charges in the protein itself. A deeper understanding of the influence of the structural properties of the. protein is gained by analysing the microscopic reduction potentials of the individual Fe ions in Fe-S centers. The latter are obtained from the electronic distribution within each type of cluster core, which, in turn, is determined from a theoretical analysis of the electron nucleus coupling derived from NMR and other spectroscopic analysis.
Abstract: We have succeeded in preparing a mutant of the High Potential Iron-Sulfur Protein (HiPIP) from Chromatium vinosum in which a cysteine ligand has been replaced by a serine (C77S). Proton chemical shift data and nuclear Overhauser effects indicate that structural perturbations induced by the C77S mutation are minimal in both the oxidized and reduced forms of the HiPIP. The reduction potential of C77S is 25 mV lower than that of the wild-type HiPIP (WT) (0.2 M ionic strength, pH 4.5-9.0, 25 degrees C). Assignment of the hyperfine shifted signals in the H-1 NMR spectrum of oxidized C77S revealed that the temperature dependences of the signals associated with cluster-ligating residues 46 and 77 are Curie and and-Curie type, respectively, and are thus the reverse of those in WT. Taken together, these observations indicate that the iron bound to Ser-77 is less reducible than the corresponding iron in WT. The results are consistent with a previous model of the electronic structure of oxidized HiPIP clusters, confirming the presence of an equilibrium between two species of differing valence distributions. The current results permit the extension of this model to predict the relative reduction potentials of the individual iron ions in the oxidized HiPIPs up to now investigated.
Abstract: The full H-1 NMR assignment of the reduced C77S mutant of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) was achieved by taking advantage of the assignment available for the wild-type protein. A total of 1565 nuclear Overhauser effect (NOE) spectroscopy cross peaks were integrated and converted into distance constraints, of which 497 were found to be irrelevant. An additional 24 dipolar constraints were obtained from one-dimensional NOE difference spectra by saturating hyperfine-shifted beta CH2 cysteine/serine protons. Forty-six (3)J(NH-H alpha) coupling constants and eight hydrogen bonds provided further constraints. Through a distance geometry approach, a family of 15 structures was calculated, which was subsequently subjected to restrained energy minimization. The root mean square deviations of the minimized structures were 0.62 +/- 0.09 and 1.09 +/- 0.11 Angstrom for backbone and heavy atoms, respectively. The resulting solution structures are very similar to those of the reduced wild-type protein (WT). An analysis of the NOEs experienced by the protons of Ser-77 in both the reduced and oxidized forms reveals that they are very similar to those experienced by Cys-77 in WT. On the basis of the hyperfine shifts observed for the Ser-77 protons and of the present structural analysis, it is concluded that the serine O gamma atom is coordinated to the polymetallic center, thus confirming the strict analogy of the electronic structures of the polymetallic center in both proteins. Capillary electrophoresis experiments demonstrate coordination of Ser-77 as an anion. Serine versus cysteine coordination in iron-sulfur proteins is briefly discussed.
Abstract: The H-1 NMR spectra of the oxidized high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila have been investigated, and the sequence-specific assignment of the protons of the cluster-bound cysteines has been performed. The spectrum of this protein is different from those of the six other proteins investigated to date in that at least one hyperfine shifted signal is missing. It is demonstrated that the ''missing'' signals of the beta CH2 protons of Cys 66 are under the diamagnetic envelope at 2.60 and 6.30 ppm (at 288 K). The hyperfine shifts of these protons and those of the beta CH2 protons of Cys 36 are consistent with the proposition that the oxidized cluster is in equilibrium between two electronic species. These species differ with respect to their valence distribution within the protein frame and are present in a molar ratio of about 4:1 at room temperature. An empirical relationship between the hyperfine shifts of the beta CH2 protons of Cys 36 and 66 and the percentage of the two electronic species is proposed.
Abstract: The H-1 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. (C) 1995 Academic Press, Inc.
Abstract: Oxidized ferredoxin from Clostridium acidi urici, containing two [Fe4S4]2+ clusters, has been investigated through H-1 NOESY and TOCSY spectroscopies. The protons of coordinated cysteines have been identified and assigned to each cluster with use of a procedure based on the assignment of two spatially close betaCH2 pairs and on the shift ratios of each betaCH2 Proton in oxidized, half-reduced, and reduced forms; each cysteine proton has been then sequence-specifically and stereospecifically assigned by looking for dipolar connectivities with amino acid residues in the vicinity of the cluster. By comparing the present data with the available spectra of the analogous protein from Clostridium pasteurianum, the sequence-specific and stereospecific assignments of cysteine protons have been obtained also for the latter protein. The natural abundance C-13 signals of the cysteine protons have been also sequence-specifically assigned. By taking advantage of the X-ray structure of a similar protein, the H-1 and C-13 hyperfine shifts have been related to the dihedral angle between the iron-sulfur-beta-carbon plane and the sulfur-beta-carbon-beta-proton or sulfur-beta-carbon-alpha-carbon planes. A parametric equation is proposed. The spin delocalization mechanism has been found to be largely dependent on unpaired spin density on the p(z) orbital of the sulfur atom. Through EXSY spectroscopy, the proton signals of the [Fe4S4]+ clusters in the reduced protein have been assigned. Their temperature dependence is compared with that of the [Fe4S4]3+ clusters present in oxidized HiPIPs and discussed in terms of the Heisenberg model for the magnetic exchange coupling within the clusters.
Abstract: H-1 two-dimensional NMR spectroscopy has been applied to the oxidized form of cytochrome c 551 from Rhodocyclus gelatinosus, which is paramagnetic with S = 1/2. The investigation has allowed a complete and unambiguous assignment of the heme protons and some residues around the heme. We have learned that: the conformation of the axial methionine is equal to that of horse heart cytochrome c and different from two isoenzymes of the same cytochrome c 551 from a different strain; pKa of 6.6 +/- 0.3 has been detected through the shift variations of seventh propionate protons. The detailed differences with other cytochromes c in the hyperfine shifts are discussed.
Abstract: Reaction of the two-[Fe4S4]-cluster ferredoxin from Clostridium acidi urici with Fe(CN)(6)(3-) yields a derivative selectively depleted of one iron ion from cluster I. The derivative binds Fe2+, Zn2+ or Co2+ yielding the native or the MFe(3)S(4) cluster, respectively. The NMR spectra of the [Fe3S4](+) species, of the Zn2+ species in the +1 state, and of the Co2+ species, in both the +1 and +2 states, are reported. The spectra of the latter show eight far hyperfine shifted signals, assigned to the cysteinic beta-CH2 protons. The hyperfine shifts and their temperature dependences are consistent with Heisenberg coupling schemes where a mixed-valence high-spin Fe3+/Fe2+ pair is antiferromagnetically coupled to a Co2+ and an Fe3+(Fe2+) ion. It is proposed that the expectation value of S-z for the Co2+ ion has a reversed sign in the total spin ground state, thus giving rise to upfield shifts of the beta-CH2 protons of the cobalt-coordinated cysteine.
Abstract: 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. (C) 1994 Academic Press, Inc.
Abstract: Solvent water H-1 NMRD profiles of hexaaquairon(III) in 1.0 M HClO4 have been obtained at several temperatures and viscosities. Dynamic parameters including the molecular rotational correlation time and the correlation time for the electron spin-lattice interaction have been obtained. The magnetic field dependence of the latter has been investigated. The present data provide a unified picture of the behavior of this system from the point of view of electron and nuclear relaxation. It is found that only part of the hyperfine coupling is significant for electron relaxation. The electron relaxation properties of iron(III) in macromolecules are critically compared to those of the hexaaqua complex.
Abstract: H-1 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.
Abstract: The nuclear magnetic relaxation dispersion (NMRD) profiles of water protons have been investigated between 0.01 and 600 MHz for lanthanide aqua complexes, where the lanthanide is Pr, Sm, Dy, Ho, Er, and Yb. The experimental profiles have been interpreted as due to contributions from proton-electron dipolar coupling and Curie relaxation mechanisms. For the former the correlation time is the electron relaxation time itself and for the latter the rotational correlation time of the aqua ion. This is the first time that a Curie contribution to longitudinal relaxation has been observed through its magnetic field dependence. The electron relaxation times are confirmed to be magnetic field independent, very short, and substantially temperature independent. Their actual values are now proposed by taking into consideration crystal field effects. The mechanisms causing electron relaxation have been discussed.
Abstract: Pseudo-contact-shifted H-1 NMR resonances of rabbit parvalbumin substituted with Yb3+ at the Ca2+ EF site have been sequence-specific assigned by a combination of 2D techniques and magnetic susceptibility tenser determination. By taking the X-ray structure of the homologous carp protein as a starting point, the sequence-specific assignment proceeded in a stepwise fashion. The first resonances assigned on the basis of their unique connectivity pattern were used to make a first coarse determination of the (chi)-tensor parameters; the latter were used to guess the identity of further hyperfine-shifted resonances, which were then checked through 2D connectivities and in turn allowed us to improve the reliability of the (chi)-tensor parameters. This cyclic procedure was repeated until virtually all pseudo-contact-shifted resonances were assigned. In the last steps the (chi)-tensor parameters did not change appreciably. The orientation of the tenser was found to be very similar to that previously proposed for the homologous Yb3+-substituted protein from carp. Modest discrepancies between some calculated and experimental shift values in the present system were found to be due mainly to structural differences between the present protein and the homologous protein whose x-ray structure was used as model and, to a lesser extent, to indetermination in the diamagnetic shift values. The success of the analysis and the univocal solution for the (chi)-tensor parameters under these conditions show that the above procedure is robust enough to permit both signal assignment and (chi)-tensor determination even when the structural data are only a rough approximation of the actual structure. These findings may open the way to the development of a systematic use of pseudo-contact shifts as constraints for MD refinement of solution structures of paramagnetic metalloproteins.
Abstract: In our efforts to characterize oxidized high-potential iron-sulfur proteins (HiPIP), we have investigated the oxidized HiPIP II from Ectothiorhodospira halophila through H-1 NMR and molecular dynamics (MD) calculations. This protein has the most symmetric isotropic shift pattern of the beta-CH2 Protons of the liganded cysteines, four signals being upfield and four downfield. H-1 NOE, NOESY, and TOCSY results have provided the necessary key connectivities to perform the assignment of the liganded cysteines, taking advantage of the structure of the HiPIP I isoprotein. It is found that the electronic distribution within the cluster is different with respect to the Chromatium vinosum and Rhodocyclus gelatinosus systems. As in the latter systems, the cluster can be described in terms of two iron(III) and two mixed valence ions, but the two pairs are oriented in a different way within the protein frame. These results are discussed in terms of structure-function relationships. An MD approach starting from the structure of HiPIP I has provided a structural model of the present protein, which is absolutely consistent with the NMR connectivities in the surroundings of the cluster.
Abstract: Water proton relaxation measurements as a function of magnetic field on solutions containing paramagnetic metalloproteins contain information on (i) number and distance of metal-coordinated water molecules, (ii) dynamics of water exchange, (iii) electron relaxation rates, and (iv) dynamics of the metal coordination sphere influencing electron relaxation. The use of appropriate theoretical tools permits us in many cases to learn about some or all of the above properties.
Abstract: A given tolerance value on the declared nutrient content of fertilizers is permitted by some national laws. A statistical method to assess systematic exploitation of tolerance values by single nutrient fertilizer producers is proposed. Production of superphosphate was selected as the case study. The method was tested on a yearly production, on randomly selected samples, and on a generated series of samples; it was found to be simple and reliable. The method is theoretically expected to be more reliable than the Wilcoxon test recommended by French law for normal distributions; it appears to fit better than the Wilcoxon test to uneven production of fertilizers.
Abstract: 2D NMR experiments performed on both the oxidized and reduced form of the high potential iron protein (HiPIP) from Chromatium vinosum, a paramagnetic iron sulfur protein for which the crystal structure is known in both oxidation states, allowed us to detect a number of scalar and dipolar connectivities of the isotropically shifted signals. On this basis it was possible to firmly identify the signals of the beta-CH2 and alpha-CH protons of the cluster-liganded cysteines and perform their sequence-specific assignments. The assignments mainly rely on the observation of NOESY cross peaks from beta or alpha-Cys protons to protons assigned to the few aromatic residues surrounding the cluster. This is the first sequence-specific assignment of Cys beta-CH2 protons for a Fe4S4 cluster. In the light of existing experimental evidence from Mossbauer data and of the theoretical model describing the magnetic coupling of the metal centers in the oxidized form, the present assignment establishes which iron ions of the oxidized cluster are in a pure ferric state and which are in a mixed valence state. These findings may be relevant as far as the actual mechanism of electron transfer is concerned. In addition, information is obtained on the angular dependence of the beta-CH2 hyperfine shifts in iron sulfur systems.
Abstract: H-1 NOE and 2D NMR studies have been performed on oxidized and reduced HiPIP from Rhodocyclus gelatinosus (formerly Rhodopseudomonas gelatinosa). By saturating the beta-CH2 Protons of the cysteines in the oxidized form which are hyperfine shifted, NOEs are measured on signals which have been then assigned to specific residues through 2D NMR. In this way the signals of the cysteines have been specifically assigned. Since we had already related the shifts of the beta-CH2 Protons to the oxidation state of each iron, we succeeded in connecting the individual cysteines with the oxidation state of each iron. Support for the assignment comes from 2D measurements of the reduced protein which is less paramagnetic. Signals in the oxidized and reduced species can be related through EXSY. These results represent a unique and successful strategy to frame an iron sulfur cluster within a protein, with the iron ions labelled according to their oxidation number.
Abstract: A new lipid component has been identified in the unsaponifiable matter of drupes of Persea americana Mill. The structure has been completely elucidated by mass and NMR spectroscopy. The compound has a cyclopent-2-enone ring with a 16 carbon side chain in position 2. Two other double bonds have been found in the side chain which are, starting from the methyl end of the chain, in the same position as in linoleic acid.
Abstract: The theoretical expectations for nuclear longitudinal relaxation rate enhancements for water protons sensing paramagnetic metal ions are shown. The cases of S = 1/2 (Cu2 + and VO2+), S = 1 (Ni2+), S = 5/2 (Mn2+ and Fe3+), S = 7/2 (Gd3+) are considered by including some factors which split the S manifold at zero field and the effect of the rotation.
Abstract: 600-MHz H-1-NMR and nuclear Overhauser enhancement spectroscopy (NOESY) spectra in (H2O)-H-2 and (H2O)-H-2, as well as truncated driven NOE difference spectra in H2O of reduced human Cu(II)2Zn(II)2 superoxide dismutase (Cu/Zn SOD) have been recorded and used to assign the active-site proton signals. A derivative with histidines selectively deuteriated in the C2 position has been used for the detection of the HC2 histidine protons. 16 out of 17 observed signals of the 18 active-site histidine ring protons have been assigned. The results are compared with previous proposals based on more limited data sets. The numerous cross peaks confirm that the structure in solution is essentially similar to the crystallographic data obtained on the oxidized form. Probably this holds also for His63 which in the reduced form is not bridging any more the two metal ions. The effects of azide binding on the exchangeable H-1-NMR signals of the reduced protein are also reported.
