Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.     

Losartan and its interaction with copper(II): biological effects

Losartan, the potassium salt of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol, is an efficient antihypertensive drug. The vibrational FTIR and Raman spectra of Losartan (its anionic and protonated forms) are discussed. In addition, the copper(II) complex of Losartan was obtained and characterized as a microcrystalline powder. The metal center is bound to the ligand through the nitrogen atoms of the tetrazolate moiety as determined by vibrational spectroscopy. The compound is a dimer with the metal centers in a tetragonal distorted environment but the presence of a monomeric impurity has been determined by EPR spectroscopy. The antioxidant properties of the complex (superoxide dismutase mimetic activity) and its effect on the proliferation and morphology of two osteoblast-like cells in culture are reported. The new compound exerted more toxic effects on tumoral cells than the copper(II) ion and Losartan.     

Antineoplastic activity of new lanthanide (cerium, lanthanum and neodymium) complex compounds

Cerium (III), lanthanum (III) and neodymium (III) complexes with 3,3'-benzylidenebis[4-hydroxycoumarin] were synthesized in view of their application as cytotoxic agents. The complexes were characterized by different physicochemical methods: elemental analysis, mass spectrometry, 1H NMR, 13C NMR and IR spectroscopy. The spectra of the complexes were interpreted on the basis of comparison with the spectrum of the free ligand. The vibrational analysis showed that in the complexes the ligand coordinated to the metal ion through both deprotonated hydroxyl groups; however, participation of the carbonyl groups in the coordination to the metal ion was also suggested. The evaluation of the cytotoxic activity of the novel lanthanide complexes on HL-60 myeloid cells revealed that they are potent cytotoxic agents. The cerium complex was found to exhibit superior activity in comparison to the lanthanum and neodymium coordination compounds, the latter being the least active. Our data give us reason to conclude that the newly synthesized lanthanide complexes should be submitted to further more detailed pharmacological and toxicological evaluation.     

Analysis of the vibrational spectra of new OH-containing E-4-arylmethylene-3-isochromanones and 3-arylcoumarins

A combined experimental and theoretical approach is presented to structural characterization of fairly large, newly synthesized organic molecules in order to enhance the effectiveness of their instrumental analysis by vibrational spectroscopy. The method consists of measurement of FT-IR and Raman spectra of the reaction products and subsequent ab initio or DFT quantum mechanical calculations (prediction) of the vibrational spectra for any anticipated structural varieties of the synthesized molecules. Comparison of the measured and computed frequencies as well as the observed and simulated spectra is performed to resolve any uncertainties in identifying the reaction products. Vibrational frequency and normal mode calculations based on scaled quantum mechanical (SQM) force fields performed at the DFT/B3LYP/6-31G* level of theory are demonstrated to provide a wealth of information that have been used in this work to ascertain the molecular structure, probable conformation and H-bond properties of three new isochromanone or coumarin derivatives, namely: 3-([2'-hydroxymethyl]-phenyl)-coumarin (1), E-4-(3'-hydroxyphenylmethylene)-3-isochromanone (2), and 2-[(2'-hydroxymethyl)phenyl]-3H-naphto[2,1-b]pyran-3-one (3).     

The role of the iron-sulfur cluster in Escherichia coli endonuclease III. A resonance Raman study.

Resonance Raman spectroscopy has been used to investigate the function and properties of the iron-sulfur cluster in Escherichia coli endonuclease III. Resonance Raman spectra in the Fe-S stretching region are indicative of a [4Fe-4S]2+ cluster with complete cysteinyl sulfur coordination, and vibrational assignments are made by analogy with bacterial ferredoxins. Minor changes in the vibrational frequencies of the modes primarily involving Fe-S(Cys) stretching accompany the binding of the inhibitor thymine glycol or an oligonucleotide containing a reduced apyrimidinic site. These changes are consistent with perturbation of the orientation of the ligating cysteinyl residues and rule out the possibility that the [4Fe-4S] cluster is directly involved with substrate or inhibitor binding. It is concluded that a structural role is most likely for the [4Fe-4S] cluster in endonuclease III.     

Studies on the conformational changes of metalloproteins induced by electrons in water-ethylene glycol solutions at low temperatures. III. Adrenal ferredoxin.

The reduction of adrenal ferredoxin (adrenodoxin) at low temperatures was investigated in order to separate local modifications of the active centre of the protein on its reduction, from the conformational transition which seems to accompany the change of the redox state of the irons; The ESR spectra of the states of the protein, where the reduced active centre is to be found by the "oxidized" conformation of the apoprotein, were obtained. The transition from the states of the protein to the state which occurs on its chemical reduction at room temperature was also investigated. The results of the work support the view that conformational changes in proteins (enzymes) which take place while they are functioning proceed after modifications of the active centres (change of the redox state, adsorption of a substrate, etc.), and are essentially caused by them. Adrenal ferredoxin was the third subject in our studies of the intermediate states of proteins which appear after reduction of their active centres by means of electrons trapped in water-ethylene glycol mixtures at the temperature of liquid nitrogen [1, 2]. In the reduced state, the active centre of the protein has an ESR signal with a g-factor of 1.94 [3, 4] which is convenient for our purposes.     

Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2

Possible origins of the different metal co-ordination topologies in the recently determined structures of rat metallothionein-2 (MT2) in single crystals and rabbit MT2 in solution were investigated. A complete structure determination for rat MT2 in solution by nuclear magnetic resonance (n.m.r.) showed that the differences in the spatial structures cannot be attributed to the different primary structures of the two species. Comparison of [113Cd7]MT2 obtained by reconstitution of the apoprotein in vitro with preparations using a different procedure showed, moreover, that the metal co-ordination observed in solution by n.m.r. is not an artefact of the protein reconstitution. Solutions of high-pressure liquid chromatographically homogeneous biosynthetic preparations of [113Cd, Zn]MT2 were obtained from rat liver following injection of 113Cd into rats in vivo, without further metal exchange after protein isolation. They contain a mixture of several forms of MT2 with different relative metal compositions, giving rise to an increased number of 113Cd resonances. For the components of the four-metal cluster, the major one of these different forms exhibits patterns in the two-dimensional [1H, 113Cd]-correlated spectra that are indistinguishable from those of [113Cd7]MT2, thereby implying identity of cluster coordination and topology. These results are discussed with regard to continued investigations into the differences between the solution structure and crystal structure of MT2.     

Chemoenzymatic Approach to Optically Active 4‐Hydroxy‐5‐alkylcyclopent‐2‐en‐1‐one Derivatives: An Application of a Combined Circular Dichroism Spectroscopy and DFT Calculations to Assignment of Absolute Configuration

A series of representative optically active derivatives of 4‐hydroxy‐5‐alkylcyclopent‐2‐en‐1‐one were prepared from the respective 2‐furyl methyl carbinols via the Piancatelli rearrangement followed by the enzymatic kinetic resolution of racemates. Applicability of chiroptical methods (experimental and calculated electronic circular dichroism [ECD] and vibrational circular dichroism [VCD] spectra) to determine the absolute configuration of both stereogenic centers in 4‐hydroxy‐5‐methylcyclopent‐2‐en‐1‐one was demonstrated. It was also demonstrated that the concurrent application of ECD and VCD spectroscopy can be used for the determination of the configuration of two stereogenic centers. Chirality 26:300–306, 2014. © 2014 Wiley Periodicals, Inc.     

Consequences of polymorphism of 5'-GMP.Na2 in the vibrational spectra of metal complexes and isotopic derivatives

The commercial mononucleotides are frequently used to obtain metal complexes and isotopic derivatives. Normally, the spectra of these new compounds are compared with the spectra of the commercial mononucleotides. Nevertheless, important variations in the vibrational spectra of the disodium 5'-guanosine monophosphate, 5'-GMP, have been observed in this work produced by submitting the commercial salt to the same general laboratory process that the obtained compounds, i.e., solving the commercial salt in water and subsequent recrystallization. These changes have been analyzed and interpreted. The variations are not significant in disodium 5'-cytosine monophosphate, 5'-CMP. It is important to take this information into account before carrying out vibrational studies with this type of molecules, since some bands attributed to isotopic substitutions or to the metal attack may be a result of manipulation of the nucleotide (solving and recrystallization) instead of the studied effect. Thus, before any work in which the nucleotide salt is manipulated (deuteration, synthesis of other isotopic derivatives or metal-nucleotide complexes), it should be noted that the process to which the sample is submitted on its own is enough to modify the vibrational spectrum. Then, attention should be paid to the changes observed in the vibrational spectra of recrystallized mononucleotides, since recrystallization may lead to a considerable phase change, and this can notably alter the vibrational spectra.     

Investigation of human erythrocyte superoxide dismutase by 1H nuclear-magnetic-resonance spectroscopy.

The 170MHZ 1 H n.m.r. spectra of the Cu(II)/Zn(II), Cu(I)/Zn(II) and apo- forms of human erythrocyte superoxide dismutase (EC 1.15.1.1) are reported. Resonances are assigned to the C-2 and C-4 protons of histidine residues in the active site, and it is suggested that five or six histidine residues serve as ligands to the metal ions in each subunit of the enzyme. The remaining assigned resonances are associated with histidine-41, N-terminal N-acetyl group, histidine- 108 and cysteine- 109. A comparison of the n.m.r. spectra of human and bovine superoxide dismutases suggests significant structural homology.     

Effect of (13)C-, (18)O- and (2)H-labeling on the infrared modes of UV-induced phenoxyl radicals

The structure and environment of redox active tyrosines present in several metalloenzymes can be studied by resonance Raman spectroscopy or Fourier transform infrared difference spectroscopy. Assignments of the vibrational modes in vivo often requires in vitro studies on model compounds. This approach is briefly reviewed. New results are shown on the influence of isotope-labeling on the infrared spectra of tyrosine, [Formula: see text] and phenol radicals obtained in vitro by UV-irradiation. The infrared spectra of the radicals are dominated by the [Formula: see text] mode at 1515-1504 cm(-1). The frequency shifts induced on this mode by (13)C- (2)H-, and (18)O-labeling are reported.     

CONSEQUENCES OF POLYMORPHISM OF 5′-GMP.Na2 IN THE VIBRATIONAL SPECTRA OF METAL COMPLEXES AND ISOTOPIC DERIVATIVES

ABSTRACT

The commercial mononucleotides are frequently used to obtain metal complexes and isotopic derivatives. Normally, the spectra of these new compounds are compared with the spectra of the commercial mononucleotides. Nevertheless, important variations in the vibrational spectra of the disodium 5′-guanosine monophosphate, 5′-GMP, have been observed in this work produced by submitting the commercial salt to the same general laboratory process that the obtained compounds, i.e., solving the commercial salt in water and subsequent recrystallization. These changes have been analyzed and interpreted. The variations are not significant in disodium 5′-cytosine monophosphate, 5′-CMP. It is important to take this information into account before carrying out vibrational studies with this type of molecules, since some bands attributed to isotopic substitutions or to the metal attack may be a result of manipulation of the nucleotide (solving and recrystallization) instead of the studied effect. Thus, before any work in which the nucleotide salt is manipulated (deuteration, synthesis of other isotopic derivatives or metal-nucleotide complexes), it should be noted that the process to which the sample is submitted on its own is enough to modify the vibrational spectrum. Then, attention should be paid to the changes observed in the vibrational spectra of recrystallized mononucleotides, since recrystallization may lead to a considerable phase change, and this can notably alter the vibrational spectra.     

EXAFS investigation of the active site of iron superoxide dismutase of Escherichia coli and Propionibacterium shermanii

The local structure of the iron site in ferric superoxide dismutase from P. shermanii was analyzed by X-ray absorption spectroscopy. The metal-ligand cluster of the enzyme is found to be similar to the crystallographically investigated ferric superoxide dismutase from E. coli. At pH 6.4 the enzyme is five-fold coordinated with three histidines, an aspartate and a water molecule. The average bond lengths between the metal and the histidines are about 2.10 Å, between metal and aspartate they are about 1.86 Å and between metal and water 1.96 Å. With an increase in pH a change in the coordination number from five to six is observed both in pre-edge peak and EXAFS spectra analysis. However, the bond lengths of the ligands do not change dramatically, they are conserved for the aspartate and increase slightly to 2.13 Å for the average metal - histidine distance at pH 9.3. The observation of the increase in coordination number is correlated with a decrease in enzymatic activity which occurs in the high pH range. The zinc EXAFS spectra of P. shermanii superoxide dismutase have shown that zinc can be incorporated in the active center instead of the iron.     

Synthesis, characterisation and antiamoebic activity of new thiophene-2-carboxaldehyde thiosemicarbazone derivatives and their cyclooctadiene Ru(II) complexes

Reaction of new thiosemicarbazones (1-4) derived from thiophene-2-carboxaldehyde and cycloalkylaminothiocarbonylhydrazine with [Ru(eta(4)-C8H12)(CH3CN)2Cl2] leads to form complexes (1a-4a) of the type [Ru(eta(4)-C8H12)(TSC)Cl2] (where TSC=thiosemicarbazone). All the compounds have been characterised by elemental analysis, IR, 1H NMR, electronic spectra and thermogravimetric analysis. It is concluded that the thionic sulphur and the azomethine nitrogen atom of the ligands are bonded to the metal ion. In vitro antiamoebic screening against (HK-9) strain of Entamoeba histolytica indicated that the Ru(II) complexes of thiophene-2-carboxaldehyde thiosemicarbazones were found more active than the thiosemicarbazones.     

Optical and EPR characterization of different species of active and inactive aconitase

It has been shown by spectroscopic (Kent, T. A., Dreyer, J-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1096-1100) and chemical (Kennedy, M.C., Emptage, M.H., Dryer, J-L., and Beinert, H. (1983) J. Biol. Chem. 258, 11098-11105) methods that interconversion of [3Fe-4S] and [4Fe-4S] clusters underlies activation and inactivation of aconitase. Since Fe-S clusters can assume different oxidation states, a number of different species of the enzyme can be expected to exist. Observations on activation-inactivation, as well as light absorption and EPR spectra, can be interpreted in terms of four species: [3Fe-4S]1+, the oxidized inactive enzyme as obtained on aerobic preparation from mitochondria; [3Fe-4S]0, the reduced inactive form as obtained on reduction in the presence of EDTA; [4Fe-4S]2+, the oxidized active form as obtained on reductive activation; and [4Fe-4S]1+, the reduced active form prepared by photoreduction of active aconitase. The light absorption spectra of each species are presented. Oxidized inactive aconitase shows EPR spectra typical of oxidized 3Fe clusters (g = 2.01), and reduced active enzyme shows spectra typical of reduced ferredoxins (g1,2,3 = 2.06, 1.93, 1.86). The EPR spectrum of the latter is drastically changed (g1,2,3 = 2.04, 1.85, 1.78) on addition of substrate. The active enzyme can be quantitatively converted to inactive enzyme by titration with ferricyanide in the presence of substrate. The correlation of EPR and optical spectra with enzymatic activity observed during titration demonstrates further that active aconitase requires an intact [4Fe-4S] cluster. A model of aconitase incorporating the four cluster species is presented, and explanations for some previous conflicting data concerning aconitase are offered.     

Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase

The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotational resonance NMR study of the active site structure in bacteriorhodopsin: conformation of the Schiff base linkage.

Rotational resonance, a new solid-state NMR technique for determining internuclear distances, is used to measure a distance in the active site of bacteriorhodopsin (bR) that changes in different states of the protein. The experiments are targeted to the active site of bR through 13C labeling of both the retinal chromophore and the Lys side chains of the protein. The time course of the rotor-driven magnetization exchange between a pair of 13C nuclei is then observed to determine the dipolar coupling and therefore the internuclear distance. Using this approach, we have measured the distance from [14-13C]retinal to [epsilon-13C]Lys216 in dark-adapted bR in order to examine the structure of the retinal-protein linkage and its role in coupling the isomerizations of retinal to unidirectional proton transfer. This distance depends on the configuration of the intervening C=N bond. The 3.0 +/- 0.2 A distance observed in bR555 demonstrates that the C=N bond is syn, and the 4.1 +/- 0.3 A distance observed in bR568 demonstrates that the C=N bond is anti. These direct distance determinations independently confirm the configurations previously deduced from solid-state NMR chemical shift and resonance Raman vibrational spectra. The spectral selectivity of rotational resonance allows these two distances to be measured independently in a sample containing both bR555 and bR568; the presence of both states and of 25% lipid in the sample demonstrates the use of rotational resonance to measure an active site distance in a membrane protein with an effective molecular mass of about 85 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman spectroscopy of Azotobacter vinelandii ferredoxin I. Vibrational features of the [3Fe-3S] cluster

Low temperature resonance Raman spectra have been obtained for Clostridium pasteurianum and Bacillus stearothermophilus ferredoxins. Several heretofore undetected fundamental bands have been observed and these data have been used to discriminate the vibrational contribution of the [3Fe-3S] cluster to the spectrum of Azotobacter vinelandii ferredoxin I. The vibrational features of the [3Fe-3S] core distinguish it from other 3-iron clusters and imply structural differences among this class of iron-sulfur clusters.     

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.     

Electrochemically induced FTIR difference spectroscopy in the mid- to far infrared (200 microm) domain: a new setup for the analysis of metal-ligand interactions in redox proteins

We report the setup of an electrochemical cell with chemical-vapor deposition diamond windows and the use of a Bruker 66 SX FTIR spectrometer equipped with DTGS and Si-bolometer detectors and KBr and mylar beam splitters, to record on the same sample, FTIR difference spectra corresponding to the structural changes associated with the change in redox state of active sites in proteins in the whole 1800-50 cm(-1) region. With cytochrome c we show that reliable reduced-minus-oxidized FTIR difference spectra are obtained, which correspond to single molecular vibrations. Redox-sensitive IR modes of the cytochrome c are detected until 140 cm(-1) with a good signal to noise. This new setup is promising to analyze the infrared spectral region where metal-ligand vibrations are expected to contribute and to extend the analysis of vibrational properties to metal sites or redox states not accessible to (resonance) Raman spectroscopy.