Biomedical subjects

Andrei V Astashkin

Publications and source records attributed to Andrei V Astashkin.

17 recordsLinked to original sources

PubMed2006

Determination of the hydration number of gadolinium(III) complexes by high-field pulsed 17O ENDOR spectroscopy.

Pulsed 17O Mims electron-nuclear double resonance (ENDOR) spectroscopy at the W band (95 GHz) and D band (130 GHz) is used for the direct determination of the water coordination number (q) of gadolinium-based magnetic resonance imaging (MRI) contrast agents. Spectra of metal complexes in frozen aqueous solutions at approximately physiological concentrations can be obtained either in the presence or absence of protein targets. This method is an improvement over the 1H ENDOR method described previously, which involved the difference ENDOR spectrum of exchangeable protons from spectra taken in H2O and D2O. In addition to exchangeable water protons, the 1H ENDOR method is also sensitive to other exchangeable protons, and it is shown here that this method can overestimate hydration numbers for complexes with exchangeable protons at GdH distances similar to that of the coordinated water, for example, from NH groups. The 17O method does not suffer from this limitation. 17O ENDOR spectroscopy is applied to Gd(III) complexes containing zero, one, or two inner-sphere water molecules. In addition, 13C and 1H ENDOR studies were performed to assess the extent of methanol coordination, since methanol is used to produce a glass in these experiments. Under the experimental conditions used for the hydration number determination (30 mol % methanol), fewer than 15 % of the coordination sites were found to be occupied by methanol.

Chemistry, Physical↗

PubMed2006

Investigation of the coordination structures of the molybdenum(v) sites of sulfite oxidizing enzymes by pulsed EPR spectroscopy.

Sulfite oxidizing enzymes (SOEs) are physiologically vital and occur in all forms of life. During the catalytic cycle the five-coordinate square-pyramidal oxo-molybdenum active site passes through the Mo(v) state, and intimate details of the structure can be obtained from pulsed EPR spectroscopy through the hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of nearby magnetic nuclei (e.g., (1)H, (2)H, (17)O, (31)P) of the ligands. By employing spectrometer operational frequencies ranging from approximately 4 to approximately 32 GHz, it is possible to make the nuclear Zeeman interaction significantly greater than the hfi and nqi, and thereby simplify the interpretations of the spectra. The SOEs exhibit three general types of Mo(v) structures which differ in the number of nearby exchangeable protons (one, two or zero). The observed structure depends upon the organism, pH, anions in the medium, and method of reduction. One type of structure has a single exchangeable Mo-OH proton approximately in the equatorial plane and a large isotropic hfi (e.g., low pH form of chicken SOE, low pH form of plant SOE reduced by Ti(iii)); the second type has two exchangeable protons with distributed orientations out of the equatorial plane and very small (or zero) isotropic hfi (e.g., high pH form of chicken SOE, high pH form of plant SOE reduced by sulfite); the third type has no nearby exchangeable protons and a coordinated oxyanion (e.g., phosphate inhibited chicken SOE, low pH form of plant SOE reduced by sulfite). An additional structural conclusion is that the orientation angle of any exchangeable equatorial ligand (OH, OH(2), PO(4)(3-)) is not uniquely fixed, but is distributed around its central value by up to +/-20 degrees (depending on pH, the type of the ligand and the type of enzyme). An unexpected finding was that the axial oxo group of SOEs exchanges with (17)O in solutions enriched in H(2)(17)O. The first determination of oxo (17)O nqi parameters for a well-characterized model compound, [Mo(17)O(SPh)(4)](-), clearly demonstrated that (17)O nqi parameters can distinguish between oxo and OH(2) ligands.

Animals↗

PubMed2006

Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle.

Pyruvate ferredoxin oxidoreductase (PFOR) is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. In the PFOR reaction, TPP reacts with pyruvate to form lactyl-TPP, which undergoes decarboxylation to form a hydroxyethyl-TPP (HE-TPP) intermediate. One electron is then transferred from HE-TPP to one of the three [4Fe-4S] clusters to form an HE-TPP radical and a [4Fe-4S]1+ intermediate. Pulsed EPR methods have been used to measure the distance between the HE-TPP radical and the [4Fe-4S]1+ cluster to which it is coupled. Computational analysis including the PFOR crystal structure and the spin distribution in the HE-TPP radical and in the reduced [4Fe-4S] cluster demonstrates that the distance between the HE-TPP radical and the medial cluster B matches the experimentally determined dipolar interaction, while one of the other two clusters is too close and the other is too far away. These results clearly demonstrate that it is the medial cluster (cluster B) that is reduced. Thus, rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step.

Catalysis↗

PubMed2005

Probing the water coordination of protein-targeted MRI contrast agents by pulsed ENDOR spectroscopy.

A novel methodology based on electron-nuclear double resonance (ENDOR) spectroscopy is used for the direct determination of the water coordination number (q) of gadolinium-based magnetic resonance imaging (MRI) contrast agents. Proton ENDOR spectra can be obtained at approximately physiological concentrations for metal complexes in frozen aqueous solutions either in the presence or absence of protein targets. It is shown that, depending on the structure of the co-ligand, the water hydration number of a complex in aqueous solution can be significantly different to when the complex is noncovalently bound to a protein. From the ENDOR spectra of the exchangeable protons, precise information on the metal-proton distance can be derived as well. These essential parameters directly correlate with the efficacy of MRI contrast agents and should therefore aid the development of novel, highly efficient compounds targeted to various proteins.

Contrast Media↗

PubMed2005

Pulsed EPR investigations of systems modeling molybdenum enzymes: hyperfine and quadrupole parameters of oxo-17O in [Mo 17O(SPh)4]-.

Ka band ESEEM spectroscopy was used to determine the hyperfine (hfi) and nuclear quadrupole (nqi) interaction parameters for the oxo-17O ligand in [Mo 17O(SPh)4]-, a spectroscopic model of the oxo-Mo(V) centers of enzymes. The isotropic hfi constant of 6.5 MHz found for the oxo-17O is much smaller than the values of approximately 20-40 MHz typical for the 17O nucleus of an equatorial OH(2) ligand in molybdenum enzymes. The 17O nqi parameter (e2qQ/h = 1.45 MHz, eta approximately = 0) is the first to be obtained for an oxo group in a metal complex. The parameters of the oxo-17O ligand, as well as other magnetic resonance parameters of [Mo 17O(SPh)4]- predicted by quasi-relativistic DFT calculations, were in good agreement with those obtained in experiment. From the electronic structure of the complex revealed by DFT, it follows that the SOMO is almost entirely molybdenum d(xy) and sulfur p, while the spin density on the oxo-17O is negative, determined by spin polarization mechanisms. The results of this work will enable direct experimental identification of the oxo ligand in a variety of chemical and biological systems.

Electron Spin Resonance Spectroscopy↗

PubMed2005

The pathogenic human sulfite oxidase mutants G473D and A208D are defective in intramolecular electron transfer.

Mutations G473D and A208D were identified in patients with isolated sulfite oxidase (SO) deficiency, and the equivalent amino acids (G451 and A186, respectively) have been localized to the vicinity of the molybdopterin active site in the X-ray structure of chicken SO [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garrett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983]. To assess the effects of these mutations in human SO, steady-state kinetic studies of enzyme turnover and laser flash photolysis measurements of intramolecular electron transfer (IET) rate constants between the reduced heme [Fe(II)] and Mo(VI) centers were carried out in the recombinant G473D, G473A, G473W, G473D/R212A, and A208D human SO mutants. In the G473D and A208D mutants, the IET rate constants at pH 6.0 are decreased by 3 orders of magnitude relative to that of the wild type. Steady-state kinetic measurements indicate that the IET process is the rate-limiting step in the catalytic cycle of these two mutants. Thus, the large decreases in the IET rate constants and the kcat values, and the large increases in the Km(sulfite) values, rationalize the fatal impact of these mutations. Far-UV CD spectra of G473D indicate that the protein backbone conformation is remarkably changed, and the sedimentation equilibrium indicates that the protein is monomeric. Furthermore, EPR studies also suggest that the active site structure of the Mo(V) form of A208D is different from that of the wild type. In contrast, similar studies on G473A show that it is dimeric, that its Mo(V) active site structure is similar to that of the wild type, and that its IET rate constant is only 2.6-fold smaller than that of the wild type. IET in G473W is severely impaired, and no IET is observed for G473D/R212A. In chicken SO, the equivalent residues (G451 and A186) are both buried inside the protein. Thus, for human SO, the mutations to charged residues at the equivalent sites most likely cause crucial global or localized structural changes, and expose an alternative docking site that may compete with the Mo domain for docking of the heme, thereby retarding IET and efficient catalytic turnover of the sulfite oxidation reaction.

Biopolymers↗

PubMed2005

Pulsed EPR studies of a bacterial sulfite-oxidizing enzyme with pH-invariant hyperfine interactions from exchangeable protons.

Variable-frequency pulsed electron paramagnetic resonance studies of the molybdenum(V) center of sulfite dehydrogenase (SDH) clearly show couplings from nearby exchangeable protons that are assigned to a Mo(V)OH(n) group. The hyperfine parameters for these exchangeable protons of SDH are the same at both low and high pH and similar to those for the high-pH forms of sulfite oxidases (SOs) from eukaryotes. The SDH proton parameters are distinctly different from the low-pH forms of chicken and human SO.

Animals↗

PubMed2005

Structures of the Mo(V) forms of sulfite oxidase from Arabidopsis thaliana by pulsed EPR spectroscopy.

The Mo(V) center of plant sulfite oxidase from Arabidopsis thaliana (At-SO) has been studied by continuous wave and pulsed EPR methods. Three different Mo(V) EPR signals have been observed, depending on pH and the technique used to generate the Mo(V) oxidation state. At pH 6, reduction by sulfite followed by partial reoxidation with ferricyanide generates an EPR spectrum with g-values similar to the low-pH (lpH) form of vertebrate SOs, but no nearby exchangeable protons can be detected. On the other hand, reduction of At-SO with Ti(III) citrate at pH 6 generates a Mo(V) signal with large hyperfine splittings from a single exchangeable proton, as is typically observed for lpH SO from vertebrates. Reduction of At-SO with sulfite at high pH generates the well-known high-pH (hpH) signal common to all sulfite oxidizing enzymes. It is proposed that, depending on the conformation of Arg374, the active site of At-SO may be in "closed" or "open" forms that differ in the degree of accessibility of the Mo center to substrate and water molecules. It is suggested that at low pH the sulfite-reduced At-SO has coordinated sulfate and is in the "closed form". Reoxidation to Mo(V) by ferricyanide leaves bound sulfate trapped at the active site, and consequently, there are no ligands with exchangeable protons. Reduction with Ti(III) citrate injects an electron directly into the active site to generate the [Mo(V)[triple bond]O(OH)]2+ unit that is well-known from model chemistry and which has a single exchangeable proton with a large isotropic hyperfine interaction. At high pH, the active site is in the "open form", and water can readily exchange into the site to generate the hpH SO.

Arabidopsis↗

PubMed2005

Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A(parallel) approximately 200 G, g(parallel) approximately 2.186, g(perpendicular) approximately 2.051). The pulsed EPR data show that one of the ligands to Cu2+ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu2+ ion is observed by pulsed electron-nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.

Binding Sites↗

PubMed2005

Determination of the g-tensors and their orientations for cis,trans-(L-N2S2)Mo(V)OX (X = Cl, SCH2Ph) by single-crystal EPR spectroscopy and molecular orbital calculations.

A single-crystal study of cis,trans-(L-N2S2)MoVOCl (1) doped into cis,trans-(N2S2)MoVIO2 (3) has enabled the g-tensor of 1 and its orientation with respect to the molecular structure to be determined. The EPR parameters (g1, 2.004; g2, 1.960; g3, 1.946; A1, 71.7 x 10(-4) cm(-1); A2, 11.7 x 10(-4) cm(-1); A3, 32.0 x 10(-4) cm(-1)) of cis,trans-(L-N2S2)MoVOCl [L-N2S2H2 = N,N'-dimethyl-N,N'-bis(mercaptophenyl)ethylenediamine] mimic those of the low-pH form of sulfite oxidase and the "very rapid" species of xanthine oxidase. The principal axis that corresponds to g1 is rotated approximately 10 degrees from the Mo[triple bond]O vector, while the principal axis that corresponds to g3 is located in the equatorial plane and approximately 38 degrees from the Mo-Cl vector. Independent theoretical calculations of the g-tensor of 1 were performed using two types of techniques: (1) the spectroscopically parametrized intermediate neglect of differential overlap technique (INDO/S) combined with single-excitation configuration interaction (CIS); (2) a scalar relativistic DFT (BP86 and B3LYP functionals) treatment using the zeroth order regular approximation to relativistic effects (ZORA) in combination with recently developed accurate multicenter mean field spin-orbit operators (RI-SOMF) and the estimation of solvent effects using dielectric continuum theory at the conductor-like screening model (COSMO) level. The excellent agreement between experiment and theory, as well as the high consistency between the INDO/S and BP86/ZORA results, provides a sound basis for analysis of the calculated orientation of the g-tensor for cis,trans-(L-N2S2)MoVO(SCH2Ph) (2), for which single-crystal EPR data are not available but which contains three equatorial sulfur donor atoms, as occurs in sulfite oxidase and xanthine oxidase. The implications of these results for the EPR spectra of the Mo(V) centers of enzymes are discussed.

Electron Spin Resonance Spectroscopy↗

PubMed2005

17O ESEEM evidence for exchange of the axial oxo ligand in the molybdenum center of the high pH form of sulfite oxidase.

A 17O ESEEM investigation of the high pH form of chicken sulfite oxidase using hyperfine sublevel correlation (HYSCORE) spectroscopy at 29.25 GHz has revealed a new type of exchangeable 17O ligand that is characterized by a significantly smaller hyperfine interaction ( approximately 5 MHz) than that previously detected by CW EPR. This new type of exchangeable oxygen ligand is assigned to the axial oxo group of the Mo(V) center.

Electron Spin Resonance Spectroscopy↗

PubMed2003

The gadolinium(III)-water hydrogen distance in MRI contrast agents.

The ion-nuclear distance of Gd(III) to a coordinated water proton, r(Gd)(-)(H), is central to the understanding of the efficacy of gadolinium-based MRI contrast agents. The dipolar relaxation mechanism operative for contrast agents has a 1/r(6) dependence. Estimates in the literature for this distance span 0.8 A (2.5-3.3 A). This study describes a direct determination of r(Gd)(-)(H) using the anisotropic hyperfine constant T( perpendicular ) determined from pulsed ENDOR spectra. Five Gd(III) complexes were examined: [Gd(H(2)O)(8)](3+), [Gd(DTPA)(H(2)O)](2)(-), [Gd(BOPTA)(H(2)O)](2)(-), MS-325, and [Gd(HP-DO3A)(H(2)O)]. The distance, r(Gd)(-)(H), was the same within error for all five complexes: 3.1 +/- 0.1 A. These distance estimates should aid in the design of new contrast agents, and in the interpretation of other molecular factors influencing relaxivity.

Algorithms↗

PubMed2003

Optically active 4- and 5-coordinate transition metal complexes of bifurcated dipeptide Schiff bases.

Symmetrical and unsymmetrical benzophenone Schiff bases of bifurcated dipeptides [e.g., Ar2C=N-CHR1- CONH-HNCO-CHR2-N=CAr2] have been synthesized using Boc methodology. These ligands may be regarded as chiral porphyrin mimics because of the alpha-carbons of the amino acids. The Schiff bases function as effective ligands for transition metals, particularly the late transition metals Ni(II), Cu(II), and Zn. Upon metal insertion, there is loss of the amide protons, resulting in N4 chelating ligands that retain the amino acid based chirality as well as newly generated metal-centered chirality, which for the Ni(II) complexes have been shown by X-ray analysis to be lambda (left-handed helix) if the amino acids are S. For Ni(II) and Cu(II), metal insertion results in highly colored complexes and is easily followed by UV-vis spectrophotometry. Several Ni(II) complexes were also characterized by 1H NMR. Co(II) and Mn(II) complexes were characterized by CW EPR. Two Cu(II) complexes, 7f.CuII and 7k.CuII, were characterized by EPR (ENDOR and ESEEM), which clearly showed the pentacoordinate nature of 7k.CuII.

Journal Article↗

PubMed2002

Pulsed EPR studies of the exchangeable proton at the molybdenum center of dimethyl sulfoxide reductase.

Electron spin echo envelope modulation (ESEEM) spectroscopy has been used to determine the hyperfine ( hfi) and quadrupole ( nqi) interactions of the exchangeable deuteron (proton) at the Mo(V) site of DMSO reductase. The data obtained have been translated into structure-related parameters. It was found that isotropic hfi constant of the proton is not unique, but is distributed within a range of 26-36 MHz. From this hfi distribution, a 30 degrees -wide distribution of the OH bond orientations due to a rotation around the Mo-O bond was estimated. The angle between the axes of the nqi and anisotropic hfi tensors was found to be anomalously small in comparison with that expected from the Mo-O-D bond geometry. This peculiarity was attributed to the effect of spin density on the hydroxyl oxygen atom. The orientation of the Mo-OH fragment with respect to the g-frame was determined from the experimental orientations of the nqi and hfi tensor axes and a theoretical evaluation of the anisotropic hfi axis direction.

Deuterium↗

PubMed2002

Pulsed EPR studies of nonexchangeable protons near the Mo(V) center of sulfite oxidase: direct detection of the alpha-proton of the coordinated cysteinyl residue and structural implications for the active site.

Pulsed electron nuclear double resonance (ENDOR) spectra of nonexchangeable protons in the vicinity of the Mo(V) center of the high pH (hpH) and low pH (lpH) forms of native chicken liver sulfite oxidase (SO) and recombinant human SO have been obtained and analyzed for the first time. The close similarity of the spectra for the chicken and human enzymes indicates that the structures of their molybdenum centers are essentially identical. For lpH SO, the closest nonexchangeable proton is found to be approximately 2.8 A from the Mo atom. To more accurately determine the distance to this proton and facilitate its assignment, the C-band electron spin-echo envelope modulation (ESEEM) spectra of lpH SO were also analyzed. From the obtained distance and comparison with the X-ray structure, this closest nonexchangeable proton is assigned to the alpha-proton of the coordinated conserved cysteinyl residue (Cys185 in chicken, Cys207 in human). The closest Mo...H distance for the nonexchangeable protons of hpH SO is found to be approximately 3.3 A. For the cysteinyl alpha-proton, such an increase in the Mo...H distance only requires a very small change in torsional angles. This study demonstrates that details of the enzyme structural rearrangements with pH can be monitored by ENDOR spectroscopy and suggests that a similar approach may be routinely used to probe the orientation of the coordinated cysteinyl residue in mutant forms of SO that are catalytically compromised.

Animals↗

PubMed2002

Models of the low-spin iron(III) hydroperoxide intermediate of heme oxygenase: magnetic resonance evidence for thermodynamic stabilization of the d(xy) electronic state at ambient temperatures.

The (13)C pulsed ENDOR and NMR study of [meso-(13)C-TPPFe(OCH(3))(OO(t)Bu)](-) performed in this work shows that although the unpaired electron in low-spin ferrihemes containing a ROO(-) ligand resides in a d(pi) orbital at 8 K, the d(xy) electron configuration is favored at physiological temperatures. The variable temperature NMR spectra indicate a dynamic situation in which a heme with a d(pi) electron configuration and planar porphyrinate ring is in equilibrium with a d(xy) electron configuration that has a ruffled porphyrin ring. Because of the similarity in the EPR spectra of the hydroperoxide complexes of heme oxygenase, cytochrome P450, and the model heme complex reported herein, it is possible that these two electron configurations and ring conformations may also exist in equilibrium in the enzymatic systems. The ruffled porphyrinate ring would aid the attack of the terminal oxygen of the hydroperoxide intermediate of heme oxygenase (HO) on the meso-carbon, and the large spin density at the meso-carbons of a d(xy) electron configuration heme suggests the possibility of a radical mechanism for HO. The dynamic equilibrium between the ruffled (d(xy)) and planar (d(pi)) conformers observed in the model complexes also suggests that a flexible heme binding cavity may be an important structural motif for heme oxygenase activity.

Carbon Isotopes↗

PubMed2001

Pulsed ELDOR spectroscopy of the Mo(V)/Fe(III) state of sulfite oxidase prepared by one-electron reduction with Ti(III) citrate.

The titration of chicken liver sulfite oxidase (SO) with the one-electron reductant Ti(III) citrate, at pH 7.0, results in nearly quantitative selective reduction of the Mo(VI) center to Mo(V), while the b-type heme center remains in the fully oxidized Fe(III) state. The selective reduction of the Mo(VI/V) couple has been established from electronic and EPR spectra. The electronic spectrum of the Fe(III) heme center is essentially unchanged during the titration, and the continuous wave (CW)-EPR spectrum shows the appearance of the well-known Mo(V) signal due to the low pH ( lpH) form of SO. Further confirmation of the selective formation of the Mo(V)/Fe(III) form of SO is provided by the approximately 1:1 ratio of the integrated intensities of the Mo(V) and low-spin Fe(III) EPR signals after addition of one equivalent of Ti(III). The selective generation of the Mo(V)/Fe(III) form of SO is unexpected, considering that previous microcoulometry and flash photolysis investigations have indicated that the Mo(VI/V) and Fe(III/II) couples of SO have similar reduction potentials at pH 7. The nearly quantitative preparation of the one-electron reduced Mo(V)/Fe(III) form of SO by reduction with Ti(III) has enabled the interaction between these two paramagnetic metal centers, which are linked by a flexible loop with no secondary structure, to be investigated for the first time by variable-frequency pulsed electron-electron double resonance (ELDOR) spectroscopy. The ELDOR kinetics were obtained from frozen solutions at 4.2 K at several microwave frequencies by pumping on the narrow Mo(V) signal and observing the effect on the Fe(III) primary echo at both higher and lower frequencies within the microwave C-band region. The ELDOR data indicate that freezing the solution of one-electron reduced SO produces localized regions where the concentration of SO approaches that in the crystal structure, which results in the interpair interactions being the dominant dipolar interaction. However, thorough analysis of the ELDOR decay curves and simulations suggests a distribution of intramolecular Mo...Fe distances, consistent with the proposal of multiple conformations in solution for the flexible loop that connects the Mo and heme domains of SO.

Animals↗