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S V Khangulov

Publications and source records attributed to S V Khangulov.

At least 19 recordsLinked to original sources
PubMed2000

"Bridging hydroxide effect" on mu-carboxylato coordination and electrochemical potentials of bimetallic centers: Mn2(II,II) and Mn2(III,III) complexes as functional models of dimanganese catalases.

Synthesis, solution structures, and electrochemistry of several dinuclear Mn2(II,II) complexes (1-4) and Mn2(III,III) complexes (6 and 8), derived from a functional catalase mimic [(L1,2)Mn2(II,II)(mu 13-O2CCH3)]2+ (1) are described that enable testing of the role of intramolecular hydroxide ligands on the redox properties. Addition of 1 equiv of hydroxide to 1 or 3 forms [(L1,2)Mn2(II,II)(mu 13-O2CCH3)(mu- OH)]+ (7A or 7B, respectively), possessing two six-coordinate Mn(II) ions bridged by hydroxide and acetato ligands. Two-electron oxidation of 7 with O2 occurs by forming [(L1,2)Mn2(III,III)(mu 1,3-O2CCH3)(mu-OH)]3+ (8) and H2O2 with no ligand rearrangements in methanol. Reaction of 8 with 2-3 equiv hydroxide forms [(L1,2)Mn2(III,III)(mu-O)(OH)(O2CCH3)]+ in which deprotonation of mu-OH- to yield mu-O2- favors subsequent addition of a terminal hydroxide ligand, accommodated by a bridging-to-terminal "carboxylate-shift". Preservation of six-coordinate Mn(II) ions throughout all hydroxide-induced transformations is observed, including oxidation by O2. Cyclic voltammetry reveals that addition of mu-OH- converts the two-electron redox couple II,II/III,III for complexes 1-4 to sequential one-electron couples at lower reduction potentials, yielding substantial stabilization of the II,III and III,III oxidation states by delta E = 440 and 730 mV, respectively. Binding of a second OH- to 7A or 7B forms (L1,2)Mn2(II,II)(mu 13-O2CCH3)(OH)2, containing two six-coordinate Mn(II) ions with two terminal hydroxides and a mu 1,3-bridging acetato. Electrochemistry reveals that displacement of the bridging hydroxide to a terminal site upon addition of the second OH- restores a two-electron redox couple II,II/III,III but now at a higher reduction potential with considerable loss of the electrochemical stabilization energy provided by the mu-OH- (delta E = 250 and 350 mV loss for Mn2(II,II) and Mn2(III,III), (respectively). These results indicate a considerably stronger influence of bridging vs terminal hydroxide ligands in stabilizing the higher oxidation states and separating the one-electron redox potentials of bimetallic centers. By contrast, in the absence of mu-OH- bridges the longer separation with the mu 1,3-carboxylato bridge in dimanganese(II,II) complexes leads to nearly complete uncoupling of the Mn(II) oxidation potentials, thus yielding a two-electron redox transition to (III,III). We hypothesize that this "bridging hydroxide effect" may be due to both greater screening of the repulsive intermetallic electric potential energy and increased resonance stabilization of the mixed-valence (II,III) oxidation state by charge delocalization . These data provide a physicochemical basis for interpretation of the catalase activity of these complexes and of dimanganese catalase enzymes (see the following manuscript).

Catalase↗
PubMed1998

L-arginine binding to liver arginase requires proton transfer to gateway residue His141 and coordination of the guanidinium group to the dimanganese(II,II) center.

Rat liver arginase contains a dimanganese(II,II) center per subunit that is required for catalytic hydrolysis of l-arginine to form urea and l-ornithine. A recent crystallographic study has shown that the Mn2 center consists of two coordinatively inequivalent manganese(II) ions, MnA and MnB, bridged by a water (hydroxide) molecule and two aspartate residues [Kanyo et al. (1996) Nature 383, 554-557]. A conserved residue, His141, is located near the proposed substrate binding region at 4.2 A from the bridging solvent molecule. The present EPR studies reveal that there is no essential alteration of the Mn2 site upon mutation of His141 to an Asn residue, which lacks a potential acid/base residue, while the catalytic activity of the mutant enzyme is 10 times lower vs wild-type enzyme. The binding affinity of l-lysine, l-arginine (substrate), and Nomega-OH-l-arginine (type 2 binders) increases inversely with the pKa of the side chain. Binding of l-lysine is more than 10 times weaker, and the substrate Michaelis constant (Km) is >6-fold greater (weaker binding) in the His141Asn mutant than in wild-type arginase. L-Lysine and Nomega-OH-L-arginine, type 2 binders, induce extensive loss of the EPR intensity, suggesting direct coordination to the Mn2 center. From these data and the pH dependence of type 2 binders, we conclude that His141 functions as the base for deprotonation of the side-chain amino group of L-lysine and the substrate guanidinium group, -NH-C(NH2)2+ and that the unprotonated side chain of these amino acids is responsible for binding to the active site. A different class of inhibitors (type 1), including L-isoleucine, L-ornithine, and L-citrulline, suppresses enzymatic activity, producing only minor change in the zero-field splitting of the Mn2 EPR signal and no change in the EPR intensity, suggestive of minimal conformational transformation. We propose that type 1 alpha-amino acid inhibitors do not bind directly to either Mn ion, but interact with the recognition site on arginase for the alpha-aminocarboxylate groups of the substrate. A new mechanism for the arginase-catalyzed hydrolysis of L-arginine is proposed which has general relevance to all binuclear hydrolases: (1) Deprotonation of substrate l-arginine(H+) by His141 permits entry of the neutral guanidinium group into the buried Mn2 region. Binding of the substrate imino group (>C=NH), most likely to MnB, is coupled to breaking of the MnB-(mu-H2O) bond, forming a terminal aquo ligand on MnA. (2) Proton transfer from the terminal MnA-aqua ligand to the substrate Ndelta-guanidino atom forms the nucleophilic hydroxide on MnA and the cationic NdeltaH2+-guanidino leaving group. Protonation of the substrate -NdeltaH2+-group is likely assisted by hydrogen bonding to the juxtaposed anionic carboxylate group of Glu277. (3) Attack of the MnA-bound hydroxide at the electrophilic guanidino C-atom forms a tetrahedral intermediate. (4) Formation of products is initiated by cleavage of the Cepsilon-NdeltaH2+ bond, yielding urea and L-ornithine(H+).

Animals↗
PubMed1998

Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer.

Formate dehydrogenase H, FDH(Se), from Escherichia coli contains a molybdopterin guanine dinucleotide cofactor and a selenocysteine residue in the polypeptide. Oxidation of 13C-labeled formate in 18O-enriched water catalyzed by FDH(Se) produces 13CO2 gas that contains no 18O-label, establishing that the enzyme is not a member of the large class of Mo-pterin-containing oxotransferases which incorporate oxygen from water into product. An unusual Mo center of the active site is coordinated in the reduced Mo(IV) state in a square pyramidal geometry to the four equatorial dithiolene sulfur atoms from a pair of pterin cofactors and a Se atom of the selenocysteine-140 residue [Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C., and Sun, P. D. (1997) Science 275, 1305-1308]. EPR spectroscopy of the Mo(V) state indicates a square pyramidal geometry analogous to that of the Mo(IV) center. The strongest ligand field component is likely the single axial Se atom producing a ground orbital configuration Mo(dxy). The Mo-Se bond was estimated to be covalent to the extent of 17-27% of the unpaired electron spin density residing in the valence 4s and 4p selenium orbitals, based on comparison of the scalar and dipolar hyperfine components to atomic 77Se. Two electron oxidation of formate by the Mo(VI) state converts Mo to the reduced Mo(IV) state with the formate proton, Hf+, transferring to a nearby base Y-. Transfer of one electron to the Fe4S4 center converts Mo(IV) to the EPR detectable Mo(V) state. The Y- is located within magnetic contact to the [Mo-Se] center, as shown by its strong dipolar 1Hf hyperfine couplings. Photolysis of the formate-induced Mo(V) state abolishes the 1Hf hyperfine splitting from YHf, suggesting photoisomerizaton of this group or phototransfer of the proton to a more distant proton acceptor group A-. The minor effect of photolysis on the 77Se-hyperfine interaction with [77Se] selenocysteine suggests that the Y- group is not the Se atom, but instead might be the imidazole ring of the His141 residue which is located in the putative substrate-binding pocket close to the [Mo-Se] center. We propose that the transfer of Hf+ from formate to the active site base Y- is thermodynamically coupled to two-electron oxidation of the formate molecule, thereby facilitating formation of CO2. Under normal physiological conditions, when electron flow is not limited by the terminal acceptor of electrons, the energy released upon oxidation of Mo(IV) centers by the Fe4S4 is used for deprotonation of YHf and transfer of Hf+ against the thermodynamic potential.

Binding Sites↗
PubMed1997

Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster.

Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe4S4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo(VI), Fe4S4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV), Fe4S4(red)] form have been determined, revealing a four-domain alphabeta structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys140 and His141 in proton abstraction and the molybdenum, molybdopterin, Lys44, and the Fe4S4 cluster in electron transfer.

Binding Sites↗
PubMed1996

Characterization of crystalline formate dehydrogenase H from Escherichia coli. Stabilization, EPR spectroscopy, and preliminary crystallographic analysis.

The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.

Crystallography, X-Ray↗
PubMed1996

Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri.

NADP(+)-coupled nicotinic acid hydroxylase (NAH) has been purified to near-homogeneity from Clostridium barkeri by an improved purification scheme that allowed the isolation of milligram amounts of enzyme of higher specific activity then previously reported. NAH is most stable at alkaline pH in the presence of glycerol. The protein which consists of four dissimilar subunits occurs in forms of different molecular masses. There are 5-7 Fe, 1 FAD, and 1 Mo per 160 kDa protein promoter. Mo in the enzyme is bound to a dinucleotide form of molybdopterin and is coordinated with selenium. Mo(V), flavin radical, and two Fe2S2 clusters could be observed with EPR spectroscopy. The Se cofactor which is essential for nicotinic acid hydroxylase activity could be released from NAH as a reactive low molecular weight compound by a number of denaturing procedures. Parallel losses of Se and catalytic activity were observed during purification and storage of the enzyme. Addition of sodium selenide or selenophosphate did not restore the catalytic activity of the enzyme. Instead, NAH is reversibly inactivated by these compounds and also by sulfide. Cyanide, a common inhibitor of Mo-containing hydroxylases, does not affect NAH catalytic activity. The "as isolated" enzyme exhibits a Mo(V) EPR signal (2.067 signal) that was detected at early stages of purification. NAH exhibits a high substrate specificity toward electron donor substrates. The ability of a nicotinate analog to reduce NAH (disappearance of 2.067 signal) correlates with the rate of oxidation of the analog in the standard assay mixture. The properties of NAH differentiate the enzyme from known Mo-containing hydroxylases.

Amino Acid Sequence↗
PubMed1995

Determination of the metal ion separation and energies of the three lowest electronic states of dimanganese (II,II) complexes and enzymes: catalase and liver arginase.

The dimanganese (II,II) catalase from Thermus thermophilus, MnCat(II,II), arginase from rat liver, Arg(II,II), and several dimanganese(II,II) compounds, LMn2XY2, which are functional catalase mimics, all possess a pair of coupled Mn(II) ions in their catalytic sites. For each of these, we have measured by EPR spectroscopy the relative energies separating the three lowest electronic states (singlet, triplet, and quintet), described a general method for extracting the individual spectra for these states by multicomponent analysis, and determined the Mn-Mn separation. The triplet-singlet and quintet-singlet energy gaps were modeled well by fitting the temperature dependence of the EPR intensities to a Boltzmann expression for a pair of Mn(II) ions coupled by isotropic Heisenberg spin exchange (-2JS1S2). This dependence indicates diamagnetic ground states with delta E10 (cm-1) = magnitude of 2J = 4 and 11.2 cm-1 for Arg-(II,II)(+borate) and MnCat(II,II)(phosphate), respectively. This large difference in magnitude of 2J reflects either a difference in the bridging ligands or, possibly, a weaker ligand field (larger ionization potential) for the Mn(II) ions in arginase. In n-butanol/CH2Cl2 the triplet-singlet energy gaps for [LMn2(CH3CO2)](C1O4)2 (1), [LMn2(CH3CO2)3] (2), and [LMn2Cl3] (3), where HL = N,N,N',N'-tetrakis(2-methylenebenzimidazole)-1,3-diaminopropan+ ++-2-ol, are 23-24 cm-1. Comparison of the Heisenberg exchange interaction constants for more than 30 dimanganese(II,II) complexes suggests a possible bridging structure of (mu-OH)(mu-carboxylate)1-2 for MnCat(II,II), while the 3-fold weaker coupling in Arg(II,II) suggests mu-aqua in place of mu-hydroxide. EPR spectra of both the triplet and quintet electronic states were extracted and found to exhibit zero-field splittings (ZFS) and resolved 55Mn hyperfine splittings indicating spin-coupled Mn2-(II,II) species. The major ZFS interaction could be attributed to the magnetic dipole-dipole interaction between the Mn(II) ions. A linear correlation is observed between the crystallographically determined Mn-Mn distance and the ZFS of the quintet state (D2) for five dimanganese pairs for which both data sets are available. Using this correlation, the Mn-Mn distance in Arg(II,II) is predicted to be 3.36-3.57 A for the native enzyme (multiple forms) and 3.59 A for MnCat(II,II)(phosphate). Addition of the inhibitor borate to Arg(II,II) simplifies the ZFS, indicative of conversion to a single species with mean Mn-Mn separation of 3.50 A. The second metal ion in dinuclear complexes possessing a shared bridging ligand has been shown to attenuate the strength of the mu-ligand field potential, as monitored by the strength of the single ion ZFS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Coordination of selenium to molybdenum in formate dehydrogenase H from Escherichia coli.

Formate dehydrogenase H from Escherichia coli contains multiple redox centers, which include a molybdopterin cofactor, an iron-sulfur center, and a selenocysteine residue (SeCys-140 in the polypeptide chain) that is essential for catalytic activity. Here we show that addition of formate to the native enzyme induces a signal typical of Mo(V) species. This signal is detected by electron paramagnetic resonance (EPR) spectroscopy. Substitution of 77Se for natural isotope abundance Se leads to transformation of this signal, indicating a direct coordination of Se with Mo. Mutant enzyme with cysteine substituted at position 140 for the selenocysteine residue has decreased catalytic activity and exhibits a different EPR signal. Since determination of the Se content of wild-type enzyme indicates approximately 1 gram atom per mol, we conclude that it is the Se atom of the SeCys-140 residue in the protein that is coordinated directly with Mo. The amino acid sequence flanking the selenocysteine residue in formate dehydrogenase H is similar to a conserved sequence found in several other prokaryotic molybdopterin-dependent enzymes. In most of these other enzymes a cysteine residue, or in a few cases a serine or a selenocysteine residue, occurs in the position corresponding to SeCys-140 of formate dehydrogenase H. By analogy with formate dehydrogenase H in these other enzymes, at least one of the ligands to Mo should be provided by an amino acid residue of the protein. This ligand could be the Se of a selenocysteine residue, sulfur of a cysteine residue, or, in the case of a serine residue, oxygen.

Amino Acid Sequence↗
PubMed1994

Nicotinic acid hydroxylase from Clostridium barkeri: electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme.

Nicotinic acid hydroxylase from Clostridium barkeri contains selenium in an unidentified form that is dissociated as a low molecular weight compound upon denaturation of the enzyme. Other cofactors of this enzyme are molybdopterin, FAD, and iron-sulfur clusters. In the current study, we show that the enzyme, as isolated, exhibits a stable Mo(V) electron paramagnetic resonance (EPR) signal ("resting" signal) and that this signal is correlated with the selenium content and nicotinate hydroxylase activity of the enzyme. Substitution of 77Se for normal selenium isotope abundance results in splitting of the Mo(V) EPR signal of the native protein without affecting the iron signals of the FeS clusters. The Mo(V) EPR signal and nicotinic acid hydroxylase activity of enzyme isolated from cells grown in selenium-deficient medium are barely detectable. In contrast, the EPR signals of the FeS clusters, the electronic absorption spectrum, the NADPH oxidase activity, and the chromatographic behavior are changed little and are typical of active selenium-containing enzyme. An EPR signal indicative of the presence of molybdenum in the selenium-deficient enzyme also is exhibited. From these results, we conclude that a dissociable selenium moiety is coordinated directly with molybdenum in the molybdopterin cofactor and, moreover, this selenium is essential for nicotinic acid hydroxylase activity.

Bacterial Proteins↗
PubMed1990

[Determining the accumulated dose of gamma radiation in the tooth enamel].

Following ideas of Japanese and Canadian scientists the authors tried to develop practical application of dental enamel for dosimetry of gamma-radiation accumulated by an individual. Human teeth were irradiated in vitro with gamma-rays of 60Co and with x-rays, and the amount of free radicals in ++non-caries enamel was measured on an ESR spectrometer. Linear regression ESR signals on dose of radiation within an interval of 0.1-20 Gy were plotted for gamma- and x-rays with regression coefficients 0.276 and 0.693, respectively. These regressions were used for estimation of accumulated doses of gamma-radiation in 85 residents of Byelorussia and 301 residents of the Tomsk region during investigation of teeth that were removed in normal dental practice.

Adult↗
PubMed1990

[Hereditary cyanosis caused by the presence of abnormal hemoglobin M in the blood: its detection, identification and properties].

A total of 17 cases of anomalous hemoglobin M (Hb M) were detected among subjects of varying nationalities in different regions of the USSR. The methods used for identification of Hb M Saskatoon, Hb M Boston, Hb M Iwate, Hb M Hyde Park have been described, among them--electron paramagnetic resonance. Spectral characteristics, electrophoretic mobility of these Hb in pH gradient, reaction with cyanides, thermal stability, in vitro reduction with methemoglobin reductase, isolated from donor's red blood cells, have been investigated. The functional parameters (log P50 and n) have been determined for hemolysates containing anomalous hemoglobin, as well as for chromatographically pure fractions of anomalous hemoglobins. The importance of the proper diagnosis of hemoglobinosis M has been stressed.

Cyanosis↗
PubMedDate not supplied

[Reduction paths of reaction centers of photosystem I of photosynthesis].

The behaviour of ESR signal I in blue-green algae and chloroplasts is considered under various illumination and with various previous treatment. It is shown that the main process determining the kinetics of P700 plus reduction is the reaction with reduced components of the electron transfer chain between the two photosystems. If the accumulation of the products is inhibited by any way, then the reduction rate depends on the reactions with reducing agents in the incubation medium accumulated under irradiation before experiment. These exogenic reducing agents may be removed from the chloroplasts with repeated washings, and from algae-by means of the dark incubation for a long time. The relation of cyclic and noncyclic electron transfer is considered with and without artificial cyclic flow mediator.

Chloroplasts↗
PubMedDate not supplied

[Kinetics of dark reduction of P700 centers in photosynthesis].

Relationship between the rate of dark fall of ESR I signal in the chloroplasts and light intensity 710 nm was studied. It is shown that the kinetics of the signal drop at sufficiently high levels of intensity is a biphase one. The fast phase (2 sec) disappears in the presence of an inhibitor of noncyclic electron transfer. It is concluded that the kinetics of electron transport is not described within the concept of the linear chain of electron carriers and a suggestion is put forward concerning a possible mechanism of this process which depends on the participation of plastoquinone in electron transport.

Chloroplasts↗
PubMedDate not supplied

[2 types of reduction of P700+ reaction centers in the leaves of green plants].

It is shown that in the leaves of Hibiscous sp. the ESR signal I rises under weak far red light after its partial inhibition with a short white flash is slower than after adaptation to 710 nm light and short dark exposition. It is suggested that this divergence in the ESR kinetics is due to the fact that only part of reduced intermediates generated by the photosystem 2 under flash is readily available to P700+, whereas other reducing reagents slowly react with the photosystem 1 reaction centres.

Binding Sites↗
PubMedDate not supplied

[Light-dependent reactions of Mn2+ and P700+ in chloroplasts during flash illumination].

The changes of the light-induced ESR signal I and exogeneous Mn2+ ions in chloroplasts of Vicia faba plants have been studied under short flash excitation and weak background far red illumination. It is shown that Mn2+ ions reactivate the noncyclic electron transport in chloroplast previously inactivated with high tris concentrations under pulse illumination. Simultaneously with P700+ reduction the ESR signal of free Mn2+ ions decays stoichometrically to P700+ reduction.

Binding Sites↗
PubMedDate not supplied

[Effect of ferricyanide, dark adaptation and aging on the properties of the EPR I signal of chloroplasts].

Ferricyanide (10-3 M) is shown to oxidize from 25 to 50% of the total P700 pool in the dark. The ferri-ferrocyanide mixture thus obtained, acting as redox buffer, accelerates the P700+ dark reduction after continuous far red illumination is turned off or after a saturating flash. The oxidized P700 yield on the flash increase in the presence of ferricyanide as a result oxidation of species in the neighborhood of P700 centers. During aging (24 h at 2--3 degrees in the dark) the yield of flash-induced P700 oxidation also increases thus indicating the loss of cyclic electron flow in aged chloroplasts.

Chemical Phenomena↗
PubMedDate not supplied

[Spin glass type structures in chloroplasts].

The nature of discovered in [2] magnetic anisotropy of ESR signal with g approximately 3 appearing in chloroplasts at helium temperatures has been studied and clarified. It was found that a signal with these properties can arise only when dark adapted chloroplasts are cooled down to temperatures lower than 23 K in an external magnetic field (Ho greater than 2.300 Oe). The results obtained indicate that the system studied belongs to the type of "spin glasses", i. e., of the magnetically diluted structures with cooperative magnetic interaction between centres. These cooperative properties can be most distinctly observed at definite temperature and magnetic field strength values.

Chloroplasts↗