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R Hille

Publications and source records attributed to R Hille.

At least 91 records · Page 5Linked to original sources
PubMed1987

On the mechanism of action of xanthine oxidase. Evidence in support of an oxo transfer mechanism in the molybdenum-containing hydroxylases.

The mechanism of action of xanthine oxidase has been investigated using single-turnover experiments in an effort to determine the primary source of the oxygen atom incorporated into product in the course of catalysis. It is found from mass spectroscopic analysis of the uric acid generated in these experiments that when 16O-labeled enzyme in [18O]H2O is reacted with substoichiometric amounts of xanthine (under conditions where no enzyme molecule is likely to react with more than one substrate molecule), the uric acid isolated from the reaction mixture contains 16O at position 8 of the purine ring. Conversely, when 18O-labeled enzyme in [16O]H2O is exposed to substoichiometric xanthine, 18O is incorporated into the product uric acid. These results strongly support a variety of chemical studies with model molybdenum complexes suggesting that the oxygen atom of the Mo = O group known to be present at the active site of xanthine oxidase is transferred to product in the course of catalysis. The mechanistic implications of the present work are discussed.

Animals↗
PubMed1986

The radical chemistry of milk xanthine oxidase as studied by radiation chemistry techniques.

The kinetics of electron transfer within the molybdoflavoenzyme xanthine oxidase has been investigated using the technique of pulse radiolysis. Subsequent to one-electron reduction of native enzyme at 20 degrees C in 20 mM pyrophosphate buffer, pH 8.5, using the CO-.2 species as reductant, a spectral change is observed having a rate constant of approximately 290 s-1. From its wavelength dependence, this spectral change is assigned to the transfer of an electron from flavin semiquinone (formed on reaction with the CO2-. species) to one of the iron-sulfur centers of the enzyme in an intramolecular equilibration process. The value for this rate constant agrees well with the 330 s-1 observed in previous stopped-flow pH-jump experiments carried out at 25 degrees C (Hille, R., and Massey, V. (1986) J. Biol. Chem. 261, 1241-1247). Experimental results with fully reduced enzyme reacting with the radiolytically generated N.3 species also support the conclusion that the equilibration of reducing equivalents among the oxidation-reduction centers of xanthine oxidase is a rapid process. Evidence is also found that xanthine oxidase possesses an unusually reactive disulfide bond that is reduced rapidly by radiolytically generated radicals. The ramifications of the present results with regard to the interpretation of experiments involving chemically reactive radical species, generated either by photolysis or radiolysis, are discussed.

Alkylation↗
PubMed1986

The equilibration of reducing equivalents within milk xanthine oxidase.

The rate at which reducing equivalents equilibrate among the several oxidation-reduction active sites in xanthine oxidase has been investigated using a pH-jump technique in which partially reduced enzyme in dilute buffer is mixed with concentrated anaerobic buffer at a different pH in a conventional stopped flow apparatus. It is found that the rate constant associated with the observed spectral change varies with pH, doubling from 155 s-1 at pH 6 to 330 s-1 at pH 8.5, but is always found to be approximately 10-fold greater than kcat at the same pH. The observation of fast rates for the equilibration of reducing equivalents within xanthine oxidase is consistent with a great deal of indirect evidence from conventional kinetic studies of both the oxidative and reductive half-reactions of xanthine oxidase and lends support to the rapid equilibrium model that has been proposed for the oxidation-reduction interactions of the several centers in xanthine oxidase (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382). The present conclusions are in conflict, however, with the interpretation of recent flash photolysis experiments with xanthine oxidase (Battacharyya, A., Tollin, G., Davis, M. D., and Edmondson, D. E. (1983) Biochemistry 22, 5270-5279). Possible sources for the apparent inconsistencies between the flash photolysis results and those of the present experiments are discussed.

Animals↗
PubMed1985

Spectroscopic studies on the iron-sulfur centers of milk xanthine oxidase.

The optical electron paramagnetic resonance and Mössbauer spectral properties of the two iron-sulfur centers present in milk xanthine oxidase have been reexamined. It is found in the case of the optical spectral change observed on reduction of the enzyme that the two centers contribute approximately equally, with a ratio of spectral contributions for Fe/S I and Fe/S II of 0.55:0.45. This conclusion is based both on the behavior of the spectral change at wavelengths where only the two iron-sulfur centers contribute to the spectral change (under experimental conditions minimizing the effect of flavin semiquinone) during reductive titrations and a comparison of the spectra of 1- and 2-electron reduced enzyme under different conditions. This very similar spectral weighting for the two centers applies throughout the visible region. In the case of the EPR spectra, it is found from computer simulation of the signals observed under nonsaturating conditions that iron-sulfur center II exhibits g values of 1.902, 1.991, and 2.110 and does not exhibit two g values above that for the free electron, as has been reported (Lowe, J., Lynden-Bell, R.M., and Bray, R. C. (1972) Biochem. J. 130, 239-249). The g values for iron-sulfur center I obtained from the simulations are 1.894, 1.932, and 2.022. Finally, Mössbauer spectra of xanthine oxidase have been obtained, and it is found that while the two iron-sulfur centers are indistinguishable in the oxidized state, the ferrous iron in one of the reduced iron-sulfur centers exhibits an unusually large quadrupole coupling.

Animals↗
PubMed1985

The reaction of arsenite-complexed xanthine oxidase with oxygen. Evidence for an oxygen-reactive molybdenum center.

The effects of arsenite on the reaction of reduced xanthine oxidase with oxygen are determined. The kinetics of the reaction monitoring the return of enzyme absorbance are investigated as are the kinetics and stoichiometries of peroxide and superoxide formation. Although some of the effects of arsenite are qualitatively consistent with expectations based on the known perturbation of the molybdenum midpoint potentials by arsenite, several results cannot be so easily explained. Specifically, arsenite introduces a very rapid phase (kobs = 110 s-1 at 125 microM oxygen) to the oxidative half-reaction which is not observed with the native enzyme. Arsenite also diminishes the amount of superoxide produced and eliminates one-electron reduced enzyme as a detectable kinetic intermediate in the reoxidation pathway. These differences appear to result from the ability of arsenite to greatly enhance the oxygen- and/or superoxide-reactivity of the reduced molybdenum center. This is reflected in the observation that reduced forms of arsenite-complexed xanthine oxidase lacking functional FAD (iodoacetamide-alkylated enzyme and deflavo enzyme) react relatively rapidly with oxygen whereas these reactions are quite slow in the absence of arsenite.

Arsenic↗
PubMed1984

Characterization of arsenite-complexed xanthine oxidase at room temperature. Spectral properties and pH-dependent redox behavior of the molybdenum-arsenite center.

Several aspects of the interaction of xanthine oxidase with arsenite are investigated. Room temperature potentiometric titrations using EPR to monitor Molybdenum reduction reveal midpoint potentials of -225 mV for the Mo(VI)-arsenite/Mo(V)-arsenite couple and -440 mV for the Mo(V)-arsenite/Mo(IV)-arsenite couple at pH 8.3. Under the same conditions, the values for native enzyme are -395 mV and -420 mV, respectively. The predicted effects of the altered Mo(VI)/Mo(V) potential on the distributions of reducing equivalents in partially reduced enzyme are compared with the experimentally observed effects in optical experiments. The bleaching that occurs on reduction of the chromophore that is generated when arsenite binds to oxidized enzyme is characterized and found to be associated with reduction of Mo(V)-arsenite to Mo(V)-arsenite. This probe enables determination of the midpoint potential for this conversion using optical data. From such data at a series of pH values ranging from 6.15 to 9.9, a pH dependence of -60 mV/pH unit increase is determined for this couple above pH 7. The ability of arsenite to bind to reduced xanthine oxidase and to desulfo enzyme are also investigated. Reduced active enzyme binds arsenite much more tightly (Kd less than 0.1 microM) and more rapidly than does oxidized active enzyme (Kd = 8 microM); oxidized desulfo enzyme binds arsenite almost as tightly (Kd = 20 microM) as does the oxidized active enzyme.

Animals↗
PubMed1984

The inhibition of xanthine oxidase by 8-bromoxanthine.

The interaction of xanthine oxidase with the substrate analog 8-bromoxanthine has been examined in an effort to determine the nature of interaction of purines with the active site of the enzyme. It is found that 8-bromoxanthine is an inhibitor of xanthine oxidase with a Ki of approximately 400 microM; inhibition is uncompetitive with respect to xanthine and noncompetitive with respect to molecular oxygen. While 8-bromoxanthine has only a slight effect on the reaction of reduced enzyme with oxygen, it dramatically slows the rate of enzyme reduction by xanthine, suggesting that inhibition does involve the interaction of 8-bromoxanthine with the molybdenum center of the enzyme. KD determinations for binding of 8-bromoxanthine to oxidized and reduced xanthine oxidase indicate that the inhibitor binds preferentially to the fully reduced form of the molybdenum center (MoIV), with dissociation constants of 1.5 mM and 18 microM for oxidized and reduced enzyme, respectively. This preferential binding to the reduced form of the enzyme is manifested in a significant increase in the oxidation-reduction potentials of the molybdenum center as determined by potentiometric titrations with 8-bromoxanthine complexed with xanthine oxidase. The shape of the Mov EPR signal observed in the course of these titrations as well as a comparison with results of reductive titrations and KD determinations with uric acid and xanthine indicate that 8-bromoxanthine interacts with the molybdenum center of xanthine oxidase in a way that is typical of purine substrates and products, despite the presence of the bulky Br group. The inhibitor thus has a potential as a probe of enzyme-substrate interactions, particularly using the technique of x-ray absorption spectroscopy.

Animals↗
PubMed1984

Purification and characterization of the Rieske iron-sulfur protein from Thermus thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine ligands.

We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mössbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mössbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mössbauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe. Within the excellent resolution of the Mössbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues. The Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian. The hyperfine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [2Fe-2S] center.

Amino Acids↗
PubMed1983

Studies of the ferredoxin from Thermus thermophilus.

The soluble ferredoxin from Thermus thermophilus was examined by Mössbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.

Amino Acids↗
PubMed1983

The interaction of arsenite with xanthine oxidase.

The binding of arsenite to the molybdenum center of milk xanthine oxidase is re-examined. The Kd for the arsenite complex has been determined to be 24 microM from equilibrium binding studies and this value has been confirmed by determination of the association and dissociation rate constants for the interaction of arsenite with xanthine oxidase. Formation of the complex is not prevented by prior reaction of the enzyme with thiol reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) or methyl methanethiosulfonate. Binding of arsenite to the enzyme perturbs both the oxidation-reduction potentials and the electron paramagnetic resonance signal of the molybdenum center observed after partial reduction of the enzyme with sodium dithionite. The EPR signal of the partially reduced arsenite-complexed enzyme is further modified in two different ways by the addition of xanthine or salicylate. Other purine and pteridine substrates and products for the enzyme yield EPR signals indistinguishable from that generated by xanthine, whereas aromatic aldehydes and carboxylic acids give signals similar to that observed in the presence of salicylate. It is thus clear that while arsenite prevents enzyme turnover, it does not preclude binding of substrate and product molecules. Binding of arsenite at the molybdenum center of xanthine oxidase does not disturb the oxidation-reduction potentials of the iron-sulfur centers of the enzyme, but evidence is presented to suggest that the midpoint potential of the FAD site is decreased by approximately 15 mV. A structure for the arsenite complex is proposed to provide a framework in which to interpret the EPR signals in a quantitative fashion.

Animals↗
PubMed1982

The presence of a reducible disulfide bond in milk xanthine oxidase.

The stoichiometry of reducing equivalents per protomer for the complex molybdoflavoprotein xanthine oxidase has been re-examined by reductive titrations with sodium dithionite and anaerobic reoxidation with cytochrome c and phenazine methosulfate of dithionite- or photo-reduced enzyme. It is found that 8.0 +/- 0.1 reducing equivalents are taken up (or given up) by the enzyme, a value of 2 eq greater than expected on the basis of the known oxidation-reduction centers in the enzyme. The reaction of reduced xanthine oxidase with [14C]iodoacetate indicates that, in the reduced form of the enzyme, additional cysteine residues are available for reaction. These results, in conjunction with the observation that reaction of oxidized enzyme with sulfite results in the appearance of an additional equivalent of thiol capable of reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) or iodoacetate, indicate the presence of a disulfide linkage in the enzyme that can be reduced by dithionite or photochemically employing EDTA and 5-deazaflavin. Neither xanthine nor lumazine, however, is capable of reducing this oxidation-reduction center, suggesting that the disulfide does not play a role in the catalytic reactions of the enzyme. These results resolve discrepancies in the literature which indicated that greater than 6 reducing equivalents were consistently needed to bring about the complete reduction of xanthine oxidase.

Animals↗
PubMed1982

Inhibition of milk xanthine oxidase by fluorodinitrobenzene.

Milk xanthine oxidase reacted with fluorodinitrobenzene resulting in the modification of two lysine residues with a 6-fold decrease in catalytic activity. Continued reaction with fluorodinitrobenzene up to a total of 11 dinitrophenyl residues/equivalent of enzyme-bound FAD resulted in no further decrease in activity. Stopped flow studies revealed that the modification perturbed the reduction of the enzyme by xanthine; this was 6-fold lower with modified than with native enzyme. The reaction of the reduced modified enzyme with oxygen was qualitatively and quantitatively the same as with native enzyme. One nitro group of each dinitrophenyl lysine residue is slowly reduced by xanthine; reduction of both nitro groups is achieved by dithionite. The two dinitrophenyl lysine reduces can be distinguished on the basis of their kinetics of reduction. One appears to be located on the protein surface and is reduced in an intermolecular reaction, while the other appears to be located in a pocket of the enzyme and is reduced in a slow intramolecular reaction.

Animals↗
PubMed1981

Studies on the oxidative half-reaction of xanthine oxidase.

The oxidative half-reaction of xanthine oxidase is reexamined with regard to the generation of the superoxide anion. By using cytochrome c reduction to monitor superoxide, it is found that the stoichiometry of superoxide produced to enzyme reoxidized is 2:1, significantly greater than previously reported (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4350-4362). Furthermore, the kinetics of superoxide-dependent cytochrome c reduction exhibits a pronounced lag during the rapid phase of enzyme reoxidation and a limiting rate identical with that of the slow phase of enzyme reoxidation. This indicates that superoxide is generated only in the last steps of the sequential removal of reducing equivalents from the enzyme by molecular oxygen. Experiments with the two-electron-reduced enzyme indicate that it too produces two superoxide ions for each molecule of enzyme reoxidized, demonstrating that it is the last two electrons to leave the enzyme in the course of reoxidation that form superoxide. The sequential scheme for the oxidative half-reaction must therefore be 6 leads to 4 leads to 2 leads to 1 leads to 0, where the numbers reflect the number of reducing equivalents in each intermediate. Using this scheme, both enzyme reoxidation and cytochrome c reduction can be accurately simulated. Reasons for the different behavior of the two-electron-reduced xanthine oxidase compared to the six- and four-electron-reduced enzyme are discussed.

Animals↗
PubMed1979

Spectral transitions of nitrosyl hemes during ligand binding to hemoglobin.

Human deoxyhemoglobin has been titrated with nitric oxide at several pH values ranging from 6.0 to 9.0, in the presence and absence of the allosteric effector inositol hexaphosphate at 25 degrees C. Samples were frozen for EPR measurements or analyzed optically within 30 s after mixing to ensure a kinetic population of intermediates. Fractions of pentacoordinate alpha-NO heme groups were determined by fitting EPR and absorbance difference spectra in terms of linear combinations of standard signals. Equivalent results were obtained by these techniques. The fraction of alpha-NO heme exhibiting pentacoordinate character in Hb4NO increases from 0.07 to 0.73 in going from pH 9 to 6. The fraction of alpha hemes which are pentacoordinate in fully saturated nitrosyl hemoglobin, Hb4(NO), increases from 0.0 to 0.41 over the same pH range. Only in the presence of bound inositol-P6 are all 4 the alpha-NO hemes pentacoordinate. Thus, the expression of modified NO heme character is not simply a reflection of the formation of low affinity quaternary conformations. Rather, within this conformation the alpha chain iron atoms exhibit an equilibrium between hexa- and pentacoordinate structures which is perturbed markedly by both proton and phosphate binding. No intermediate coordination structure of the type suggested by Chevion et al. (Chevion, M., Stern, A., Peisach, J., Blumberg, W.E., and Simon, S. (1978) Biochemistry 17, 1745-1750) appears to occur since the observed alpha-NO heme spectra can always by represented quantitatively as a linear combination of the normal hexacoordinate and pentacoordinate signals. The formation of pentacoordinate alpha-NO causes this subunit to exhibit a higher affinity for nitric oxide. Thus on standing at low levels of saturation, there is a slow (t1/2 approximately equal to 8 min at pH 7, 25 degrees C) re-equilibration of ligand from beta to alpha subunits. The final ratio of alpha-NO to beta-NO is 2 to 1 in the absence of phosphates and greater than 10 to 1 in the presence of inositol hexaphosphate.

Electron Spin Resonance Spectroscopy↗
PubMed1977

Chain equivalence in reaction of nitric oxide with hemoglobin.

Mixtures of nitric oxide and hemoglobin were prepared in a rapid freeze apparatus and analyzed by EPR spectroscopy. Spectra from samples at various degrees of saturation showed that the two subunits bound NO at equal rates. Identical results were observed in 0.1 M phosphate at pH 6.5 and 0.1 M 2,2'-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol, 0.1 M NaCl at pH 7.0, both in the presence and absence of inositol hexaphosphate at either buffer condition. At subsaturating levels of NO (less than 60%), or at all levels of saturation in the presence of inositol hexaphosphate, it was found that the EPR spectrum of nitrosylhemoglobin varied with the length of time before freezing. This change was characterized by the development of a hyperfine structure at g = 2.01 which appeared with a half-time of approximately 0.4 s. Maxwell and Caughey (Maxwell, J. C., and Caughey, W. S. (1976) Biochemistry 15, 388-395) have attributed this three-line EPR hyperfine structure to the formation of a pentacoordinate ferroheme-NO complex. Corresponding slow changes were observed in the visible absorption spectrum following the binding of low levels of NO to deoxyhemoglobin or inositol hexaphosphate to fully saturated nitrosylhemoglobin. Thus it appears that NO binding to the alpha and beta subunits of deoxyhemoglobin takes place at equal rates and, under conditions favoring the T quaternary state (low saturation, presence of inositol hexaphosphate), a further slow structural change takes place, resulting in the cleavage of the iron--proximal histidine bond.

Electron Spin Resonance Spectroscopy↗