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R Hille

Publications and source records attributed to R Hille.

At least 55 records · Page 3Linked to original sources
PubMed1997

Spectroscopic and kinetic characterization of the recombinant cytochrome c reductase fragment of nitrate reductase. Identification of the rate-limiting catalytic step.

The recombinant NADH-cytochrome c reductase fragment of spinach NADH-nitrate reductase (EC 1.6.6.1), consisting of the contiguous heme-containing cytochrome b domain and flavin-containing NADH-cytochrome b reductase fragment, has been characterized spectroscopically and kinetically. Reductive titration with sodium dithionite indicates heme reduction takes place prior to flavin reduction, which correlates well with the reduction potentials for enzyme-bound heme (15 mV) and FAD (-280 mV). Reductive titration with NADH also indicates that the reduced enzyme forms a charge-transfer complex with NAD+. The circular dichroism spectrum of the oxidized fragment is primarily due to the flavin, whereas the ferrous heme dominates the circular dichroism spectrum of reduced enzyme. Three kinetic phases are observed in the course of the reaction of the enzyme with NADH, each with a distinct spectral signature. The fast phase represents flavin reduction, concomitant with the formation of a charge-transfer complex between reduced flavin and NAD+, and exhibits hyperbolic dependence on NADH concentration with a Kd of 3 microM and a limiting rate constant of 560 s-1. Electron transfer from reduced flavin to heme with a rate constant of 12 s-1 is the intermediate phase, which is rate-limited by breakdown of the charge-transfer complex between NAD+ and reduced flavin. The slow phase is dismutation of a pair of molecules of two-electron reduced enzyme (generated at the end of the second phase of the reaction) to give one molecule each of one- and three- electron reduced enzyme, with a second order rate constant of 2 x 10(6) M-1 s-1. In the presence of excess NADH, this dismutation reaction is followed by the rapid reaction of the one-electron reduced enzyme with a second equivalent of NADH to generate fully reduced enzyme. On the basis of this work, it appears that dissociation of NAD+ from the reduced flavin site rate limits electron transfer to the cytochrome and likely represents the overall rate-limiting step of catalysis.

Binding Sites↗
PubMed1997

An exposed tyrosine on the surface of trimethylamine dehydrogenase facilitates electron transfer to electron transferring flavoprotein: kinetics of transfer in wild-type and mutant complexes.

In wild-type trimethylamine dehydrogenase, tyrosine-442 is located at the center of a concave region on the surface of the enzyme that is proposed to form the docking site for the physiological redox acceptor, electron transferring flavoprotein. The intrinsic rate constant for electron transfer in the reoxidation of one-electron dithionite-reduced wild-type trimethylamine dehydrogenase (modified with phenylhydrazine) by electron transferring flavoprotein was investigated by stopped-flow spectroscopy. Analysis of the temperature dependence of the reaction rate by electron transfer theory yielded values for the reorganizational energy of 1.4 eV and the electronic coupling matrix element of 0.82 cm-1. The role played by residue Tyr-442 in facilitating reduction of ETF by TMADH was investigated by isolating three mutant forms of the enzyme in which Tyr-442 was exchanged for a phenylalanine, leucine, or glycine residue. Rates of electron transfer from these mutants of TMADH to ETF were investigated by stopped-flow spectroscopy. At 25 degrees C, modest reductions in rate were observed for the Y442F (1.4-fold) and Y442L (2.2-fold) mutant complexes, but a substantial decrease in rate (30.5-fold) and an elevated dissociation constant for the complex were seen for the Y442G mutant enzyme. Inspection of the crystal structure of wild-type TMADH reveals that Tyr-442 is positioned along one side of a small cavity on the surface of the enzyme: Val 344, located at the bottom of this cavity, is the closest surface residue to the 4Fe-4S center of TMADH and is likely to be positioned on a major electron transfer pathway to ETF. The reduced electron transfer rates in the mutant complexes are probably brought about by decreases in electronic coupling between the electron transfer donor and acceptor within the complex, either directly or indirectly due to unfavorable change in the orientation of the two proteins with respect to one another.

Bacteria↗
PubMed1996

Characterization of an autoreduction pathway for the [Fe4S4]3+ cluster of mutant Chromatium vinosum high-potential iron proteins. Site-directed mutagenesis studies to probe the role of phenylalanine 66 in defining the stability of the [Fe4S4] center provide evidence for oxidative degradation via a [Fe3S4] cluster.

A number of point mutations of the conserved aromatic residue phenylalanine 66 (Phe66Tyr, -Asn, -Cys, -Ser) in Chromatium vinosum high-potential iron sulfur protein have been examined with the aim of understanding the functional role of this residue. Nonconservative replacements with polar residues have a minimal effect on the midpoint potential of the [Fe4S4]3+/2+ cluster, typically < +25 mV, with a maximum change of +40 mV for Phe66Asn. With the exception of the Phe66Tyr mutant, the oxidized state was found to be unstable relative to the recombinant native, with regeneration of the reduced state. The pathway for this transformation involves degradation of the cluster in a fraction of the sample, which provides the reducing equivalents required to bring about reduction of the remainder of the sample. This degradative reaction proceeds through a transient [Fe3S4]+ intermediate that is characterized by typical g values and power saturation behavior and is prompted by the increased solvent accessibility of the cluster core in the nonconservative Phe66 mutants as evidenced by 1H-15N HMQC NMR experiments. These results are consistent with a model where the critical role of the aromatic residues in the high-potential iron proteins is to protect the cluster from hydrolytic degradation in the oxidized state.

Bacterial Proteins↗
PubMed1996

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.

Aldehyde Oxidoreductases↗
PubMed1996

Reaction of the C30A mutant of trimethylamine dehydrogenase with diethylmethylamine.

The role played by the 6-S-cysteinyl-FMN bond of trimethylamine dehydrogenase in the reductive half-reaction of the enzyme has been studied by following the reaction of the slow substrate diethylmethylamine with a C30A mutant of the enzyme lacking the covalent flavin attachment to the polypeptide. Removal of the 6-S-cysteinyl-FMN bond diminishes the limiting rate for the first of the three observed kinetic phases of the reaction by a factor of 6, but has no effect on the rate constants for the two subsequent kinetic phases. The flavin in the C30A enzyme recovered from the reaction of the C30A enzyme with excess substrate is found to have been converted to the 6-hydroxy derivative, rendering the enzyme inactive. The noncovalently bound FMN of the C30A mutant enzyme is also converted to 6-hydroxy-FMN and rendered inactive upon reduction with excess trimethylamine, but not by reduction with dithionite, even at high pH or in the presence of the effector tetramethylammonium chloride. These results suggest that one significant role of the 6-S-cysteinyl-FMN bond is to prevent the inactivation of the enzyme during catalysis. A reaction mechanism is proposed whereby OH- attacks C-6 of a flavin-substrate covalent adduct in the course of steady-state turnover to form 6-hydroxy-FMN.

Binding Sites↗
PubMed1996

The reductive half-reaction of xanthine oxidase. The involvement of prototropic equilibria in the course of the catalytic sequence.

The pH dependence and solvent isotope sensitivity of three discrete steps in the reductive half-reaction of xanthine oxidase have been investigated. The pH dependence of both kcat/Km from steady-state experiments and kred/Kdfrom rapid reaction experiments with xanthine as substrate indicate that enzyme reacts preferentially with the neutral form of substrate and that an ionizable group in the active site having a pKa of approximately 6.6 must be unprotonated for reaction to take place. The solvent kinetic isotope effect on kred/Kd is 2.4, once a uniform shift on going to D2O of approximately 1 unit for both pKa values is taken into account. The pH dependence of the formation and decay of Ered-P formed in the course the reaction of xanthine oxidase with lumazine has also been examined. Formation of this complex exhibits bell-shaped pH dependence, with pKa values of 6.5 and 7.8, consistent with the results obtained with xanthine. Decay of the Ered-P complex is base-catalyzed with a pKa > 11 and exhibits a small solvent kinetic isotope effect of 1.7 at pH/D 8.5. By contrast, the catalytic intermediate giving rise to the "very rapid" EPR signal that is transiently observed in the course of the reaction of enzyme with the substrate 2-hydroxy-6-methylpurine is found to undergo acid-catalyzed breakdown with an associated pKa < 6. Formation and decay of this species exhibit solvent kinetic isotope effects of 2.0 and 3.5 at pH 10. The results are discussed in the context of a specific reaction mechanism for the reductive half-reaction of xanthine oxidase, in which discrete ionizations associated with the molybdenum center of the active site play critical roles in determining the magnitude of the rate constants by which the Mo(IV)-P and Mo(V)-P intermediates form and decay.

Catalysis↗
PubMed1996

Resonance Raman spectroscopic identification of a histidine ligand of b595 and the nature of the ligation of chlorin d in the fully reduced Escherichia coli cytochrome bd oxidase.

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.

Cytochrome a Group↗
PubMed1996

Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL.

BACKGROUND: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two-component regulators. Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins. We have probed the size and chemistry of the FixL heme pocket by measuring the affinites, on rates and off rates for a wide variety of ferric heme ligands. Cyanide, but not fluoride, regulates the kinase activity. To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding. RESULTS: The affinities of ferric FixL for heme ligands follow the same trend as their pKa values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate. The association rates follow the reverse trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands. The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained. CONCLUSIONS: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

Azides↗
PubMed1996

Purification of xanthine dehydrogenase and sulfite oxidase from chicken liver.

Xanthine dehydrogenase and sulfite oxidase from chicken liver are oxomolybdenum enzymes which catalyze the oxidation of xanthine to uric acid and sulfite to sulfate, respectively. Independent purification protocols have been previously described for both enzymes. Here we describe a procedure by which xanthine dehydrogenase and sulfite oxidase are purified simultaneously from the same batch of fresh chicken liver. Also, unlike the protocols described earlier, this procedure avoids the use of acetone extraction as well as a heat step, thus minimizing damage to the molybdenum centers of the enzymes.

Acetone↗
PubMed1995

The reaction of chicken liver sulfite oxidase with dimethylsulfite.

We have undertaken a steady-state and rapid kinetic study of the reaction of enzyme with sulfite and dimethylsulfite. Methylation of sulfite results in a significant increase in Km and Kd for the substrate in the course of steady-state and rapid reaction kinetics, respectively, but kcat and the limiting rate constant for enzyme reduction (kred) are essentially unchanged. This indicates that while substrate oxyanion groups are effective in stabilizing the Eox.S complex, the breakdown of this complex proceeds at the same rate even in their absence. The critical element of the substrate required for reactivity is a suitable lone-pair available to undertake nucleophilic attack on a Mo = O group of the active site.

Animals↗
PubMed1995

The reaction of trimethylamine dehydrogenase with electron transferring flavoprotein.

The kinetics of electron transfer between trimethylamine dehydrogenase (TMADH) and its physiological acceptor, electron transferring flavoprotein (ETF), has been studied by static and stopped-flow absorbance measurements. The results demonstrate that reducing equivalents are transferred from TMADH to ETF solely through the 4Fe/4S center of the former. The intrinsic limiting rate constant (klim) and dissociation constant (Kd) for electron transfer from the reduced 4Fe/4S center of TMADH to ETF are about 172 s-1 and 10 microM, respectively. The reoxidation of fully reduced TMADH with an excess of ETF is markedly biphasic, indicating that partial oxidation of the iron-sulfur center in 1-electron reduced enzyme significantly reduces the rate of electron transfer out of the enzyme in these forms. The interaction of the two unpaired electron spins of flavin semiquinone and reduced 4Fe/4S center in 2-electron reduced TMADH, on the other hand, does not significantly slow down the electron transfer from the 4Fe/4S center to ETF. From a comparison of the limiting rate constants for the oxidative and reductive half-reactions, we conclude that electron transfer from TMADH to ETF is not rate-limiting during steady-state turnover. The overall kinetics of the oxidative half-reaction are not significantly affected by high salt concentrations, indicating that electrostatic forces are not involved in the formation and decay of reduced TMADH-oxidized ETF complex.

Electron Transport↗
PubMed1995

Spectroscopic and kinetic characterization of the recombinant wild-type and C242S mutant of the cytochrome b reductase fragment of nitrate reductase.

Spectroscopic and kinetic studies comparing the behavior of the recombinant cytochrome b reductase fragment of corn leaf nitrate reductase and a mutant in which cysteine 242 is replaced with a serine residue (C242S) have been carried out. The visible and circular dichroism spectra of the wild-type and mutant protein are virtually identical and compare well with those reported for nitrate reductases from other sources. The reduced wild-type protein forms a charge-transfer complex with NAD+ that has an absorption envelope that extends into the near infrared, with a maximum around 800 nm. The C242S mutant forms a similar charge-transfer complex with NAD+ but to a lesser extent than the wild-type. The reduction potential of the flavin for the wild-type protein is -287 mV, and that for the mutant is -279 mV. The rate of reduction by NADH of the C242S mutant is 7-fold slower than that for the wild-type protein, and the Kd is larger by a factor of 2. These results indicate that the cysteine 242 residue plays a role principally in facilitating electron transfer from NADH to the flavin rather than in binding of NADH to the enzyme.

Circular Dichroism↗
PubMed1995

Prototropic control of intramolecular electron transfer in trimethylamine dehydrogenase.

The pH dependence of static optical/EPR spectra of trimethylamine dehydrogenase reduced to the level of two equivalents (TMADH2eq) has been examined and indicates the existence of three different states for this iron-sulfur flavoprotein. At pH 6, TMADH2eq exists principally in a form possessing flavin mononucleotide hydroquinone, with its iron-sulfur center oxidized. At pH 8, the enzyme principally contains flavin mononucleotide semiquinone and reduced iron-sulfur, but despite the proximity of the two centers to one another, their magnetic moments do not interact. At pH 10, TMADH2eq exhibits the EPR spectrum that is diagnostic of a previously characterized spin-interacting state in which the magnetic moments of the flavin semiquinone and reduced iron-sulfur center are strongly ferromagnetically coupled. The kinetics of the interconversion of these three states have been investigated using a pH jump technique in both H2O and D2O. The observed kinetics are consistent with a reaction mechanism involving sequential protonation/deprotonation and intramolecular electron transfer events. All reactions studied show a normal solvent kinetic isotope effect. Proton inventory analysis indicates that at least one proton is involved in the reaction between pH 6 and 8, which principally controls intramolecular electron transfer, whereas at least two protons are involved between pH 8 and 10, which principally control formation of the spin-interacting state. The results of these and previous studies indicate that for TMADH2eq, between pH 10 and 6, at least three protonation/deprotonation events are associated with intramolecular electron transfer and formation of the spin-interacting state, with estimated pK alpha values of 6.0, 8.0, and approximately 9.5. These pK alpha values are attributed to the flavin hydroquinone, flavin semiquinone, and an undesignated basic group on the protein, respectively.

Bacterial Proteins↗
PubMed1995

The molybdenum centers of xanthine oxidase and xanthine dehydrogenase. Determination of the spectral change associated with reduction from the Mo(VI) to the Mo(IV) state.

The UV-visible absorbance change associated with reduction of the molybdenum centers of xanthine oxidase and xanthine dehydrogenase has been determined using a double-difference technique. At pH 8.5, the Mo(VI) minus Mo(IV) difference spectrum seen with xanthine oxidase exhibits a positive feature at 420 nm, having an extinction change of approximately 3,000 M-1 cm-1 as well as evidence for a negative feature below 340 nm. In xanthine oxidase this change is found to exhibit a marked pH dependence, implicating protonation/deprotonation events associated with changes in the molybdenum oxidation state. Application of the double-difference protocol to the respective circular dichroism spectra of xanthine oxidase and xanthine dehydrogenase reveals appreciable CD changes at 420 and 580 nm associated with the reduction of the molybdenum center. The present results demonstrate a direct spectroscopic handle on the molybdenum centers of both xanthine oxidase and xanthine dehydrogenase.

Animals↗
PubMed1995

Inactivation of xanthine oxidase by oxidative radical attack.

Steady-state radiolysis, pulse radiolysis and epr studies, combined with enzyme activity measurements, were carried out on the mechanism by which radical attack, through one-electron oxidation, inactivates xanthine oxidase. Electron transfer to both the N3 and Br2- radical species was used to initiate oxidative damage on the enzyme. Inactivation was found to occur to a greater extent at low than at high pH and is associated with the initial formation of a tryptophanyl radical which converts by a known intramolecular pathway to a tyrosyl radical with a rate constant of 5 x 10(3) S-1. The tyrosyl radical in turn slowly loses around half of its absorbance at an intramolecular rate constant of 350S-1 and is consistent with the establishment of a radical equilibrium with cysteine residue(s). The sequence of reactions could be repeated several times on the same irradiated sample implying that restitution of the implied cysteinyl radical occurs leading to other damage in the protein. N3+Trp/N-->Trp/N-->Tyr/O<-->Cys/S-->?. Epr evidence implies that inactivation of the enzyme from the above sequence of reactions arises in part from alternations to Fe/S center I in the enzyme.

Hydrogen-Ion Concentration↗
PubMed1995

Flavoprotein structure and mechanism. 4. Xanthine oxidase and xanthine dehydrogenase.

Xanthine oxidase and xanthine dehydrogenase are enzymes involved in the metabolism of purines and pyrimidines in various organisms. Their relationship to one another has been the subject of considerable debate, primarily because of their proposed roles in ischemia/reperfusion damage in tissues. Differences in the kinetics and oxidation-reduction behavior of the two forms are accounted for by the presence in the dehydrogenase of a binding site for NAD+, as well as a substantially lower reduction potential for the flavin FADH./FADH2 couple of the dehydrogenase relative to the oxidase. This review presents recent advances of our understanding of the biochemistry and molecular biology of these systems, including a model for the overall morphology of xanthine oxidizing enzymes. The evidence that the two enzymes represent alternate forms of the same gene product, in some cases reversibly interconvertible between one another, is discussed.

Animals↗