246 research outputs found
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Crystallographic, Kinetic and Computational Studies on the Reaction Mechanism of Xanthine Oxidoreductase
No full textXanthine oxidoreductase is a molybdenum-containing enzyme which catalyzes the hydroxylation on sp2 hybridized carbon centers of a broad family of substrates including purines, aldehydes and various other heterocycles. It catalyzes the sequential hydroxylation of physiological substrate hypoxanthine to uric acid. Deposition of uric acid crystals in human joints with accompanying inflammation is the major cause of gout. The production of reactive oxygen species by xanthine oxidase is implicated in the pathology of various inflammatory and cardiovascular diseases. The current study mainly involves: (1) X-ray crystallography to elucidate the orientations of various substrates at the active site of bovine xanthine oxidase. Our observation of a single dominant productive orientation of xanthine, alternative orientations of hypoxanthine, a single nonproductive orientation of guanine and the dominant nonproductive orientation of indole-3-aldehyde correlates well with different catalytic activities of xanthine oxidase with these substrates and suggests the existence of dynamic sampling of substrate orientations at enzyme active site. (2) X-ray crystallography to reveal the orientations of inhibitors arsenite and quercetin at the active site of xanthine oxidase. The binding modes of these inhibitors provide structural basis for the mechanism of inhibition and insights into inhibitor design for potential therapeutics. (3) UV-visible spectroscopy to quantitatively characterize the kinetics and catalytic specificity in the reactions of sequential hydroxylation of hypoxanthine to uric acid. We conclude that the hydroxylation of hypoxanthine by xanthine oxidase is strictly specific toward C-2 over C-8, although 6,8-dihydroxypurine is an effective substrate as xanthine both of which can be converted to uric acid by xanthine oxidase. (4) Primary deuterium kinetic isotopic effect (KIE) study on the wild-type enzyme and Gln197 mutants of R. capsulatus xanthine dehydrogenase. The small apparent KIE on kcat suggests that hydrogen transfer step is neither rate-limiting for the wild-type enzyme nor for the mutants. We identify gain-of-function Q197A and loss-of-function Q197E mutants. (5) Computation of pKa of Glu802/232 of xanthine oxidoreductase and kinetic characterization of E232Q mutant. Our data suggest that Glu802/232 has an acidic pKa upon binding of xanthine or hypoxanthine which supports its catalytic role in facilitating proton tautomerization
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Structural and Kinetic Studies of Xanthine Oxidase and the Xanthine Oxidase Family of Enzymes
No full textXanthine dehydrogenase/oxidase (XDH/XO) is a molybdenum-containing enzyme which is involved with hydroxylation a number of sp2-hybridized centers including purines, heterocycles, and aldehydes. Its main role in the cell is to convert hypoxanthine to xanthine and xanthine to uric acid. Although xanthine oxidase has been studied for decades, details of its mechanism and how the active site allows for substrate specificity and catalysis are still not completely known. In the present work, several methods have been used to investigate the mechanism of xanthine oxidase and the roles of the active site residues towards substrate binding and catalysis. 1.) The kinetic rates of bovine xanthine oxidase and variants of the homologous Rhodobacter capsulatus xanthine dehydrogenase toward various substrates have been observed. Investigation of the E232Q variant of R. capsulatus xanthine dehydrogenase revealed that removal of the ionizable Glutamate 232 resulted in a dramatic loss in activity at higher pH, as compared to wild-type enzyme, providing insight into the role of Glu 232. 2.) We used X-ray crystallography to investigate interactions of the enzyme with the slow substrate indole-3-aldehyde and the non-substrate guanine. The dominant nonproductive orientations of the molecules correlate with the observed kinetic rates. 3.) The effects of active site residues on the chemical step of the reaction were investigated utilizing kinetic isotope effect studies. With the primary deuterium isotope effects, previously described by Dr. Cao, and intrinsic isotope effects Dk, derived from the tritium isotope effect studies conducted for bovine xanthine oxidase and bacterial xanthine dehydrogenase, the extent that the chemical step is rate-limiting was calculated for each. Comparison of the enzymes with amino acid substitution variants allowed for insight into the role of the active site residues by the monitoring of changes (or lack of change) in the rate of the chemical step and it's comparison to changes in the overall rate of reaction. To examine how the molybdenum cofactor matures and is incorporated into the xanthine oxidase family of enzymes, computational structural studies have been performed looking at the enzymes involved in the sulfuration of the molybdenum cofactor and incorporation of the enzyme into members of the xanthine oxidase family of enzymes. Identification of a conserved ~125 amino acid motif was identified whose connectivity to the remainder of the polypeptide makes possible a "hinge" movement to act as a target for cofactor insertion machinery. Potential open conformations have also been computationally simulated. Homologs to NifS-4, which is involved in cofactor sulfuration, and XdhC, which is involved in sulfuration and incorporation into the apo-enzyme were analyzed and docked with bacterial xanthine dehydrogenase to provide a structural basis for how the sulfuration and incorporation of cofactor occurs
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Kinetic, Mechanistic, and Spectroscopic Studies of the Mo/Cu Containing CO dehydrogenase of Oligotropha carboxidovorans
No full textCarbon monoxide dehydrogenase from Oligotropha carboxidovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are passed intramolecularly through two [2Fe-2S] clusters and finally to a FAD cofactor. From FAD the electrons are ultimately passed to the electron transport chain of the Gram-negative organism.In the current study we have examined a variety of aspects of this enzyme in the oxidative- and reductive-half reactions and propose mechanisms for the oxidation of carbon monoxide and the proximal electron acceptor of the enzyme. First, we have identified the proximal acceptor of reducing equivalents. We have found CO dehydrogenase passes electrons directly to the quinone pool without using a cytochrome as an intermediary as had previously been proposed. This establishes a new category of redox-partner for the xanthine oxidase family of enzymes.Next, we examined the active site and find silver can be replaced for the active site copper. Cyanide effectively removes the copper and a Ag(I)-thiourea solution can reactive the enzyme, albeit at a lower turnover rate. The silver reconstitution can be verified by EPR, evident by the lack of coupling to the copper I=3/2 nucleus and in its place the sliver I=1/2 nucleus. This altered but active form of the protein is used to compare and contrast with the native copper- containing enzyme to develop a mechanism for CO oxidation.We then examined the EPR of CO dehydrogenase reduced by CO by electron nuclear double resonance spectroscopy (ENDOR). The ENDOR spectra of this state confirm that the 63,65Cu exhibits strong and almost entirely isotropic coupling, show that this coupling atypically has a positive sign, aiso = +148 MHz. When the intermediate is generated using 13CO, coupling to the 13C is observed, with aiso = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase leads us to conclude that the intermediate contains a partially reduced, Mo(V)/Cu(I), center with CO bound at the copper. We next further characterized the kinetics and mechanisms of hydrogenase activity previously reported and find CO dehydrogenase effectively catalyzes H2 oxidation to protons. This activity is found to be independent of pH and does not appear to be reversible. A new EPR signal was found and is attributed to the H2 bound state with the molybdenum in an oxidation state, Mo(V), that prevents further catalysis.Finally, we have examined the inhibition of the enzyme by n-butylisonitrile and bicarbonate. We find that n-butylisonitrile reduces the and irreversibly inhibits the enzyme as is suggested by the crystal structure and computational studies previously reported. Bicarbonate acts as an uncompetitive inhibitor, reducing vmax and Km, while also producing a new EPR signal of the bicarbonate complex
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Kinetic and Mechanistic Characterization of Formate Dehydrogenase DABG (FdsDABG) from Cupriavidus necator and NAD+-Dependent NADPH:Ferredoxin Oxidoreductase (NfnI) from Pyrococcus furiosus
No full textFlavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are utilized by all three domains of life. The versatility of the flavin cofactor allows for many complicated reductive and oxidative reactions to occur within the cell. Being able to transfer one or two electrons at a time is advantageous when used for metabolic pathway. Many redox-active enzymes function via a ping-pong mechanism allowing for the reductive and the oxidative half-reactions to be studied separately to characterize the observed rate of electron transfer within the enzyme. This present work focuses on the kinetic and mechanistic characterization of two distinct types of flavoprotein, the cytosolic formate dehydrogenase (FdsDABG) from Cupriavidus necator and the NAD+-dependent NADPH:ferredoxin oxidoreductases NfnI and NfnII from Pyroccocus furiosus. Both types of systems are thought to have either been present in the last universal common ancestor (LUCA). The C. necator FdsDABG is a cytosolic enzyme that is capable of performing the reversible conversion of formate to carbon dioxide (CO2), ultimately reducing NAD+ for use in the native organism. The enzyme belongs to the DMSO reductase family of molybdenum-containing enzymes with two equivalents of a pyranopterin cofactor coordinated to the metal via an enedithiolate side chain. The remainder of the molybdenum coordination sphere consists of a cysteinate ligand provided by the polypeptide and a terminal Mo=S sulfido group that is thought accept a hydride in the course of formate oxidation. That the reaction proceeds via direct hydride transfer from formate to this catalytically essential sulfur widely accepted to be a universal trait among the metal dependent formate dehydrogenase family. Such a hydride transfer mechanism is supported by rapid reaction kinetics and electron paramagnetic resonance (EPR) studies. In addition, the kinetic studies of a histidine, that is highly conserved, variant have also been studied under similar conditions. NfnI is from the newly discovered class of flavin based electron bifurcating (FBEB) class of enzymes. This class of enzymes utilize a unique flavin site that is able to separate the two electrons accepted from a median potential donor and send them down thermodynamically distinct high- and low-potential pathways, ultimately reducing high-and low-potential acceptors, respectively. The high-potential pathway is exergonic in nature due to the more positive reduction potential of the acceptor, thus allowing the first electron to be transferred out of the bifurcating flavin to be exergonic. After electron transfer into the high-potential pathway the bifurcating flavin is left in a one-electron reduced semiquinone oxidation state that is strongly reducing due to its thermodynamically unstable nature. The low-potential electron thus generated is then transferred along the low-potential pathway to reduce the low-potential acceptor ferredoxin. It is through the generation of the unstable and strongly reducing semiquinone state that the enzyme is able to couple the reduction of the low-potential acceptor to the high-potential acceptor and drive the electron transfer from the median donor to the low-potential acceptor in a favorable fashion. The thermodynamics of NfnI are well understood, however how the enzyme is able to maintain the fidelity of the electrons and prevent low-potential electrons from being transferred into the exergonic branch is still not well understood and is the aim of the research presented. NfnII, a paralog to NfnI, from the same organism does not harbor bifurcation activity, seemingly losing the ability to pass electrons to ferredoxin. Interestingly, NfnII has also lost its NAD+/NADH specific activity due to structural changes at the S-FAD interface. The opportunity to compare a bifurcating and non-bifurcating enzyme paralogs presents a novel case to examine how discrete changes in amino acid residues can play a large role in facilitating bifurcation activity in these enzymes. Study into both these systems also harbors some mechanistic work into how bifurcation is gated as a whole within the enzyme and why the pair of electrons are not transferred into the high-potential pathway. In the present work, the rapid-reaction kinetics of these systems have been studied through a combination of UV/visible and electron paramagnetic resonance (EPR) spectroscopic methods. It is hoped that the results of this work will lay the foundation for better bio-inspired synthetic catalysts to be utilized in conjunction with renewable energy sources
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Structural Characterization and Kinetic Analysis of Formate Dehydrogenase DABG From C. Necator and Formate Dehydrogenase F From P. atrosepticum
No full textCarbon dioxide (CO2) is a potent greenhouse gas that has been building up in the Earth’s atmosphere since the beginning of the industrial revolution, resulting in anthropogenic climate change that constitutes an existential threat to human society. Adoption of renewable energies alone is likely to be insufficient to tackle this threat and current methods of capturing CO2 rely on the challenging and costly practice of burying trapped CO2 underground. In recent years, biological systems that can capture and convert CO2 to a much more practical compound have been the focus of many studies. Molybdenum-containing formate dehydrogenases are very interesting as they interconvert CO2 with the so-called feedstock chemical formate. Elucidation of the highly efficient catalytic mechanism by which enzymes catalyze this interconversion under mild conditions is expected to lead to the development of new bio-inspired catalysts, providing a means to effectively capture CO2. Moreover, such catalysts will lead to an attractive means to store energy in the form of chemical bonds. In the present work, the structure and function of the FdsDABG formate dehydrogenase from Cupriavidus necator, a cytosolic NAD+-dependent enzyme, and the FdhF formate dehydrogenase from Pectobacterium atrosepticum, an NAD+ independent formate dehydrogenase also found in the cytosol, have been investigated. Various techniques have been employed, including kinetic steady-state assays, rapid reaction kinetics, X-ray crystallography, electron paramagnetic resonance, extended X-ray absorption fine structure and electrochemical methods to investigate these formate dehydrogenases.
This work has revealed that for FdsDABG, acid/base catalysis does not have a significant impact on the mechanism of formate oxidation, consistent with this enzyme specifically catalyzing a hydride transfer reaction utilizing CO2 as the substrate for reverse catalysis. In addition, inactivation of FdsDABG in air is shown to occur through a superoxide-mediated process which can be prevented by superoxide dismutase. X-ray crystal analysis of FdsBG has yielded information regarding the position and arrangement of its redox-active cofactors, including details of the NAD+/NADH binding site. Finally, UV/Visible absorption and formate reduction experiments of FdhF confirm the recombinant enzyme’s functionality and provide insights on how the electron transfer process occurs between the molybdenum center and its sole iron-sulfur cluster. The robust findings of our investigations provide compelling evidence supporting a hydride transfer mechanism for molybdenum containing formate dehydrogenases
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Spectroscopic and Mechanistic Studies of the Mo/Cu Carbon Monoxide Dehydrogenase From Oligotropha carboxidovorans
No full textThe molybdenum- and copper-containing enzyme carbon monoxide dehydrogenase from Oligotropha carboxidovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, bioremediating about 400 million tons of CO from the atmosphere annually. During catalysis, the substrate is oxidized at a binuclear metal center containing Mo and Cu, with electrons passed via two [2Fe-2S] clusters to a FAD cofactor before ultimately being transferred to the quinone pool of the electron transport chain. Our studies have examined different aspects of catalysis, from the nature of the electron flow through the system to an examination of the binuclear center during enzyme turnover.First, we have identified the formation of the FADH● semiquinone species during catalysis. This is the first confirmed appearance of the neutral radical species in CO dehydrogenase, revealed from enzyme-monitored turnover, quench flow, and reductive titration experiments.Next, we have determined that pH effects in CO dehydrogenase are unique to the enzyme and associated with its FAD. pH jump and reductive titration experiments at pH 6 and 10 reveal pH-dependent UV/visible spectra. Upon covalent modification of the flavin by diphenyliodonium chloride, which leads to its covalent modification and inactivation at the FAD, spectral differences at the two pH extremes are abolished. Similar experiments involving xanthine oxidase and xanthine dehydrogenase show no pH-dependent spectral differences, implying that the pH effects are unique to CO dehydrogenase.Lastly, electron nuclear double resonance (ENDOR) experiments have been performed to further characterize the binuclear center of the partially-reduced enzyme. ENDOR data of 12C and 13C bicarbonate-bound enzyme reveal that bicarbonate is bound to the copper, rather than the molybdenum of the binuclear center, and so is unlikely to be an intermediate during catalysis. Analysis of 16O and 17O Mims ENDOR indicate that the equatorial ligand in the molybdenum coordination sphere is not a Mo=O but Mo-OH, and is catalytically-labile, being incorporated into the product CO2 and regenerated from solvent in the course of each catalytic sequence
Xanthine Oxidase—A Personal History
No full textA personal perspective is provided regarding the work in several laboratories, including the author’s, that has established the reaction mechanism of xanthine oxidase and related enzymes
Structure of the Molybdenum Site in YedY, a Sulfite Oxidase Homologue from <i>Escherichia coli</i>
No full textYedY from Escherichia coli is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a MoIV model complex. The K-edge energy was indicative of MoV in YedY, in agreement with X-and Q-band electron paramagnetic resonance results, whereas the hSO protein contained MoVI. In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of∼2.4 A), and one (axial) oxo ligand (Mo=O,∼1.7 A). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (∼2.1 A) or possibly to a shorter Mo-O-bond. Yet another oxygen (or nitrogen) at a∼2.6 A distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a∼2.6 Ã distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism
An extension of the Hille-Hardy formula
No full textWhile attempting to give extensions of the well-known Hille-Hardy formula for the generalized Laguerre polynomials
{
L
n
(
α
)
(
x
)
}
\{ {L_n}^{(\alpha )}(x)\}
defined by
, the author applies here certain operational techniques and the method of finite mathematical induction to derive several bilinear generating functions associated with various classes of generalized hypergeometric polynomials. It is observed that the earlier works of Brafman [2], [3], [4], Chaundy [5], Meixner [12], Weisner [16], and others quoted in the literature, are only specialized or limiting forms of the results presented here.</p
Nature of the catalytically labile oxygen at the active site of xanthine oxidase
No full textIn this paper we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized active site of xanthine oxidase at pH 6 and 10. These results indicate that the active site possesses one terminal oxygen ligand (Mo=O), two thiolate ligands (Mo-S), one terminal sulfido ligand (Mo=S), and one Mo-OH moiety. EXAFS analysis demonstrates that the Mo-OH bond shortens from 1.97 A at pH 6 to 1.75 A at pH 10, which is consistent with the generation of a Mo-O- moiety. This study provides convincing structural evidence that the catalytic oxygen donor at the oxidized active site of xanthine oxidase is Mo-OH rather than the Mo-OH2 ligation previously suggested by X-ray crystallography. These results support a mechanism initiated by base-assisted nucleophilic attack of the substrate by Mo-OH.Christian J. Doonan, Amy Stockert, Russ Hille, and Graham N. Georg
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