RESUMEN
Hydrogenases (H2 ase) catalyze the oxidation of dihydrogen and the reduction of protons with remarkable efficiency, thereby attracting considerable attention in the energy field due to their biotechnological potential. For this simple reaction, [NiFe] H2 ase has developed a sophisticated but intricate mechanism with the heterolytic cleavage of dihydrogen, where its Ni-Fe active site exhibits various redox states. Recently, new spectroscopic and crystal structure studies of [NiFe] H2 ases have been reported, providing significant insights into the catalytic reaction mechanism, hydrophobic gas-access tunnel, proton-transfer pathway, and electron-transfer pathway of [NiFe] H2 ases. In addition, [NiFe] H2 ases have been shown to play an important role in biofuel cell and solar dihydrogen production. This concept provides an overview of the biocatalytic reaction mechanism and biochemical application of [NiFe] H2 ases based on the new findings.
Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Electrones , Hidrógeno/química , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Biocatálisis , Fuentes de Energía Bioeléctrica , Dominio Catalítico , Cupriavidus necator/química , Cupriavidus necator/enzimología , Desulfovibrio gigas/química , Desulfovibrio gigas/enzimología , Desulfovibrio vulgaris/química , Desulfovibrio vulgaris/enzimología , Humanos , Hidrógeno/metabolismo , Hidrogenasas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Hierro-Azufre/metabolismo , Methanosarcina barkeri/química , Methanosarcina barkeri/enzimología , Oxidación-Reducción , Protones , Energía SolarRESUMEN
The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.
Asunto(s)
Aldehído Oxidorreductasas/química , Hidrogenasas/química , Simulación de Dinámica Molecular , Complejos Multienzimáticos/química , Mioglobina/química , Aldehído Oxidorreductasas/genética , Sitios de Unión , Biocatálisis , Dominio Catalítico , Clostridiales/química , Clostridiales/enzimología , Análisis por Conglomerados , Desulfovibrio gigas/química , Desulfovibrio gigas/enzimología , Difusión , Humanos , Hidrogenasas/genética , Cinética , Ligandos , Cadenas de Markov , Complejos Multienzimáticos/genética , Mutación , Mioglobina/genética , Unión Proteica , TermodinámicaRESUMEN
Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a homodimeric molybdenum-containing protein that catalyzes the hydroxylation of aldehydes to carboxylic acids and contains a Mo-pyranopterin active site and two FeS centers called FeS 1 and FeS 2. The electron transfer reaction inside DgAOR is proposed to be performed through a chemical pathway linking Mo and the two FeS clusters involving the pyranopterin ligand. EPR studies performed on reduced as-prepared DgAOR showed that this pathway is able to transmit very weak exchange interactions between Mo(V) and reduced FeS 1. Similar EPR studies but performed on DgAOR samples inhibited with glycerol and ethylene glycol showed that the value of the exchange coupling constant J increases ~2 times upon alcohol inhibition. Structural studies in these DgAOR samples have demonstrated that the Mo-FeS 1 bridging pathway does not show significant differences, confirming that the changes in J observed upon inhibition cannot be ascribed to structural changes associated neither with pyranopterin and FeS 1 nor with changes in the electronic structure of FeS 1, as its EPR properties remain unchanged. Theoretical calculations indicate that the changes in J detected by EPR are related to changes in the electronic structure of Mo(V) determined by the replacement of the OHx labile ligand for an alcohol molecule. Since the relationship between electron transfer rate and isotropic exchange interaction, the present results suggest that the intraenzyme electron transfer process mediated by the pyranopterin moiety is governed by a Mo ligand-based regulatory mechanism.
Asunto(s)
Aldehído Oxidorreductasas/química , Desulfovibrio gigas/enzimología , Molibdeno/química , Conformación Proteica , Aldehído Oxidorreductasas/antagonistas & inhibidores , Aldehído Oxidorreductasas/metabolismo , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Cinética , Ligandos , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Desulfovibrio gigas aldehyde oxidoreductase (DgAOR) is a mononuclear molybdenum-containing enzyme from the xanthine oxidase (XO) family, a group of enzymes capable of catalyzing the oxidative hydroxylation of aldehydes and heterocyclic compounds. The kinetic studies reported in this work showed that DgAOR catalyzes the oxidative hydroxylation of aromatic aldehydes, but not heterocyclic compounds. NMR spectroscopy studies using (13)C-labeled benzaldehyde confirmed that DgAOR catalyzes the conversion of aldehydes to the respective carboxylic acids. Steady-state kinetics in solution showed that high concentrations of the aromatic aldehydes produce substrate inhibition and in the case of 3-phenyl propionaldehyde a suicide substrate behavior. Hydroxyl-substituted aromatic aldehydes present none of these behaviors but the kinetic parameters are largely affected by the position of the OH group. High-resolution crystallographic structures obtained from single crystals of active-DgAOR soaked with benzaldehyde showed that the side chains of Phe425 and Tyr535 are important for the stabilization of the substrate in the active site. On the other hand, the X-ray data of DgAOR soaked with trans-cinnamaldehyde showed a cinnamic acid molecule in the substrate channel. The X-ray data of DgAOR soaked with 3-phenyl propionaldehyde showed clearly how high substrate concentrations inactivate the enzyme by binding covalently at the surface of the enzyme and blocking the substrate channel. The different reactivity of DgAOR versus aldehyde oxidase and XO towards aromatic aldehydes and N-heterocyclic compounds is explained on the basis of the present kinetic and structural data.
Asunto(s)
Aldehído Oxidorreductasas/química , Aldehídos/química , Desulfovibrio gigas/enzimología , Conformación Proteica , Aldehído Oxidorreductasas/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Molibdeno/química , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Unión Proteica , Especificidad por SustratoRESUMEN
Anaerobic organisms have molecular systems to detoxify reactive oxygen species when transiently exposed to oxygen. One of these systems is superoxide reductase, which reduces O2 (.-) to H2 O2 without production of molecular oxygen. In order to complete the reduction of superoxide anion, superoxide reductase requires an electron, delivered by its redox partners, which in Desulfovibrio gigas are rubredoxin and/or desulforedoxin. In this work, we characterized the interaction of Desulfovibrio gigas superoxide reductase with both electron donors by using steady-state kinetics, 2D NMR titrations, and backbone relaxation measurements. The rubredoxin surface involved in the electron transfer complex with superoxide reductase comprises the solvent-exposed hydrophobic residues in the vicinity of its metal center (Cys9, Gly10, Cys42, Gly43, and Ala44), and a Kd of 3 µM at 59 mM ionic strength was estimated by NMR. The ionic strength dependence of superoxide-mediated rubredoxin oxidation by superoxide reductase has a maximum kapp of (37 ± 12) min(-1) at 157 mM. Relative to the electron donor desulforedoxin, its complex with superoxide reductase was not detected by chemical shift perturbation, though this protein is able to transfer electrons to superoxide reductase with a maximum kapp of (31 ± 7) min(-1) at an ionic strength of 57 mM. Competition experiments using steady-state kinetics and NMR spectroscopy (backbone relaxation measurements and use of a paramagnetic relaxation enhancement probe) with Fe-desulforedoxin in the presence of (15) N-Zn-rubredoxin showed that these two electron donors compete for the same site on the enzyme surface, as shown in the model structure of the complex generated by using restrained molecular docking calculations. These combined strategies indicate that the two small electron donors bind in different manners, with the desulforedoxin complex being a short lived electron transfer complex or more dynamic, with many equivalent kinetically competent orientations.
Asunto(s)
Proteínas Hierro-Azufre/química , Oxidorreductasas/química , Rubredoxinas/química , Desulfovibrio gigas/enzimología , Transporte de Electrón , Peróxido de Hidrógeno/química , Proteínas Hierro-Azufre/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular , Concentración Osmolar , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rubredoxinas/metabolismo , Superóxidos/químicaRESUMEN
Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Desulfovibrio gigas/enzimología , Regulación Bacteriana de la Expresión Génica/fisiología , Hidrogenasas/clasificación , Hidrogenasas/metabolismo , Proteínas Bacterianas/genética , Desulfovibrio gigas/genética , Desulfovibrio gigas/metabolismo , Fermentación , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/fisiología , Hidrógeno/metabolismo , Hidrogenasas/genética , Ácido Láctico/metabolismo , Datos de Secuencia Molecular , Ácido Pirúvico/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
Fluorescence activity has been used to identify Desulfovibrio and has been termed the 'desulfoviridin test'. This fluorescence is attributed to the prosthetic group of bisulfite reductase, a key enzyme in dissimilatory sulfate reduction. We have pursued the use of fluorescence measurements to quantify sulfate-reducing bacteria. Cells of D. desulfuricans and D. gigas were treated with NaOH and produced two fluorescence spectra: one with maximum fluorescence with an excitation at 395 nm and an emission at 605 nm and another with an excitation at 320 nm and emission at 360 nm. Using the fluorescence with excitation at 395 nm and emission at 605 nm, we explored a series of parameters to measure Desulfovibrio in pure cultures and environmental samples. Fluorescence measurements are reliable provided the cells are treated with 1.75 N NaOH and the chromophore released from the cells is not exposed to strong light intensity, and is not exposed to temperatures greater than 20 °C, and measurements are done within a few minutes of extraction. Bleaching of fluorescence was attributed to metal ions in solution which was not observed until metal concentrations reached 1.5mM. We propose that D. desulfuricans is appropriate as the reference organism for measurement of sulfate-reducing bacteria by fluorescence and by using fluorescence intensity, 10(5) cells/ml can be readily detected in environmental samples.
Asunto(s)
Carga Bacteriana/métodos , Desulfovibrio desulfuricans/aislamiento & purificación , Desulfovibrio gigas/aislamiento & purificación , Fluorescencia , Sulfatos/metabolismo , Desulfovibrio desulfuricans/enzimología , Desulfovibrio gigas/enzimología , Luz , Oxidación-Reducción , Manejo de Especímenes/métodos , Temperatura , Factores de TiempoRESUMEN
Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59-Asp63 and Asp95-Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners.
Asunto(s)
Desulfovibrio gigas/enzimología , Flavodoxina/química , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión/genética , Cristalografía por Rayos X , Desulfovibrio gigas/genética , Transporte de Electrón , Mononucleótido de Flavina/química , Mononucleótido de Flavina/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η(2) fashion inhibiting the enzyme activity.
Asunto(s)
Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Desulfovibrio gigas/enzimología , Aldehído Oxidorreductasas/antagonistas & inhibidores , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Bovinos , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Peróxido de Hidrógeno/farmacología , Cinética , Ligandos , Modelos Moleculares , Conformación Proteica , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/química , Xantina Oxidasa/metabolismoRESUMEN
Understanding the interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is important in the field of biotechnology and, in particular, for the development of biofuel cells. In this study, we investigated the adsorption behavior of the standard [NiFe] hydrogenase of Desulfovibrio gigas on amino-terminated alkanethiol self-assembled monolayers (SAMs) with different levels of protonation. Classical all-atom molecular dynamics (MD) simulations revealed a strong correlation between the adsorption behavior and the level of ionization of the chemically modified electrode surface. While the hydrogenase undergoes a weak but stable initial adsorption process on SAMs with a low degree of protonation, a stronger immobilization is observable on highly ionized SAMs, affecting protein reorientation and conformation. These results were validated by complementary surface-enhanced infrared absorption (SEIRA) measurements on the comparable [NiFe] standard hydrogenases from Desulfovibrio vulgaris Miyazaki F and allowed in this way for a detailed insight into the adsorption mechanism at the atomic level.
Asunto(s)
Proteínas Bacterianas/química , Desulfovibrio gigas/química , Desulfovibrio vulgaris/química , Hidrogenasas/química , Proteínas Inmovilizadas/química , Protones , Adsorción , Alcanos/química , Desulfovibrio gigas/enzimología , Desulfovibrio vulgaris/enzimología , Electrodos , Oro/química , Concentración de Iones de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Compuestos de Sulfhidrilo/química , TermodinámicaRESUMEN
[NiFe]-hydrogenases are enzymes that catalyze the reversible interconversion of protons and hydrogen at a heterobimetallic site containing Ni and Fe. This organometallic site has served as an inspiration for the synthesis of a number of biomimetic complexes, but, unfortunately, most close structural mimics have shown little to no reactivity with either of the substrates for hydrogenases. This suggests that interactions between the metallo-active site and the protein scaffold are crucial in tuning reactivity. As a first step toward development of peptide-based models, in this paper we demonstrate a synthetic strategy for construction of peptide coordinated, cysteinyl thiolate bridged Ni-M complexes in which M is a hetero-organometallic fragment. We utilize the seven amino acid peptide ACDLPCG as a scaffold for construction of these peptide-coordinated metallocenters. This peptide binds Ni in an N(2)S(2) environment consisting of the amino terminus, an amide nitrogen, and the two cysteinyl thiolates. We show that these thiolates serve as reactive sites for formation of heterometallic complexes in which they serve as bridging ligands. The method is general, and a number of heterometallic fragments including Ru(η(6)-arene)(2+), M(CO)(4)(piperidine) for M = Mo and W, and Fe(2)(CO)(6) were successfully incorporated, and the resulting metallopeptides characterized via a range of spectroscopic techniques. This methodology serves as the first step to construction of hydrogenase peptidomimetics that incorporate defined outer coordination sphere interactions intended to tune reactivity.
Asunto(s)
Hidrogenasas/metabolismo , Compuestos Organometálicos/metabolismo , Péptidos/metabolismo , Dominio Catalítico , Desulfovibrio gigas/enzimología , Hidrogenasas/química , Modelos Moleculares , Conformación Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Péptidos/químicaRESUMEN
Superoxide reductases are involved in relevant biological electron transfer reactions related to protection against oxidative stress caused by reactive oxygen species. The electrochemical features of metalloproteins belonging to the three different classes of enzymes were studied by potentio-dynamic techniques (cyclic and square wave voltammetry): desulfoferrodoxin from Desulfovibrio vulgaris Hildenborough, class I superoxide reductases and neelaredoxin from Desulfovibrio gigas and Treponema pallidum, namely class II and III superoxide reductases, respectively. In addition, a small protein, designated desulforedoxin from D. gigas, which has high homology with the N-terminal domain of class I superoxide reductases, was also investigated. A comparison of the redox potentials and redox behavior of all the proteins is presented, and the results show that SOR center II is thermodynamically more stable than similar centers in different proteins, which may be related to an intramolecular electron transfer function.
Asunto(s)
Electroquímica/métodos , Oxidorreductasas/química , Proteínas Bacterianas/química , Carbono/química , Desulfovibrio gigas/enzimología , Desulfovibrio vulgaris/enzimología , Electroquímica/instrumentación , Electrodos , Ferredoxinas/química , Vidrio/química , Oro/química , Proteínas de Unión a Hierro/química , Proteínas Hierro-Azufre/química , Superóxido Dismutasa/química , Propiedades de SuperficieRESUMEN
Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H(2)-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H(2) â 2H(+) + 2e(-) (ref. 1). In contrast to the well-studied O(2)-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O(2)-tolerant H(2) oxidation activity; however, the mechanism of O(2) tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues--in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes--and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O(2)-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O(2) tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O(2) in preventing the formation of an unready, inactive state of the enzyme.
Asunto(s)
Hidrogenasas/química , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/química , Hierro/química , Oxígeno/metabolismo , Piscirickettsiaceae/enzimología , Azufre/química , Biocatálisis , Cristalografía por Rayos X , Cisteína/química , Desulfovibrio gigas/enzimología , Proteínas Hierro-Azufre/metabolismo , Modelos Químicos , Modelos Moleculares , Oxidación-Reducción , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Protones , Relación Estructura-ActividadRESUMEN
Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.
Asunto(s)
Formiato Deshidrogenasas/química , Formiato Deshidrogenasas/aislamiento & purificación , Formiatos/química , Modelos Moleculares , Molibdeno/química , Tungsteno/química , Dióxido de Carbono/química , Catálisis , Simulación por Computador , Desulfovibrio/enzimología , Desulfovibrio/metabolismo , Desulfovibrio desulfuricans/enzimología , Desulfovibrio desulfuricans/metabolismo , Desulfovibrio gigas/enzimología , Desulfovibrio gigas/metabolismo , Electrones , Cinética , Conformación Molecular , Oxidación-Reducción , Conformación ProteicaRESUMEN
Xanthine oxidoreductase is a molybdenum-containing enzyme that catalyzes the hydroxylation reaction of sp(2)-hybridized carbon centers of a variety of substrates, including purines, aldehydes, and other heterocyclic compounds. The complex of arsenite-inhibited xanthine oxidase has been characterized previously by UV-vis, electron paramagnetic resonance, and X-ray absorption spectroscopy (XAS), and the catalytically essential sulfido ligand of the square-pyrimidal molybdenum center has been suggested to be involved in arsenite binding through either a µ-sulfido,µ-oxo double bridge or a single µ-sulfido bridge. However, this is contrary to the crystallographically observed single µ-oxo bridge between molybdenum and arsenic in the desulfo form of aldehyde oxidoreductase from Desulfovibrio gigas (an enzyme closely related to xanthine oxidase), whose molybdenum center has an oxo ligand replacing the catalytically essential sulfur, as seen in the functional form of xanthine oxidase. Here we use X-ray crystallography to characterize the molybdenum center of arsenite-inhibited xanthine oxidase and solve the structures of the oxidized and reduced inhibition complexes at 1.82 and 2.11 Å resolution, respectively. We observe µ-sulfido,µ-oxo double bridges between molybdenum and arsenic in the active sites of both complexes. Arsenic is four-coordinate with a distorted trigonal-pyramidal geometry in the oxidized complex and three-coordinate with a distorted trigonal-planar geometry in the reduced complex. The doubly bridged binding mode is in agreement with previous XAS data indicating that the catalytically essential sulfur is also essential for the high affinity of reduced xanthine oxidoreductase for arsenite.
Asunto(s)
Arsenitos/farmacología , Desulfovibrio gigas/enzimología , Inhibidores Enzimáticos/farmacología , Molibdeno/química , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/química , Dominio Catalítico , Cristalografía por Rayos X , Desulfovibrio gigas/química , Modelos Moleculares , Molibdeno/metabolismo , Xantina Oxidasa/metabolismoRESUMEN
Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn(2+), Zn-AK; Co(2+), Co-AK; and Fe(2+), Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.
Asunto(s)
Adenilato Quinasa/química , Cobalto/química , Desulfovibrio gigas/enzimología , Hierro/química , Compuestos Organometálicos/química , Zinc/química , Adenilato Quinasa/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Cobalto/metabolismo , Cristalografía por Rayos X , Humanos , Hierro/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Compuestos Organometálicos/metabolismo , Alineación de Secuencia , Zinc/metabolismoRESUMEN
The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr-I and Dsr-II, are compared at 1.76 and 2.05 Å resolution respectively. The dimeric α2ß2γ2 structure of Dsr-I contains eight [4Fe-4S] clusters, two saddle-shaped sirohaems and two flat sirohydrochlorins. In Dsr-II, the [4Fe-4S] cluster associated with the sirohaem in Dsr-I is replaced by a [3Fe-4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr-I and Dsr-II confirm the co-factor structures, whereas EPR of a third but inactive form, Dsr-III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe-4S] cluster replaced by a [3Fe-4S] centre. In Dsr-I and Dsr-II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ-subunit C-terminus is inserted into a positively charged channel formed between the α- and ß-subunits, with its conserved terminal Cys104 side-chain covalently linked to the CHA atom of the sirohaem in Dsr-I. In Dsr-II, the thioether bond is broken, and the Cys104 side-chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S3O6(2-), S2O3(2-) and S2-.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Desulfovibrio gigas/enzimología , Hidrogenosulfito Reductasa/química , Hidrogenosulfito Reductasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Catálisis , Dominio Catalítico , Desulfovibrio gigas/química , Desulfovibrio gigas/genética , Hidrogenosulfito Reductasa/genética , Conformación Molecular , Datos de Secuencia MolecularRESUMEN
Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 A resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 A resolution, respectively. Zn(2+)-AK and Fe(2+)-AK crystallized in space group I222 with similar unit-cell parameters, whereas Co(2+)-AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn(2+)-AK and Fe(2+)-AK forms and a dimer was present for the Co(2+)-AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes.
Asunto(s)
Adenilato Quinasa/química , Adenilato Quinasa/aislamiento & purificación , Cobalto/metabolismo , Desulfovibrio gigas/enzimología , Hierro/metabolismo , Difracción de Rayos X , Zinc/metabolismo , Cristalización , Cristalografía por Rayos X , Oxidación-Reducción , Sulfatos/metabolismoRESUMEN
It has been over a decade now since it was revealed that the metal containing active sites of hydrogenases possess carbonyl and cyanide ligands bound to iron. The presence of these ligands in hydrogenases came as a surprise and to-date these ligands have not been observed to be associated with any other enzymatic metallocenter. The elucidation of the structures of these unique metalloenzymes and their associated metal clusters created opportunity for a number of different lines of research. For synthetic chemists, the structures of hydrogenase active sites have provided attractive targets for syntheses that advance our understanding of the electronic structure and reactivity of these unique enzyme active sites. These efforts contribute to the synthesis of first row transition metal catalysts for hydrogen oxidation and hydrogen production that could have significant impacts on alternative and renewable energy solutions. Although effective synthetic approaches have been identified to generate models with a high degree of similarity to these active sites, the details of how these metal clusters are synthesized biochemically have not been resolved. Since hydrogen metabolism is presumed to be an early feature in the energetics of life and hydrogen metabolizing organisms can be traced very early in molecular phylogeny, the metal clusters at hydrogenase active sites are presumed to be among the earliest of known co-factors. Comparison of mineral based precursors and synthetic cluster analog chemistry to what is observed in contemporary biological systems may shed light on how proto-metabolically relevant catalysts first arose prebiotically by the processes of adoption of pre-existing functionality and ligand assisted catalysis.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Biomimética , Hidrogenasas/química , Compuestos Organometálicos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Cristalografía por Rayos X , Desulfovibrio gigas/enzimología , Hidrogenasas/genética , Hidrogenasas/metabolismo , Minerales/química , Modelos Moleculares , Compuestos Organometálicos/síntesis químicaRESUMEN
Structural analysis of the resting state of [NiFe]hydrogenase ([NiFe]H(2)ase) shows that the active site has a characteristic bis(mu-thiolato)NiFe unit, where the Ni atom and the Fe atom are bridged by an undetermined oxygen-bearing ligand. This ligand probably derives from the aqueous solvent and is therefore most likely to be H(2)O, OH(-) or O(2-). Here, we compare the reactivities of a NiFe and a NiRu complex when bearing either acetonitrile or aqueous ligands and demonstrate the critical role of an aqueous ligand in hydrogenase and its mimics. We also make observations on the necessity of organometallic metal-carbon bonds to the supporting frameworks.