Structural and biochemical characterization of the M405S variant of Desulfovibrio vulgaris formate dehydrogenase.

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO2 reduction, together with an increased sensitivity to oxygen inactivation.

Bioelectrocatalytic Activity of W-Formate Dehydrogenase Covalently Immobilized on Functionalized Gold and Graphite Electrodes.

The decrease of greenhouse gases such as CO2 has become a key challenge for the human kind and the study of the electrocatalytic properties of CO2-reducing enzymes such as formate dehydrogenases is of importance for this goal. In this work, we study the covalent bonding of Desulfovibrio vulgaris Hildenborough FdhAB formate dehydrogenase to chemically modified gold and low-density graphite electrodes, using electrostatic interactions for favoring oriented immobilization of the enzyme. Electrochemical measurements show both bioelectrocatalytic oxidation of formate and reduction of CO2 by direct electron transfer (DET). Atomic force microscopy and quartz crystal microbalance characterization, as well as a comparison of direct and mediated electrocatalysis, suggest that a compact layer of formate dehydrogenase was anchored to the electrode surface with some crosslinked aggregates. Furthermore, the operational stability for CO2 electroreduction to formate by DET is shown with approximately 100% Faradaic yield.

Towards cryogenic neutron crystallography on the reduced form of [NiFe]-hydrogenase.

A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni-Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni-Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni-Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

Exploring the gas access routes in a [NiFeSe] hydrogenase using crystals pressurized with krypton and oxygen.

Hydrogenases are metalloenzymes that catalyse both H2 evolution and uptake. They are gas-processing enzymes with deeply buried active sites, so the gases diffuse through channels that connect the active site to the protein surface. The [NiFeSe] hydrogenases are a special class of hydrogenases containing a selenocysteine as a nickel ligand; they are more catalytically active and less O2-sensitive than standard [NiFe] hydrogenases. Characterisation of the channel system of hydrogenases is important to understand how the inhibitor oxygen reaches the active site to cause oxidative damage. To this end, crystals of Desulfovibrio vulgaris Hildenborough [NiFeSe] hydrogenase were pressurized with krypton and oxygen, and a method for tracking labile O2 molecules was developed, for mapping a hydrophobic channel system similar to that of the [NiFe] enzymes as the major route for gas diffusion.

Mechanism and Application of the Catalytic Reaction of [NiFe] Hydrogenase: Recent Developments.

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.

Characterization of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough.

Short and branched chain fatty acid kinases participate in both bacterial anabolic and catabolic processes, including fermentation, through the reversible, ATP-dependent synthesis of acyl phosphates. This study reports biochemical properties of a predicted butyrate kinase from Desulfovibrio vulgaris str. Hildenborough (DvBuk) expressed heterologously and purified from Escherichia coli. Gel filtration chromatography indicates purified DvBuk is active as a dimer. The optimum temperature and pH for DvBuk activity is 44°C and 7.5, respectively. The enzyme displays enhanced thermal stability in the presence of substrates as observed for similar enzymes. Measurement of kcat and KM for various substrates reveals DvBuk exhibits the highest catalytic efficiencies for butyrate, valerate and isobutyrate. In particular, these measurements reveal this enzyme's apparent high affinity for C4 fatty acids relative to other butyrate kinases. These results have implications on structure and function relationships within the ASKHA superfamily of phosphotransferases, particularly regarding the acyl binding pocket, as well as potential physiological roles for this enzyme in Desulfovibrio vulgaris str. Hildenborough.

Suppressing hydrogen peroxide generation to achieve oxygen-insensitivity of a [NiFe] hydrogenase in redox active films.

Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as hydrogenases for their use in fuel cells. However, the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day. Here, we employ operando confocal microscopy to elucidate the deactivation processes. The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst. We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] hydrogenase to over one week, approaching the experimental anaerobic half-life. Altogether, our data support the theory that redox films make the hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress.

Long-range electron-electron interaction and charge transfer in protein complexes: a numerical approach.

With application to the nitrite reductase hexameric protein complex of Desulfovibrio vulgaris, NrfH2A4, we suggest a strategy to compute the energy landscape of electron transfer in large systems of biochemical interest. For small complexes, the energy of all electronic configurations can be scanned completely on the level of a numerical solution of the Poisson-Boltzmann equation. In contrast, larger systems have to be treated using a pair approximation, which is verified here. Effective Coulomb interactions between neighbouring sites of excess electron localization may become as large as 200 meV, and they depend in a nontrivial manner on the intersite distance. We discuss the implications of strong Coulomb interactions on the thermodynamics and kinetics of charging and decharging a protein complex. Finally, we turn to the effect of embedding the system into a biomembrane.

Structural insight into metallocofactor maturation in carbon monoxide dehydrogenase.

The nickel-dependent carbon monoxide dehydrogenase (CODH) employs a unique heterometallic nickel-iron-sulfur cluster, termed the C-cluster, to catalyze the interconversion of CO and CO2 Like other complex metalloenzymes, CODH requires dedicated assembly machinery to form the fully intact and functional C-cluster. In particular, nickel incorporation into the C-cluster depends on the maturation factor CooC; however, the mechanism of nickel insertion remains poorly understood. Here, we compare X-ray structures (1.50-2.48 Å resolution) of CODH from Desulfovibrio vulgaris (DvCODH) heterologously expressed in either the absence (DvCODH-CooC) or presence (DvCODH+CooC) of co-expressed CooC. We find that the C-cluster of DvCODH-CooC is fully loaded with iron but does not contain any nickel. Interestingly, the so-called unique iron ion (Feu) occupies both its canonical site (80% occupancy) and the nickel site (20% occupancy), with addition of reductant causing further mismetallation of the nickel site (60% iron occupancy). We also demonstrate that a DvCODH variant that lacks a surface-accessible iron-sulfur cluster (the D-cluster) has a C-cluster that is also replete in iron but lacks nickel, despite co-expression with CooC. In this variant, all Feu is in its canonical location, and the nickel site is empty. This D-cluster-deficient CODH is inactive despite attempts to reconstitute it with nickel. Taken together, these results suggest that an empty nickel site is not sufficient for nickel incorporation. Based on our findings, we propose a model for C-cluster assembly that requires both CooC and a functioning D-cluster, involves precise redox-state control, and includes a two-step nickel-binding process.

Energetics for the Mechanism of Nickel-Containing Carbon Monoxide Dehydrogenase.

Nickel-containing carbon monoxide (CO) dehydrogenase is an enzyme that catalyzes the important reversible carbon dioxide reduction. Several high-resolution structures have been determined at various stages of the reduction, which can be used as good starting points for the present computational study. The cluster model is used in combination with a systematic application of the density functional theory as recently described. The results are in very good agreement with experimental evidence. There are a few important results. To explain why the X-ray structure for the reduced Cred1 state has an empty site on nickel, it is here suggested that the cluster has been over-reduced by X-rays and is therefore not the desired reduced state, which instead contains a bound CO on nickel. After an additional reduction, a hydride bound to nickel is suggested to play a role. In order to obtain energetics in agreement with experiments, it is concluded that one sulfide bridge in the Ni-Fe cluster should be protonated. The best test of the accuracy obtained is to compare the computed rate for reduction using -0.6 V with that for oxidation using -0.3 V, where good agreement was obtained. Obtaining a mechanism that is easily reversible is another demanding aspect of the modeling. Nickel oscillates between nickel(II) and nickel(I), while nickel(0) never comes in.

New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates.

Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox-dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non-invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H2 and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase-mediated reactions simultaneously. Using the proposed system, we confirmed that H2 (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D2 /H2 O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (kout ) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.

Reaction Mechanism of [NiFe] Hydrogenase Studied by Computational Methods.

[NiFe] hydrogenases catalyze the reversible conversion of molecular hydrogen to protons and electrons. This seemingly simple reaction has attracted much attention because of the prospective use of H2 as a clean fuel. In this paper, we have studied the full reaction mechanism of this enzyme with various computational methods. Geometries were obtained with combined quantum mechanical and molecular mechanics (QM/MM) calculations. To get more accurate energies and obtain a detailed account of the surroundings, we performed big-QM calculations with 819 atoms in the QM region. Moreover, QM/MM thermodynamic cycle perturbation calculations were performed to obtain free energies. Finally, density matrix renormalisation group complete active space self-consistent field calculations were carried out to study the electronic structures of the various states in the reaction mechanism. Our calculations indicate that the Ni-L state is not involved in the reaction mechanism. Instead, the Ni-C state is reduced by one electron and then the bridging hydride ion is transferred to the sulfur atom of Cys546 as a proton and the two electrons transfer to the Ni ion. This step turned out to be rate-determining with an energy barrier of 58 kJ/mol, which is consistent with the experimental rate of 750 ± 90 s-1 (corresponding to ∼52 kJ/mol). The cleavage of the H-H bond is facile with an energy barrier of 33 kJ/mol, according to our calculations. We also find that the reaction energies are sensitive to the size of the QM system, the basis set, and the density functional theory method, in agreement with previous studies.

Characterization of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough.

The [NiFeSe] hydrogenases are a subgroup of the well-characterized family of [NiFe] hydrogenases, in which a selenocysteine is a ligand to the nickel atom in the binuclear NiFe active site instead of cysteine. These enzymes display very interesting catalytic properties for biological hydrogen production and bioelectrochemical applications: high H2 production activity, bias for H2 evolution, low H2 inhibition, and some degree of O2 tolerance. Here we describe the methodologies employed to study the [NiFeSe] hydrogenase isolated from the sulfate-reducing bacteria D. vulgaris Hildenborough and the creation of a homologous expression system for production of variant forms of the enzyme.

Photoreduction of CO2 with a Formate Dehydrogenase Driven by Photosystem II Using a Semi-artificial Z-Scheme Architecture.

Solar-driven coupling of water oxidation with CO2 reduction sustains life on our planet and is of high priority in contemporary energy research. Here, we report a photoelectrochemical tandem device that performs photocatalytic reduction of CO2 to formate. We employ a semi-artificial design, which wires a W-dependent formate dehydrogenase (FDH) cathode to a photoanode containing the photosynthetic water oxidation enzyme, Photosystem II, via a synthetic dye with complementary light absorption. From a biological perspective, the system achieves a metabolically inaccessible pathway of light-driven CO2 fixation to formate. From a synthetic point of view, it represents a proof-of-principle system utilizing precious-metal-free catalysts for selective CO2-to-formate conversion using water as an electron donor. This hybrid platform demonstrates the translatability and versatility of coupling abiotic and biotic components to create challenging models for solar fuel and chemical synthesis.

Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase.

The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris, providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging paradigm of metallocluster plasticity.

Influence of cytochrome charge and potential on the cathodic current of electroactive artificial biofilms.

An electroactive artificial biofilm has been optimized for the cathodic reduction of fumarate by Shewanella oneidensis. The system is based on the self-assembly of multi-walled carbon nanotubes with bacterial cells in the presence of a c-type cytochrome. The aggregates are then deposited on an electrode to form the electroactive artificial biofilm. Six c-type cytochromes have been studied, from bovine heart or Desulfuromonas and Desulfuvibrio strains. The isoelectric point of the cytochrome controls the self-assembly process that occurs only with positively-charged cytochromes. The redox potential of the cytochrome is critical for electron transfer reactions with membrane cytochromes of the Mtr pathway. Optimal results have been obtained with c3 from Desulfovibrio vulgaris Hildenborough having an isoelectric point of 10.2 and redox potentials of the four hemes ranging between -290 and -375 mV vs SHE. A current density of 170 µA cm-2 could be achieved in the presence of 50 mM fumarate. The stability of the electrochemical response was evaluated, showing a regular decrease of the current within 13 h, possibly due to the inactivation or leaching of loosely-bound cytochromes from the biofilm.

Reactivation of standard [NiFe]-hydrogenase and bioelectrochemical catalysis of proton reduction and hydrogen oxidation in a mediated-electron-transfer system.

Standard [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF-H2ase) catalyzes the uptake and production of hydrogen (H2) and is a promising biocatalyst for future energy devices. However, DvMF-H2ase experiences oxidative inactivation under oxidative stress to generate Ni-A and Ni-B states. It takes a long time to reactivate the Ni-A state by chemical reduction, whereas the Ni-B state is quickly reactivated under reducing conditions. Oxidative inhibition limits the application of DvMF-H2ase in practical devices. In this research, we constructed a mediated-electron-transfer system by co-immobilizing DvMF-H2ase and a viologen redox polymer (VP) on electrodes. The system can avoid oxidative inactivation into the Ni-B state at high electrode potentials and rapidly reactivate the Ni-A state by electrochemical reduction of VP. H2 oxidation and H+ reduction were realized by adjusting the pH from a thermodynamic viewpoint. Using carbon felt as a working-electrode material, high current densities-up to (200 ±â€¯70) and -(100 ±â€¯9) mA cm-3 for the H2-oxidation and H+-reduction reactions, respectively-were attained.

Glycoside hydrolase DisH from Desulfovibrio vulgaris degrades the N-acetylgalactosamine component of diverse biofilms.

Biofilms of sulfate-reducing bacteria (SRB) produce H2 S, which contributes to corrosion. Although bacterial cells in biofilms are cemented together, they often dissolve their own biofilm to allow the cells to disperse. Using Desulfovibrio vulgaris as a model SRB, we sought polysaccharide-degrading enzymes that disperse its biofilm. Using a whole-genome approach, we identified eight enzymes as putative extracellular glycoside hydrolases including DisH (DVU2239, dispersal hexosaminidase), an enzyme that we demonstrated here, by utilizing various p-nitrooligosaccharide substrates, to be an N-acetyl-ß-D-hexosaminidase. For N-acetyl-ß-D-galactosamine (GalNAc), Vmax was 3.6 µmol of p-nitrophenyl/min (mg protein)-1 and Km was 0.8 mM; the specific activity for N-acetyl ß-D-glucosamine (GlcNAc) was 7.8 µmol of p-nitrophenyl/min (mg protein)-1 . Since GalNAc is one of the three exopolysaccharide matrix components of D. vulgaris, purified DisH was found to disperse 63 ± 2% biofilm as well as inhibit biofilm formation up to 47 ± 4%. The temperature and pH optima are 60°C and pH 6, respectively; DisH is also inhibited by copper and is secreted. In addition, since polymers of GalNAc and GlcNAc are found in the matrix of diverse bacteria, DisH dispersed biofilms of Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis. Therefore, DisH has the potential to inhibit and disperse a wide-range of biofilms.

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation.

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I and can be an indication of capacity for electron bifurcation.