Molybdenum Active Site Research Articles

Design of high performance nitrogen reduction electrocatalysts by doping defective polyoxometalate with a single atom promoter.

Single-atom catalysts (SACs) are emerging as promising candidates for electrochemical nitrogen reduction reaction (NRR). Previous studies have shown that the single-atom centers of SACs can not only serve as active sites, but also act as promoters to affect the catalytic properties. However, the use of single metal atoms as promoters in electrocatalysis has rarely been studied. In this work, the defective Keggin-type phosphomolybdic acid (PMA) is used as a substrate to support the single metal atoms. We aim to tune the electronic structures of the exposed molybdenum active sites on defective PMA by using these supported single atoms as promoters for efficient NRR. Firstly, the stability and N2 adsorption capacity were studied to screen for an effective catalyst capable of activating N2. Most of the SACs were found to have good stability and N2 adsorption capacity. Then, we compared the selectivity and NRR activity of the catalysts and found that catalysts with metal atom promoters have improved NRR selectivity and activity. Finally, electronic structure analysis was carried out to understand the promoting effect of the promoter on N2 activation and the activity of the NRR process. This work provides a new strategy for designing efficient catalysts for electrocatalytic reactions by introducing promoters.

Arsenite oxidase in complex with antimonite and arsenite oxyanions: Insights into the catalytic mechanism

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.

The Importance of Molybdenum(IV) Active Sites in Promoting Electrochemical Reduction of N2 to NH3 with MoFe Bimetallic Catalysts

To overcome the low faradaic efficiency (FE) of single Mo or Fe based electrocatalysts in nitrogen reduction reactions (NRR) due to the competition from the hydrogen evolution reaction (HER), a series of bimetallic MoFe compound catalysts were prepared under an NH3 atmosphere through a facile precipitation-pyrolysis method. The formed tetravalent Mo was found to be capable of inducing better electronic interactions between the surface nitrogen species and the Fe metal groups, thus improving the FE. It was demonstrated that the prepared ternary MoFe-N catalyst exhibited a remarkable FE of 33.26 % and a high NH3 yield rate of 33.31 μg h−1 mg−1 cat. for NRR, which was believed to have been caused by an obvious change in the valence of Mo that resulted in a lower HER activity. X-ray photoelectron spectroscopy analysis further revealed that thermal processing under an NH3 atmosphere formed the Mo(IV) active sites in Mo–N bond, which led to a significant suppression in HER activity. Finally, through the study of the surface hydrogenation mechanism, it was concluded that the synergistic effect of the adsorbed H* and Mo active sites was the main reason for the improved performance of NRR.

The Inactivation of Human Aldehyde Oxidase 1 by Hydrogen Peroxide and Superoxide.

Mammalian aldehyde oxidases (AOX) are molybdo-flavoenzymes of pharmacological and pathophysiologic relevance that are involved in phase I drug metabolism and, as a product of their enzymatic activity, are also involved in the generation of reactive oxygen species. So far, the physiologic role of aldehyde oxidase 1 in the human body remains unknown. The human enzyme hAOX1 is characterized by a broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into their corresponding carboxylic acids, and hydroxylating various heteroaromatic rings. The enzyme uses oxygen as terminal electron acceptor to produce hydrogen peroxide and superoxide during turnover. Since hAOX1 and, in particular, some natural variants produce not only H2O2 but also high amounts of superoxide, we investigated the effect of both ROS molecules on the enzymatic activity of hAOX1 in more detail. We compared hAOX1 to the high-O2 .--producing natural variant L438V for their time-dependent inactivation with H2O2/O2 .- during substrate turnover. We show that the inactivation of the hAOX1 wild-type enzyme is mainly based on the production of hydrogen peroxide, whereas for the variant L438V, both hydrogen peroxide and superoxide contribute to the time-dependent inactivation of the enzyme during turnover. Further, the level of inactivation was revealed to be substrate-dependent: using substrates with higher turnover numbers resulted in a faster inactivation of the enzymes. Analysis of the inactivation site of the enzyme identified a loss of the terminal sulfido ligand at the molybdenum active site by the produced ROS during turnover. SIGNIFICANCE STATEMENT: This work characterizes the substrate-dependent inactivation of human aldehyde oxidase 1 under turnover by reactive oxygen species and identifies the site of inactivation. The role of ROS in the inhibition of human aldehyde oxidase 1 will have a high impact on future studies.

Probing Molybdenum Active Sites during In Situ Photoreduction of the Mo6+/SiO2 Catalyst.

The photoreduction of the Mo6+/SiO2 system with CO was investigated in situ, employing a recently developed experimental setup allowing for the acquisition of transmission FT-IR spectra under simultaneous UV irradiation. Carbon monoxide, besides acting as a reducing agent in such processes, is also a useful probe molecule able to detect coordinatively unsaturated sites exposed on the surface. The unprecedented quality of the spectroscopic data, obtained as a function of the reduction time, allowed us to better rationalize the different mechanisms previously proposed for the photoreduction process. These results, coupled with UV-Vis spectroscopic data, shed light on the oxidation state and surface structure of supported molybdenum species, which are key active sites for several important reactions, such as selective oxidation, polymerization, hydrodesulfurization, epoxidation and olefin metathesis.

Oxygen and nitrite reduction by heme‐deficient sulphite oxidase in a patient with mild sulphite oxidase deficiency

Isolated sulphite oxidase deficiency (iSOD) is an autosomal recessive inborn error in metabolism characterised by accumulation of sulphite, which leads to death in early infancy. Sulphite oxidase (SO) is encoded by the SUOX gene and forms a heme- and molybdenum-cofactor-dependent enzyme localised in the intermembrane space of mitochondria. Within SO, both cofactors are embedded in two separated domains, which are linked via a flexible 11 residue tether. The two-electron oxidation of sulphite to sulphate occurs at the molybdenum active site. From there, electrons are transferred via two intramolecular electron transfer steps (IETs) via the heme cofactor and to the physiologic electron acceptor cytochrome c. Previously, we reported nitrite and oxygen to serve as alternative electron acceptors at the Moco active site, thereby overcoming IET within SO. Here, we present evidence for these reactions to occur in an iSOD patient with an unusual mild disease representation. In the patient, a homozygous c.427C>A mutation within the SUOX gene leads to replacement of the highly conserved His143 to Asn. The affected His143 is one of two heme-iron-coordinating residues within SO. We demonstrate, that the H143N SO variant fails to bind heme in vivo leading to the elimination of SO-dependent cytochrome c reduction in mitochondria. We show, that sulphite oxidation at the Moco domain is unaffected in His143Asn SO variant and demonstrate that nitrite and oxygen are able to serve as electron acceptors for sulphite-derived electrons in cellulo. As result, the patient H143N SO variant retains residual sulphite oxidising activity thus ameliorating iSOD progression.

Identifying Catalytic Active Sites of Trimolybdenum Phosphide (Mo3P) for Electrochemical Hydrogen Evolution

AbstractSolid‐state electrocatalysis plays a crucial role in the development of renewable energy to reshape current and future energy needs. However, finding an inexpensive and highly active catalyst to replace precious metals remains a big challenge for this technology. Here, tri‐molybdenum phosphide (Mo3P) is found as a promising nonprecious metal and earth‐abundant candidate with outstanding catalytic properties that can be used for electrocatalytic processes. The catalytic performance of Mo3P nanoparticles is tested in the hydrogen evolution reaction (HER). The results indicate an onset potential of as low as 21 mV, H2 formation rate, and exchange current density of 214.7 µmol s−1 g−1cat (at only 100 mV overpotential) and 279.07 µA cm−2, respectively, which are among the closest values yet observed to platinum. Combined atomic‐scale characterizations and computational studies confirm that high density of molybdenum (Mo) active sites at the surface with superior intrinsic electronic properties are mainly responsible for the remarkable HER performance. The density functional theory calculation results also confirm that the exceptional performance of Mo3P is due to neutral Gibbs free energy (ΔGH*) of the hydrogen (H) adsorption at above 1/2 monolayer (ML) coverage of the (110) surface, exceeding the performance of existing non‐noble metal catalysts for HER.

The design and catalytic performance of molybdenum active sites on an MCM-41 framework for the aerobic oxidation of 5-hydroxymethylfurfural to 2,5-diformylfuran

The catalytic activity decreases as –(SiO)3Mo(OH)(O) > –(SiO)2Mo(O)2 > –(O)4–MoO.

SBA-15 supported molybdenum oxide towards efficient catalytic oxidative desulfurization: effect of calcination temperature of catalysts

ABSTRACTThe phosphomolybdic acid has been successfully supported on functionalized ionic liquid (IL) modified mesoporous silica SBA-15. Subsequently, the obtained products were calcined at different temperatures. The oxidative desulfurization of model fuel containing benzothiophene, dibenzothiophene or 4,6-dimethyldibenzothiophene has been carried out with HPMo-IL/SBA-15 calcined at various temperatures. Based on desulfurization rate and cost, the optimum calcination temperature of HPMo-IL/SBA-15 catalyst was around 400 °C. More molybdenum oxide active sites were shown by the decomposition of the IL at the higher temperature, which caused better desulfurization performance.

Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes.

Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a "closed" conformation, suitable for internal electron transfer, and an "open" conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.

The active site structure and catalytic mechanism of arsenite oxidase

Arsenite oxidase is thought to be an ancient enzyme, originating before the divergence of the Archaea and the Bacteria. We have investigated the nature of the molybdenum active site of the arsenite oxidase from the Alphaproteobacterium Rhizobium sp. str. NT-26 using a combination of X-ray absorption spectroscopy and computational chemistry. Our analysis indicates an oxidized Mo(VI) active site with a structure that is far from equilibrium. We propose that this is an entatic state imposed by the protein on the active site through relative orientation of the two molybdopterin cofactors, in a variant of the Rây-Dutt twist of classical coordination chemistry, which we call the pterin twist hypothesis. We discuss the implications of this hypothesis for other putatively ancient molybdopterin-based enzymes.

Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR, 14,15N HYSCORE, and DFT Study.

Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in 98Mo and 15N nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively 15N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.

The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.

Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarG-His(1098)). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of -88 and -36 mV, respectively). Ala variants of His(1092) and His(1098) also elicit large ΔEm values of -143 and -101 mV, respectively. An Arg variant of His(1092) elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.

Synergism of Extruded and Monolithic Co–Mo/γ-AL2O3–Sepiolite Catalytic Systems in the Hydrodesulphurization Reaction

This study evaluates the synergism of γ-alumina supported cobalt-molybdenum samples prepared by impregnation in excess of solvent. These materials were then agglomerated with sepiolite in order to obtain samples of extruded cylinders and honeycomb monoliths shaped. The activity of the gas–oil hydrodesulphurization reaction was measured under reaction conditions similar to those used in industry, and the samples were characterised by using electron probe micro analysis and scanning electron microscopy. It was observed that catalytic activity and synergism are higher in both, the extruded and the monolithic samples, when both, the cobalt and molybdenum were impregnated on separate surfaces (Co//Mo), where it was also possible to observe synergism due to hydrogen spillover. The lowest level of activity observed in the extruded and monolithic bimetallic catalysts (CoMo) may be attributed to the cobalt covering the molybdenum active sites.

Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2

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 η2 fashion inhibiting the enzyme activity.

Oxo–Mo(IV)(dithiolene)thiolato Complexes: Analogue of Reduced Sulfite Oxidase

A series of [Mo(IV)O(mnt)(SR)(N-N)](-) (mnt = maleonitriledithiolate; R = Ph, nap, p-Cl-Ph, p-CO2H-Ph, and p-NO2-Ph; N-N = 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)) complexes analogous to the reduced active site of enzymes of the sulfite oxidase family has been synthesized and their participation in electron transfer reactions studied. Equatorial thiolate and dithiolene ligations have been used to closely simulate the three sulfur coordinations present in the native molybdenum active site. These synthetic analogues have been shown to participate in electron transfer via a pentavalent EPR-active Mo(V) intermediate with minimal structural change as observed electrochemically by reversible oxidative responses. The role of the redox-active dithiolene ligand as an electron transfer gate between external oxidants and the molybdenum center could be envisaged in one of the analogue systems where the initial transient EPR signal with <g> = 2.008 is replaced by the appearance of a typical Mo(V)-centered EPR (<g> = 1.976) signal. The appearance of such a ligand-based transient radical at the initial stage has been supported by the ligand-centered frontier orbital from DFT calculation. A stepwise rationale has been provided by computational study to show that the coupled effects of the diimine bite angle and the thiolato dihedral angle determine the metal- or ligand-based frontier orbital occupancy. DFT calculation has further supported the similarity between the reduced, semireduced, and oxidized resting state of the molybdenum center in Moco of SO with the synthesized complexes and their corresponding one-electron and fully oxidized species.

Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis

BackgroundYedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity.ResultsIn this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher.ConclusionOur study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.

The Crystal Structure of Cupriavidus necator Nitrate Reductase in Oxidized and Partially Reduced States

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe–4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C 4mim]Cl −. In addition, UV–Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.

DFT and PIO study of the influences of Mo valance state and surface hydroxyl on supported-MoOx catalysts for ethylene polymerization

Phillips catalyst (CrOx/SiO2) is an important industrial catalyst for ethylene polymerization, but the highly toxic chromium may contaminate the environment and do harm to human's health. Supported-MoOx catalyst with low toxicity has potential to replace Phillips catalyst if its catalytic performance could be improved. In this work, models of molybdenum active sites with different valence states (5+, 4+, 3+, 2+) supported on Al2O3 and SiO2, respectively, were established to investigate the effects of the valence states and surface hydroxyl on the catalyst activity using the combination of density functional theory (DFT) and paired interacting orbitals (PIO) methods. DFT results showed that supported Mo2+ center had the lowest energy barrier of ethylene insertion and thus possessed the highest activity for ethylene polymerization. PIO method additionally elucidated the orbital interaction and electron transformation between Mo2+ center and ethylene monomer. Hydroxyl on the support surface could poison the active center by coordination with Mo center. It had been demonstrated that pre-reduction of hexa-valent molybdenum into lower valence state +2 and elimination of surface hydroxyl groups of support were the key factors to obtain highly efficient ethylene polymerization catalyst. The molecular modeling results of this work provided theoretical basis for further experimental developments of green and highly efficient supported Mo-based polyethylene catalysts.