Reductive Half-reaction Research Articles

Nonlinear Time‐course of Aldehyde Oxidase

Many promising drug candidates metabolized by Aldehyde Oxidase (AOX) fail during the clinical trial due to underestimation of their clearance. AOX is species specific and this makes traditional allometric studies a poor choice for estimating human clearance. In this study, we have monitored metabolite formation of six AOX substrates, O6‐benzylguanine (O6BG), DACA, zaleplon, phthalazine, BIBX1382 and zoniporide in purified expressed human aldehyde oxidase (HAO) using LC/MS/MS over a 240‐minute time period. Numerical data fitting to three different kinetic schemes was performed using Mathematica software and the goodness of fit was assessed by Akaike value. Figure 1 shows the comparison between fitting of data for the substrate O6BG to three different schemes, namely Michaelis‐Menten (MM), Dead model and Modulated activity model (MAM). Based on the Akaike values the third kinetic scheme fit the data more closely and therefore was the model that we chose for our further studies.Our data shows that the total velocity for substrates becomes slower than the initial velocity by 3.1, 6.5, 2.9, 32.2, 2.7 and 0.2 fold for O6BG, DACA, zaleplon, phthalazine, BIBX1382 and zoniporide respectively in HAO while the Km remains constant. Two different hypothesis were considered for this decrease in enzymatic linearity over time. First, reactive oxygen species produced during the catalytic cycle may cause enzyme inactivation. Second, the enzyme's reductive half reaction is slow to reduce oxygen, and the fast rate is an initial rate prior to the reduction of the enzyme.The first hypothesis was tested by using Super oxide dismutase (SOD) and catalase to remove the reactive oxygen species. However, the results showed no significant effect on enzyme linearity with or without SOD and catalase. The second hypothesis was tested by using 5‐Nitroquinoline (5NQ), an oxidative and reductive substrate of AOX. Recently it has been proposed that the substrate reduction happens on a different site in AOX and it is assumed to be in the Flavin site. Therefore, by using 5NQ, we have introduced competition between oxygen and the reductive substrate for the Flavin site and therefore disrupting the electron shuttling. The data is consistent with this hypothesis and using 5NQ reduces the enzymatic activity and increases enzyme's nonlinearity.If the oxidation of the enzyme is the rate limiting step, then all the k5 values in the Modulate activity model should be the same but our data shows up to 3‐fold difference between k5 values of different substrates. This observation can be traced back to substrate inhibition that happens in substrates such as DACA and phthalazine. We are proposing that for such substrates, the second substrate molecule would bind to the reduction site and therefore lowering the k5 value further down.Overall, the main goal of this paper is to show that almost all AOX‐catalyzed reactions are nonlinear over time, and that ignoring this leads to an underestimation of clearance. We believe that the constant under‐prediction of drug clearance by AOX is due to the rapid slowing of the reaction rate once the reaction is started and that even short incubations would not lead to an accurate determination of the kcat.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences [GM100874].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The reactive form of a C–S bond–cleaving, CO2-fixing flavoenzyme

2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.

Mechanistic Studies on Bioinspired Aerobic C-H Oxidation of Amines with an ortho-Quinone Catalyst.

We report herein our mechanistic studies of the ortho-quinone-catalyzed aerobic oxidation of primary, secondary, and tertiary amines. Two different catalytic pathways were discovered for the reductive half reactions: for primary amines, the reaction was found to proceed via a transamination pathway, while the reactions with secondary amines and tertiary amines proceeded via hydride transfer. We also found that the amine substrates could significantly promote the regeneration of the ortho-quinone catalyst in the oxidative half reaction, in which a proton transfer occurs between the amine substrates and catechol derivatives (the reduced form of the ortho-quinone catalyst).

Performing anaerobic stopped-flow spectrophotometry inside of an anaerobic chamber.

The catalytic cycle of most flavin-dependent enzymes can be divided into oxidative and reductive half-reactions. Although some enzymes are oxidized by electron carrier proteins or organic compounds, many use oxygen as the final electron acceptor. In order to properly study the reductive half-reaction of flavin-dependent enzyme that react with oxygen, as in the case of oxidases and monooxygenases, it is necessary to establish anaerobic conditions that will only allow the reduction process to be monitored. The reduced flavoenzyme can be further studied by exposing it to oxygen to monitor the oxidative half-reaction. Anaerobic chambers provide an ideal environment for performing these experiments as they reliably maintain an anaerobic atmosphere inside a large workspace. A common tool used to study flavin-dependent enzymes is the stopped-flow spectrophotometry. This chapter describes methods for performing stopped-flow experiments in an anaerobic chamber. We include information about the chamber components, setting up a stopped-flow spectrophotometer inside of a chamber, preparing anaerobic solutions, and performing experiments to measure the reductive and oxidative half-reactions of flavin-dependent monooxygenases.

A detailed mechanism of the oxidative half-reaction of d-amino acid oxidase: another route for flavin oxidation.

d-Amino acid oxidase (DAAO) is a flavoenzyme whose inhibition is expected to have therapeutic potential in schizophrenia. DAAO catalyses hydride transfer from the substrate to the flavin in the reductive half-reaction, and the flavin is reoxidized by O2 in the oxidative half-reaction. Quantum mechanical/molecular mechanical calculations were performed and their results together with available experimental information were used to elucidate the detailed mechanism of the oxidative half-reaction. The reaction starts with a single electron transfer from FAD to O2, followed by triplet-singlet transition. FAD oxidation is completed by a proton coupled electron transfer to the oxygen species and the reaction terminates with H2O2 formation by proton transfer from the oxidized substrate to the oxygen species via a chain of water molecules. The substrate plays a double role by facilitating the first electron transfer and by providing a proton in the last step. The mechanism differs from the oxidative half-reaction of other oxidases.

Investigations of two-component flavin-dependent monooxygenase systems.

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems.

Tyrosine substitution of a conserved active-site histidine residue activates Plasmodium falciparum peroxiredoxin 6.

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro- and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so-called Prx6-type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady-state kinetics with tert-butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four- to six-fold in vitro. Stopped-flow kinetics with reduced PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y revealed a preference for H2 O2 as an oxidant with second order rate constants for H2 O2 and tBuOOH around 2.5 × 107 M-1 s-1 and 3 × 106 M-1 s-1 , respectively. Differences between the oxidation kinetics of PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y were observed during a slower second-reaction phase. Our kinetic data support the interpretation that the reductive half-reaction is the rate-limiting step for PfPrx6 catalysis in steady-state measurements. Whether the increased activity of PfPrx6H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6-type enzymes is non-essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain-of-function mutant PfPrx6H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.

High Performance Si-Based Photoanodes Using a Conformal Co-Based Catalysts By Electrodeposition

Converting and storing solar energy has been regarded as a promising approach to address the current global reliance on fossil fuels. The generation of hydrogen from water using sunlight can form potentially the basis of a clean and renewable source of energy. Solar water splitting system requires stable light-absorbing electrodes for both the oxidative and reductive half-reactions. However, the kinetically slow oxygen evolution reaction (OER) is a bottle-neck process for producing sustainable solar-driven fuels using water. Silicon has a suitable band gap (1.12 eV) to absorb the wide range of the solar spectrum and has high carrier mobility. For this reason, silicon is an attractive candidate among the various materials such as transition metal oxides and semiconductors. Since the silicon photoanode is unstable at electrolyte and suffers from fast photocorrosion due to the position of thermodynamic redox potentials, the solar to hydrogen conversion efficiency of bare silicon photoanode is largely suppressed. To solve those problems of silicon, recent studies on silicon photoanode has focused on designing a surface passivation layer that protects against chemical and photo-induced corrosion without degrading the inherent photoactive ability of silicon and promotes the chemical adsorption of water molecules to lower the overpotential at the solid/liquid interface. Herein, we report the synthesis and photoelectrochemical properties of multifunctional cobalt based catalysts on a n-type silicon photoanode by using facile electrodeposition. This method not only significantly reduces the synthesis time and costs, but also easily control reaction by changing applied voltage or current. The solution-processed n-Si photoanode showed large photovoltage and superior catalytic behavior. By using efficient oxygen evolution catalysts, onset potential shifted to cathodic direction, leading to highly efficient Si-based photoanodes.

Probing the mechanism of xanthine oxidase and 2-amino xanthine: an implication of energy, charge bond order and wave function.

Xanthine oxidase (XO) is an important molybdenum-containing enzyme catalyzing the hydroxylation of hypoxanthine to xanthine and xanthine to uricacid. The mechanistic action by which xanthine oxidase oxidizes purine derivatives is not well understood. A better understanding of the overall mechanism is supposed to enhance our ability to control the metabolic properties of potential drug molecules metabolized by this enzyme. In this work a model substrate, 2-Amino Xanthine has been used to study the mechanistic action of the enzyme. For this reason, the present theoretical work was intended to probe a unified mechanism for the oxidation of 2-Amino Xanthine by xanthine oxidase. Parameters like total electronic energy, Mulliken atomic charges, wave functions, and percent contribution of chemical fragments were generated using a DFT method employing B3LYP level of theory with 6-31G(d',p') basis set for nonmetals and LanL2DZ basis set for molybdenum. AOmix software package that employs single point energy output as an input file was employed for wave function and percent fragment analysis. From these result new reaction intermediates and plausible reaction mechanism root has been reported for reductive and oxidative half reaction using 2-Amino Xanthine as model substrate. In this work it can be concluded that a stepwise mechanistic route with hydrogen bonding reaction complex and active site resemble very rapid Mo (V) intermediate is most plausible.    Â

Controlling the process of denitrification in flooded rice soils by using microbial fuel cell applications

Controlling the denitrification rate could help reduce the losses of applied nitrogen (N) fertilizer in fields. The applicability of microbial fuel cell (MFC) for controlling denitrification in flooded rice soils was investigated based on MFC theory coupled with redox changes. Because the soil about 10–20 cm beneath water and the soil near the water surface are anaerobic and aerobic, respectively, gradients of electric potentials could be generated between them, upon connecting them through insulated wires. Electrons released through the oxidation of organic matter during microbial metabolism can be utilized by this set-up, generating electricity. This can reduce the availability of electrons for reductive half-reactions of nitrate, to suppress denitrification. We studied the N losses in soil using planting pots with gas chamber experiment under three conditions: MFC systems, MFC systems with an externally applied voltage, and non-MFC systems as a control. Each system was set in triplicate, supplied with the same N fertilizer amounts, and flooded with automatic irrigation. Soil redox potential, N2O flux, and inorganic nitrogen concentration in soil pore water were periodically monitored. The redox potentials of both MFC systems and MFC systems with externally applied voltage were significantly higher than that of non-MFC systems, while N2O flux levels were significantly lower than that of non-MFC systems. The rice reproductive stage was the most effective on suppressing N2O flux with MFC application. However, the effect of externally applied voltage on suppressing N2O flux remains unclear. Inorganic nitrogen retention efficiencies in pore water were higher in MFC systems, which is consistent with the N2O flux difference. While the proportion of denitrified N estimated for MFC systems was 2.3%, that of non-MFC systems was 6.6%. We confirmed the applicability of MFCs to control soil redox potential and thereby suppress the denitrification based on planting pot experiments.

Kinetic Characterization of PA1225 from Pseudomonas aeruginosa PAO1 Reveals a New NADPH:Quinone Reductase.

The pa1225 gene of Pseudomonas aeruginosa strain PAO1 was cloned, and the resulting enzyme (PA1225) was purified and revealed to be an NADPH:quinone reductase. By using kinetics, fluorescence, and mass spectrometric analyses, PA1225 was shown to utilize FAD to transfer a hydride ion from NADPH to quinones. The enzyme could also use NADH, but with an efficiency that was 40-fold lower than that of NADPH as suggested by the kcat/ Km values at pH 6.0. Similar initial rates of reaction were determined with 1,4-benzoquinone and 2,6-dimethoxy-1,4-benzoquinone in the range between 25 and 200 μM, suggesting a low Km value for the quinone-oxidizing substrate. The lack of inhibition by NADP+ versus NADPH at saturating concentrations of 1,4-benzoquinone was consistent with a ping-pong bi-bi mechanism. The reductive half-reaction at pH 6.0 had Kd values of 0.07 mM with NADPH and 1.8 mM with NADH; the kred for flavin reduction was independent of pH with values of ∼10 s-1 with NADPH and ∼5 s-1 with NADH. Thus, the enzyme specificity for the reducing substrate arises primarily from a tighter binding of NADPH than of NADH. At pH 6.0, the kcat value with NADPH and 1,4-benzoquinone was 10.1 s-1, consistent with the hydride transfer from NADPH to FAD being fully rate limiting for the overall turnover of the enzyme. The enzyme showed negligible NADPH oxidase and azoreductase activities. This study enables annotation of the pa1225 gene as NADPH:quinone reductase, elucidates the enzymatic function of PA1225 in P. aeruginosa PAO1, and establishes that PA1225 is not an azoreductase as previously proposed.

Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase.

Diamine oxidase (DAO) is an enzyme involved in the regulation of cell proliferation and the immune response. This enzyme performs oxidative deamination in the catabolism of biogenic amines, including, among others, histamine, putrescine, spermidine, and spermine. The mechanistic details underlying the reductive half-reaction of the DAO-catalyzed oxidative deamination which leads to the reduced enzyme cofactor and the aldehyde product are, however, still under debate. The catalytic mechanism was proposed to involve a prototropic shift from the substrate–Schiff base to the product–Schiff base, which includes the rate-limiting cleavage of the Cα–H bond by the conserved catalytic aspartate. Our detailed mechanistic study, performed using a combined quantum chemical cluster approach with empirical valence bond simulations, suggests that the rate-limiting cleavage of the Cα–H bond involves direct hydride transfer to the topaquinone cofactor—a mechanism that does not involve the formation of a Schiff base. Additional investigation of the D373E and D373N variants supported the hypothesis that the conserved catalytic aspartate is indeed essential for the reaction; however, it does not appear to serve as the catalytic base, as previously suggested. Rather, the electrostatic contributions of the most significant residues (including D373), together with the proximity of the Cu2+ cation to the reaction site, lower the activation barrier to drive the chemical reaction.

A bacterial flavin‐dependent oxidoreductase that captures carbon dioxide into biomass

Atmospheric carbon dioxide is used as a carbon source for building biomass in plants and photosynthetic microbes. Non‐photosynthetic processes that also fix carbon dioxide have more recently been discovered. This research focuses on a microbial mechanism for coupling acetone to CO2 to make a central metabolite, acetoacetate. The key reaction is catalyzed by NADPH‐2‐ketopropyl‐coenzyme M oxidoreductase/carboxylase (2‐KPCC), a bacterial enzyme that is part of the flavin and cysteine‐disulfide containing oxidoreductase family (DSORs) which are best known for reducing metallic or disulfide substrates. Our research asks: how has nature repurposed a DSOR to, uniquely, break a C‐S bond and then trap and fix CO2? 2‐KPCC lacks a conserved, catalytically essential acid‐base histidine possessed by all other DSOR enzymes, having instead a phenylalanine (F501) at the same position. Mutagenesis showed that a F501H mutant has a similar rate of catalytic turnover; however, the product is acetone instead of acetoacetate (Figure 1). We hypothesized that F501 is important for both the reductive half reaction – which generates the reactive, C‐S bond breaking form of the active site – and for the oxidative half, in which an enolacetone intermediate reacts with either the correct (CO2) or incorrect (H+) electrophile. In this study, we used real‐time and spectroscopic methods to examine the reductive half reaction. In typical DSORs, this reaction generates a Cys/FAD charge transfer species. However, we showed that 2‐KPCC generates an electronically unique form of the active site, in which the flavin is oxidized and a pair of active site histidines are reduced and protonated. We hypothesize that this form of the active site generates the substrate‐reactive Cys in a more nucleophilic form, where it is capable of cleaving relatively strong C‐S bonds. The resulting enolacetone carbanion is an extremely potent nucleophile that is capable of directly attacking CO2. Research on 2‐KPCC and other biological CO2 fixation methods adds to our arsenal of strategies for carbon dioxide capture and use.Support or Funding InformationDOE:DE‐FG02‐04ER15563This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Hydrogen Bond Donors on Reactivity of Sulfite Oxidase

Sulfite oxidase (SO) catalyzes the conversion of sulfite to sulfate as the last step in the degradation of cysteine and methionine [1]. Severe neonatal neurological problems are caused by SO deficiency in humans [2]. The overall mechanism of SO suggested that the reductive half reaction of SO is the rate limiting step for enzyme activity [3]. Using a computational approach, first I will study the elementary steps for the half reaction to determine the rate limiting step, then by introducing mutations in the active site of SO I will determine the effects of the hydrogen donors present in the active site.SO is part of the Molybdenum cofactor family of enzymes as it contains a mononuclear Molybdenum (Mo) that is penta‐coordinated [4]. At the Mo active site, the reductive half reaction of sulfite to sulfate takes place in two steps. The first step is a nucleophilic attack by the sulfite ion to the equatorial oxygen group. The second step is the release of sulfate facilitated by the spectator oxygen group [4]. By determining the energy profiles for both steps in this reaction I will determine which step is rate limiting and determine the route, associative or dissociative interchange, of the reaction.Model enzyme complexes have suggested that hydrogen donors in the active site play a key role in mediating this reaction. In previous studies performed we determined that the product release step is facilitated by pyridinium a hydrogen donor for the reaction. Pyridinium lowers the activation energy for the product release step (ΔH~ 30 kJ/mol) and promotes a dissociative interchange reaction route. In this study I will determine if hydrogen donors in the Mo active site behave as pyridinium does in the model enzyme complexes.Using Gaussian ONIOM to perform QM/MM calculations for the reaction of sulfite to sulfate catalyzed by SO, I will determine the energy profile of the reaction to identify the rate limiting step and the role of possible hydrogen donors in the active site of SO by comparing the reaction mechanism of two variants of SO with wild type SO. The variants will demonstrate the role as possible hydrogen bond donors of Tyrosine and water that are located in the active site of MO.The role of sulfite oxidase is crucial to the degradation of sulfite in the body and prevention of the accumulation of toxic sulfite in the body. Understanding how SO functions at a molecular level is crucial for the design and implementation of treatment for SO deficiency. The elementary steps of the enzyme activity can help determine specific roles of key residues in the enzyme. These steps cannot be seen in large scale enzyme kinetics studies, but computational studies allow the study of small movements and allow the study of these small‐scale reaction mechanics.Support or Funding InformationLouis Stokes Alliance for Minority Participation (LSAMP) HRD # 1305011This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structure and Enzymatic Properties of an Unusual Cysteine Tryptophylquinone-Dependent Glycine Oxidase from Pseudoalteromonas luteoviolacea.

Glycine oxidase from Pseudoalteromonas luteoviolacea (PlGoxA) is a cysteine tryptophylquinone (CTQ)-dependent enzyme. Sequence analysis and phylogenetic analysis place it in a newly designated subgroup (group IID) of a recently identified family of LodA-like proteins, which are predicted to possess CTQ. The crystal structure of PlGoxA reveals that it is a homotetramer. It possesses an N-terminal domain with no close structural homologues in the Protein Data Bank. The active site is quite small because of intersubunit interactions, which may account for the observed cooperativy toward glycine. Steady-state kinetic analysis yielded the following values: kcat = 6.0 ± 0.2 s-1, K0.5 = 187 ± 18 μM, and h = 1.77 ± 0.27. In contrast to other quinoprotein amine dehydrogenases and oxidases that exhibit anomalously large primary kinetic isotope effects on the rate of reduction of the quinone cofactor by the amine substrate, no significant primary kinetic isotope effect was observed for this reaction of PlGoxA. The absorbance spectrum of glycine-reduced PlGoxA exhibits features in the range of 400-650 nm that have not previously been seen in other quinoproteins. Thus, in addition to the unusual structural features of PlGoxA, the kinetic and chemical reaction mechanisms of the reductive half-reaction of PlGoxA appear to be distinct from those of other amine dehydrogenases and amine oxidases that use tryptophylquinone and tyrosylquinone cofactors.

A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH2-H4folate) to produce NAD+ and 5-methyltetrahydrofolate (CH3-H4folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes Ered(wild-type)•NADH and Eox(Glu28Gln)•CH3-H4folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6–10 fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme.

Computational exploration of reactive fragment for mechanism-based inhibition of xanthine oxidase

The xanthine oxidase (XO) family is a group of molybdenum-containing enzymes that catalyze the transfer of an oxygen atom from water to a substrate, such as xanthine or aldehyde. The proposed mechanism for the reductive half-reaction of xanthine oxidase involves nucleophilic addition of Mo-bound hydroxide to the substrate and hydride transfer from the substrate to sulfido group (Mo=S) at the molybdenum cofactor (Moco) reaction center. A DFT study of a mechanism involving the near-by glutamic acid (Glu) residue for the oxidation of several model substrates found that the direct involvement of the Glu residue side chain significantly increases the reactivity of the cofactor. The reaction is stepwise with a hydride transfer as the rate determining step. On the basis of mechanistic studies, a QM model was established to search the potential reactive fragments for mechanism-based inhibitors. Two important parameters were designed to evaluate each fragment: the activation enthalpy (ΔH≠) which estimates how effectively an inhibitor competes with xanthine to react with the Moco site, and the enthalpy associated with release of the product (ΔH) which estimates how easily the product in product complex (PRO) could be replaced by a water molecule. The computational findings add to our understanding of the catalytic mechanism of xanthine oxidase, and also assist the screening of mechanism-based inhibitors for this important drug target.

Modeling the Transition State Structures of the Reductive-Half Reaction Active Site of Xanthine Oxidase Bound to Guanine Analogues: A Density Functional Theory Approach

Modeling and characterization the transition state structure of enzyme catalyzed reactions is essential. A DFT method employing B3LYP level of theory with 6-31G (d',p') basis set for non-metals and LanL2DZ basis set for molybdenum have been used. The bond orders of chemical fragments were calculated using AOmix softaware. The effect of chalcogen replacement, amine group and methyl group in the parent structure of xanthine bound to xanthine oxidase active site were compared. The transition state structure of model substrates (2AX, 2A6TP, 2A6SP and 2A6MP) bound to the truncated form of XO active site has been confirmed by the presence of one negative imaginary frequencies (s-1) (-60), (-140), (-230) and (-270), respectively. The corresponding normalized energy barriers (kcal/mol) from pre-transition state to the transition state, respectively, are (13.869), (21.753), (23.109) and (0.212). In this work, 2A6SP and 2A6TP substrates were found to be potential xanthine oxidase inhibitors. The large bond distances and minimum bond order for CRH-HRH bond, and small bond distances and maximum bond order for SMo-HRH bond at the transition state for chalcogen replaced 2AX confirms early transition state structure. Methyl substituted 2AX analog found to have post transition state structure. A potential xanthine oxidase inhibitor can be designed from purine family enzymes using DFT approach.

Cocatalysts in Semiconductor-based Photocatalytic CO2 Reduction: Achievements, Challenges, and Opportunities.

Ever-increasing fossil-fuel combustion along with massive CO2 emissions has aroused a global energy crisis and climate change. Photocatalytic CO2 reduction represents a promising strategy for clean, cost-effective, and environmentally friendly conversion of CO2 into hydrocarbon fuels by utilizing solar energy. This strategy combines the reductive half-reaction of CO2 conversion with an oxidative half reaction, e.g., H2 O oxidation, to create a carbon-neutral cycle, presenting a viable solution to global energy and environmental problems. There are three pivotal processes in photocatalytic CO2 conversion: (i) solar-light absorption, (ii) charge separation/migration, and (iii) catalytic CO2 reduction and H2 O oxidation. While significant progress is made in optimizing the first two processes, much less research is conducted toward enhancing the efficiency of the third step, which requires the presence of cocatalysts. In general, cocatalysts play four important roles: (i) boosting charge separation/transfer, (ii) improving the activity and selectivity of CO2 reduction, (iii) enhancing the stability of photocatalysts, and (iv) suppressing side or back reactions. Herein, for the first time, all the developed CO2 -reduction cocatalysts for semiconductor-based photocatalytic CO2 conversion are summarized, and their functions and mechanisms are discussed. Finally, perspectives in this emerging area are provided.

Acid-facilitated product release from a Mo(IV) center: relevance to oxygen atom transfer reactivity of molybdenum oxotransferases.

We report that pyridinium ions (HPyr+) accelerate the conversion of [Tp*MoIVOCl(OPMe3)] (1) to [Tp*MoIVOCl(NCCH3)] (2) by 103-fold, affording 2 in near-quantitative yield; Tp*=hydrotris(3,5-dimethyl-1-pyrazolyl)borate. This novel reactivity and the mechanism of this reaction were investigated in detail. The formation of 2 followed pseudo-first-order kinetics, with the observed pseudo-first-order rate constant (k obs) linearly correlated with [HPyr+]. An Eyring plot revealed that this HPyr+-facilitated reaction has a small positive value of ∆S ‡ indicative of a dissociative interchange (Id)mechanism, different from the slower associative interchange (Ia)mechanism in the absence of HPyr+ marked with a negative ∆S ‡. Interestingly, log(k obs) was found to be linearly correlated to the acidity of substituted pyridinium ions. This novel reactivity is further investigated using combined DFT and ab initio coupled cluster methods. Different reaction pathways, including Id, Ia, and possible alternative routes in the absence or presence of HPyr+, were considered, and enthalpy and free energies were calculated for each pathway. Our computational results further underscored that the Id route is energetically favored in the presence of HPyr+, in contrast with the preferred Ia-NNO pathway in the absence of HPyr+. Our computational results also revealed molecular-level details for the HPyr+-facilitated Id route. Specifically, HPyr+ initially becomes hydrogen-bonded to the oxygen atom of the Mo(IV)-OPMe3 moiety, which lowers the activation barrier for the Mo-OPMe3 bond cleavage in a rate-limiting step to dissociate the OPMe3 product. The implications of our results were discussed in the context of molybdoenzymes, particularly the reductive half-reaction of sulfite oxidase.