Active Site Model Research Articles

Development of Potential Multi-Target Inhibitors for Human Cholinesterases and Beta-Secretase 1: A Computational Approach.

Alzheimer's disease causes chronic neurodegeneration and is the leading cause of dementia in the world. The causes of this disease are not fully understood but seem to involve two essential cerebral pathways: cholinergic and amyloid. The simultaneous inhibition of AChE, BuChE, and BACE-1, essential enzymes involved in those pathways, is a promising therapeutic approach to treat the symptoms and, hopefully, also halt the disease progression. This study sought to identify triple enzymatic inhibitors based on stereo-electronic requirements deduced from molecular modeling of AChE, BuChE, and BACE-1 active sites. A pharmacophore model was built, displaying four hydrophobic centers, three hydrogen bond acceptors, and one positively charged nitrogen, and used to prioritize molecules found in virtual libraries. Compounds showing adequate overlapping rates with the pharmacophore were subjected to molecular docking against the three enzymes and those with an adequate docking score (n = 12) were evaluated for physicochemical and toxicological parameters and commercial availability. The structure exhibiting the greatest inhibitory potential against all three enzymes was subjected to molecular dynamics simulations (100 ns) to assess the stability of the inhibitor-enzyme systems. The results of this in silico approach indicate ZINC1733 can be a potential multi-target inhibitor of AChE, BuChE, and BACE-1, and future enzymatic assays are planned to validate those results.

Understanding the promotion effect of Pt in the reaction system of water gas shift reaction catalyzed by Pt/α-MoC from theoretical perspectives

Pt/α-MoC is an outstanding catalyst for the low-temperature water gas shift (WGS, CO + H2O → H2 + CO2) reaction, exhibiting elevated activity, stability, and selectivity. In this study, three significant active site models (α-MoC-111, Pt-111, and Pt1/α-MoC-111) in Pt/α-MoC catalyst were established to investigate the promotion effect of Pt on the WGS reaction catalyzed by Pt/α-MoC. The results indicate that a synergistic effect at the metal-carbide interface enhances chemical activity compared to pure Pt-111 and α-MoC-111. Firstly, the surface of Pt1/α-MoC-111 exhibits strong metal-support interaction between Pt single atom and α-MoC-111 surface. Compared to the α-MoC-111 surface, the introduction of Pt strengthens the Mo−Mo bond while weakening the Mo−C bond. Secondly, adding a Pt atom significantly increases adsorption strength for CO and H2O, while decreasing it for products (CO2), oxides (O atom), and by-products (C atom). The advantage of suppressing surface carbon deposition and oxidation is observed on the surface of Pt1/α-MoC-111. Furthermore, elementary reactions from the three mainstream reaction pathways of the WGS reaction are systematically calculated to evaluate catalytic performance among these three active sites. On the Pt1/α-MoC-111 surface, activation energy barriers for H2O dissociation and CO oxidation are reduced. This provides theoretical ideas for designing high-efficiency α-MoC based catalysts in the future.

Synthesis of Transition Metal-sulfur Complexes by Si-S Bond Cleavage

Abstract: This mini-review summarizes the methods available for the synthesis of transition metalsulfur complexes, which have been used as models of metalloprotein active sites, desulfurization catalysts, and organometallic functional materials. All the methods use silicon-sulfur compounds as starting materials, exploiting the selective cleavage of Si—S bond and the subsequent incorporation of sulfur fragments into the metal complex. Mechanistic considerations are also provided. Period covered: 1968 to date.

Highly Active Single-Atom Nanozymes with High-Loading Iridium for Sensitive Detection of Pesticides.

Single-atom nanozymes (SAzymes) are novel mimic-enzyme materials with atomically doped active sites. They play a pivotal role in the field of nanozymes because of their excellent catalytic activities, high utilization efficiency of the metal atoms, and simple model of active sites. Herein, the peroxidase (POD)-like SAzymes with high-loading iridium (Ir) (5.31%) on graphene oxide (GO) nanosheets [Ir(III)/GO] were prepared through a coordination reaction between the Ir(III) complex and the oxygen-containing groups in GO. The preparation strategy avoids nitrogen doping and pyrolysis procedures which are the usually used strategies to improve the GO-based enzyme mimic activity. Ascribed to the highly active Ir atoms, Ir(III)/GO SAzymes demonstrate outstanding POD-like activity without the oxidase-like activity. In advantage of the excellent POD-like activity, a simple and sensitive colorimetric pesticide detection platform is established. The developed sensing platform offers an excellent "switch-on" pirimicarb (PIB) detection in the linear range of 10-300 nM with a limit of detection (LOD) of 2.81 nM. Moreover, the detection platform was fabricated into a portable test kit, which is composed of a test swab and sample processing tube. In the aid of a color-reading APP, the test kit can detect PIB with the LOD of 3.31 nM. It is astonishing to get this excellent detection sensitivity just using the simple colorimetric strategy. This work not only provides a novel strategy to synthesize Ir-based SAzymes but also exhibits the super capability of Ir(III)/GO in the biosensing field.

Hybrids of [FeFe]- and [NiFe]-H2ase Active Site Models.

Complexes of the type (diphosphine)Ni(μ-SR)2Fe(CO)3 are investigated with azadithiolate (adt, HN(CH2S-)2) as the dithiolate. The resulting complexes are hybrid models for the active sites of the [NiFe]- and [FeFe]-hydrogenases. The key complex (dppv)Ni(μ-adt)Fe(CO)3 (3) was prepared from the complex Ni[(SCH2)2NCbz](dppv), which contains a Cbz-protected adt ligand (Cbz = C(O)OCH2Ph, dppv = cis-1,2-(Ph2P)2C2H2). This complex combines with Fe2(CO)9 to give (dppv)Ni[(μ-SCH2)2NCbz]Fe(CO)3, which is readily deprotected to give 3. Complex 3 undergoes protonation at both Fe and N to give successively [(dppv)Ni(μ-adt)FeH(CO)3]+ ([H3]+) and [(dppv)Ni(μ-adtH)FeH(CO)3]2+ ([H3H]2+). The redox properties and dynamics of these complexes resemble previously reported analogues with propanedithiolate. Solutions of [H3]+ readily degrade to [(dppv)Ni[(μ-SCH2)2NCH2]Fe(CO)3]+ ([4]+), which features a methylene group linking N and Fe. Complex [4]+ can be made in high yield by reaction of [H3]+ with CH2O, and this conversion was also demonstrated with 13CH2O. Complex [4]+ undergoes hydrogenolysis by photochemical reaction with H2 to give [(dppv)Ni[(μ-SCH2)2NMe]FeH(CO)3]+, the N-methylated analogue of [H3]+. Upon treatment ith Me3O+, [4]+ undergoes quaternization, giving [(dppv)Ni[(μ-SCH2)2N(Me)CH2]Fe(CO)3]2+. In contrast with the lability of [H3]+, the phosphine-substituted derivative [(dppv)Ni(μ-adt)FeH(CO)2(PPh3)]+ did not degrade. Most complexes were characterized by X-ray crystallography.

NO Generation from Nitrite at Zinc(II): Role of Thiol Persulfidation in the Presence of Sulfane Sulfur.

Nitrite-to-NO transformation is of prime importance due to its relevance in mammalian physiology. Although such a one-electron reductive transformation at various redox-active metal sites (e.g., Cu and Fe) has been illustrated previously, the reaction at the [ZnII] site in the presence of a sacrificial reductant like thiol has been reported to be sluggish and poorly understood. Reactivity of [(Bn3Tren)ZnII-ONO](ClO4) (1), a nitrite-bound model of the tripodal active site of carbonic anhydrase (CA), toward various organic probes, such as 4-tert-butylbenzylthiol (tBuBnSH), 2,4-di-tert-butylphenol (2,4-DTBP), and 1-fluoro-2,4-dinitrobenzene (F-DNB), reveals that the ONO-moiety in the [ZnII]-nitrite coordination motif of complex 1 acts as a mild electrophile. tBuBnSH reacts mildly with nitrite at a [ZnII] site to provide S-nitrosothiol tBuBnSNO prior to the release of NO in 10% yield, whereas the phenolic substrate 2,4-DTBP does not yield the analogous O-nitrite compound (ArONO). The presence of sulfane sulfur (S0) species such as elemental sulfur (S8) and organic polysulfides (tBuBnSnBntBu) during the reaction of tBuBnSH and [ZnII]-nitrite (1) assists the nitrite-to-NO conversion to provide NO yields of 65% (for S8) and 76% (for tBuBnSnBntBu). High-resolution mass spectrometry (HRMS) analyses on the reaction of [ZnII]-nitrite (1), tBuBnSH, and S8 depict the formation of zinc(II)-persulfide species [(Bn3Tren)ZnII-Sn-BntBu]+ (where n = 2, 3, 4, 5, and 6). Trapping of the persulfide species (tBuBnSS-) with 1-fluoro-2,4-dinitrobenzene (F-DNB) confirms its intermediacy. The significantly higher nucleophilicity of persulfide species (relative to thiol/thiolate) is proposed to facilitate the reaction with the mildly electrophilic [ZnII]-nitrite (1) complex. Complementary analyses, including multinuclear NMR, electrospray ionization-MS, UV-vis, and trapping of reactive S-species, provide mechanistic insights into the sulfane sulfur-assisted reactions between thiol and nitrite at the tripodal [ZnII]-site. These findings suggest the critical influential roles of various reactive sulfur species, such as sulfane sulfur and persulfides, in the nitrite-to-NO conversion.

C-S cleavage of dibenzothiophenes with or without steric hindrance by the interface between PtSx and ZnO

Desulfurization plays a critical role in refining crude oil to produce fuel and the manufacture of fine chemicals. However, its low efficiency becomes a great challenge for the present desulfurization if only the C-S cleavage is considered a promising approach to reducing hydrogen consumption under atmospheric pressure. Accordingly, we investigate the critical factors necessary to achieve high C-S cleavage of dibenzothiophenes under mild conditions of 0.1 MPa and H2/oil = 100. The mechanism of reactive adsorption desulfurization of dibenzothiophenes with or without steric hindrance at atmospheric pressure on Pt/ZnO is proposed by studying the adsorption model of active metal sites, the Pt efficiency in C-S cleavage influenced by the steric hindrance and the structural evolution of Pt active centers in the presence of sulfur adsorption/deposition. Based on both experimental and DFT results, we report C-S cleavage of dibenzothiophenes with (TOF = 15 h−1) or without steric hindrance (TOF = 128 h−1) that can be highly efficiently fractured (99.9%) by the interface between PtSx and ZnO. This work has great practical significance for developing atmospheric industrial adsorbents for ultra-deep reactive adsorption desulfurization.

Active Site Identification for Glycerol Hydrodeoxygenation over the Oxygen Modified Molybdenum Carbide Surface

Density functional theory and microkinetic reactor modeling were used to investigate the hydrodeoxygenation (HDO) mechanism of glycerol on the oxygenated Mo2C catalyst surface to understand the activity and product selectivity under practically relevant reaction conditions. Reactor simulations with multiple active site models predicted that a fully oxygenated surface with acid–base (OH,O) pairs is active for glycerol dehydration and it can selectively cleave one C–O bond to produce 3-hydroxypropanal (HPA). The acid sites are not directly involved in the C–O bond scission process; however, surface oxygen vacancy formation is promoted in the presence of acid sites. The rate-limiting C–O bond cleavage process occurs on the exposed Mo sites with a concerted β-hydrogen transfer to the nearest conjugate base (Mo–O) via an E2 elimination mechanism. Dehydration of HPA to acrolein was observed at longer residence times. Our analysis revealed that reaction conditions, such as temperature and partial pressure of H2, can be tuned to promote further deoxygenation of HPA and acrolein to produce propanal and propylene.

Micro-mechanism insights into the adsorption of anionic dyes using quaternary ammonium-functionalised chitosan aerogels

The development of adsorbents with outstanding adsorption capacities, wide versatility, and excellent recyclability for the removal of organic dyes remains a challenge. In this study, a quaternised chitosan-based aerogel (QCSA) was fabricated via a facile method to effectively treat concomitant anionic dyes. Porous QCSA with high hydrophilicity, nontoxicity, excellent thermal stability, and sustainability exhibits adsorption properties superior to most previously reported adsorbents. The equilibrium adsorption capacities for Congo red, Sunset yellow, and Methyl orange were 1259.6, 550.2, and 607.5 mg/g, respectively. Notably, the spent QCSA exhibits excellent cyclic performance. The multilayer adsorption, external–internal mass transfer resistance, and adsorption on the active site models were employed to enable a more accurate description of the dynamic characteristics, confirming that double-layer chemisorption was the dominant process. A quantitative analysis of the electrostatic potential and the independent gradient model further verified that electrostatic interactions, hydrogen bonding, and van der Waals forces led to the highly efficient adsorption of dye molecules. Therefore, the eco-friendly and recyclable QCSA is a promising adsorbent for trapping anionic dyes from aquatic systems.

Metagenomic Type IV Aminotransferases Active toward (R)-Methylbenzylamine

Aminotransferases (ATs) are pyridoxal 5′-phosphate-dependent enzymes that catalyze the reversible transfer of an amino group from an amino donor to a keto substrate. ATs are promising biocatalysts that are replacing traditional chemical routes for the production of chiral amines. In this study, an in silico-screening of a metagenomic library isolated from the Curonian Lagoon identified 11 full-length fold type IV aminotransferases that were successfully expressed and used for substrate profiling. Three of them (AT-872, AT-1132, and AT-4421) were active toward (R)-methylbenzylamine. Purified proteins showed activity with L- and D-amino acids and various aromatic compounds such as (R)-1-aminotetraline. AT-872 and AT-1132 exhibited thermostability and retained about 55% and 80% of their activities, respectively, even after 24 h of incubation at 50 °C. Active site modeling revealed that AT-872 and AT-4421 have an unusual active site environment similar to the AT of Haliscomenobacter hydrossis, while AT-1132 appeared to be structurally related to the AT from thermophilic archaea Geoglobus acetivorans. Thus, we have identified and characterized PLP fold type IV ATs that were active toward both amino acids and a variety of (R)-amines.

Rationally designed Au-ZrOx interaction for boosting 5-hydroxymethylfurfural oxidation

Upgrading bioderived 5-hydroxymethylfurfural (HMF) to high value-added bioplastic monomer 2,5-furandicarboxylic acid (FDCA) is a promising route for biomass conversion. Developing an efficient supported metal catalyst and clarifying the structure–activity relationship between the support and active sites is the key to boost FDCA production. Herein, ZrO2 coated on natural and abundant halloysite nanotubes (HNTs) was synthesized and employed as a support for Au nanoparticles loading. The Au-ZrOx interface active sites were successfully engineered, and the active site amount can be rationally tailored by adjusting the crystal phases of ZrO2 via changing calcination temperature. A combination study of catalysts surface physicochemical properties and catalytic activity reveals that oxygen vacancies (Ov) play a vital role in enhancing the adsorption of O2. Meanwhile, interface active sites facilitate the chemisorption and activation of O2 molecules, which makes it easier to produce oxygen active species (superoxide radicals). More importantly, in-situ infrared spectroscopy and adsorption experiments also demonstrate that the catalyst with a higher Ov concentration not only contributes to a stronger Lewis acid intensity, but also enhances the adsorption capacity of a key intermediate 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), thus leading to an acceleration of the rate-determining step of hydroxyl group oxidation. The highest 99.4 % of FDCA yield can be obtained in 3.0 h. This newly proposed model of interface active sites may shed a light on the design of novel heterogeneous catalysts for the related biomass oxidation reactions.

Reduction of Nitric Oxide to Nitrous Oxide in Flavodiiron Proteins: Catalytic Mechanism and Plausible Intermediates

The flavin dependent nonheme diiron proteins comprise a family of enzymes, which can act as scavengers for both molecular oxygen and nitric oxide. The reduction of nitric oxide to nitrous oxide and water in flavodiiron proteins (FDPs) has been studied both experimentally and computationally, but the reaction mechanism is far from well understood. From experiments, it is known that two NO molecules can bind to the reduced active site, forming an observable diferrous dinitrosyl complex. A main question has been whether nitrous oxide can be formed directly from the diferrous dinitrosyl complex or if further reduction and/or protonation is needed to make this step feasible. Experiments have shown that nitrous oxide can be formed in a deflavinated form of the enzyme, indicating that further reduction is not needed. In the present study, hybrid density functional theory calculations are performed on a cluster model of the Thermotoga maritima FDP active site. We show that nitric oxide can be reduced to nitrous oxide and water using a direct coupling mechanism, i.e., without further additions to the reduced active site. The diferrous dinitrosyl complex can form an unstable N–N bridging hyponitrite intermediate, which can rotate into an N–O bond bridging hyponitrite with a low barrier. From this intermediate, the N–O bond cleavage leading to release of nitrous oxide is energetically feasible. An energy profile for the entire catalytic cycle of such a direct coupling mechanism is presented, and it is shown that the suggested mechanism agrees with data on FDP variants. Finally, an energy profile for the entire process starting with the fully reduced enzyme turning over four NO equivalents is constructed. This energy profile suggests explanations to experimentally observed states, such as the dihydroxyl form of the fully oxidized diferric state, and the difference with respect to returning to the original oxidized state after NO reduction between the flavinated and the deflavinated form of the enzyme.

Hyperbranched polyethyleneimine-functionalised chitosan aerogel for highly efficient removal of melanoidins from wastewater

Melanoidins are hazardous dark-coloured substances contained in molasses-based distillery wastewater. Adsorption is an effective approach to eliminate melanoidins from wastewater. However, melanoidin adsorption capacities of available adsorbents are unsatisfactory, which seriously limits their practical application. A hyperbranched polyethyleneimine-functionalised chitosan aerogel (HPCA) was fabricated as an effective adsorbent for melanoidin scavenging. HPCA demonstrated superior melanoidin adsorption efficiency because of its high specific surface area, abundant amino functional groups, and high hydrophilicity. Melanoidin removal rate of HPCA was 94.95%, which remained at 91.45% after 5 cycles. Notably, using the Langmuir isothermal model, the maximum melanoidin adsorption capacity of HPCA was determined to be 868.36 mg/g, surpassing those of most of previously reported adsorbents. Toxicity experiments indicated that HPCA can be considered a safe adsorbent with excellent biocompatibility that hardly threatens aquatic organisms. The efficient melanoidin removal of HPCA was attributed to electrostatic attraction, H-bonding, and van der Waals force. However, the adsorption might be predominantly controlled by electrovalent interaction between protonated amino groups of HPCA and carboxyl/carboxylate groups of melanoidins. Two novel models, namely, external diffusion resistance-internal diffusion resistance mixed model and adsorption on active site model, were employed to describe the dynamic mass transfer characteristics of melanoidin adsorption by HPCA.

Insights into mass transfer mechanism and micro-interaction of melanoidin adsorption on polyethyleneimine-functionalised pomelo-peel-derived aerogel

Melanoidins are recalcitrant colouring and polluting polymers present in the effluent streams of molasses-based distilleries. Adsorption is the most prospective technology with ease of operation and high efficiency. Herein, the removal of melanoidins from spent wash was investigated by synthesising a polyethyleneimine (PEI) functionalised pomelo-peel (PP) derived aerogel (PEI-PPA). The PEI-PPA is distinguished by the porous network structure and abundant accessible functional groups (mainly protonated primary amine) that are conducive to the elimination of colouring contaminants (melanoidins). The incorporation of PEI into supporting matrices (i.e., PP–derived aerogel) effectively improved the melanoidin adsorption capacity (from a previous-best research of 230 mg/g to the current 615.70 mg/g), while the melanoidin removal rate reached 92.36 %. The independent gradient model and Hirshfeld surface analysis were used to investigated the possible electroweak interaction relationship between PEI-PPA and melanoidin. By analysing the number of melanoidin molecules bonded to each active site using a novel double-layer adsorption model, a mixed inclination (parallel and non-parallel orientation) of melanoidin onto the PEI-PPA surface was found. For the two mass transfer models fitted results, the adsorption onto the active sites (AOAS) model better described the behaviour of melanoidin adsorption by PEI-PPA than the external diffusion resistance–internal diffusion resistance (EDR–IDR) mixed model under the same conditions, revealing the AOAS was rate-controlling during the mass transfer process. AOAS both contained chemisorption and physisorption. The calculated rates of chemisorption were far higher than those of physisorption before equilibration, indicating chemisorption was main the rate-determining step. Altogether, this work proposed an effective conversion for PP into bio-adsorbent and provided a detailed analysis of mass transfer mechanism.

EFEITO DOS DEFEITOS DE SUPERFÍCIE E DO pH NA ADSORÇÃO E NA CATÁLISE DA ELETRO-OXIDAÇÃO DE CO EM SUPERFÍCIES MODELOS DE PLATINA

SURFACE DEFECTS AND pH EFFECTS ON ADSORPTION AND CATALYSIS OF CO ELECTRO-OXIDATION ON MODEL PLATINUM SURFACES. Platinum is one of the well-known catalytic materials for which the electro-oxidation of carbon monoxide better behaves as a sensitive reaction to the catalyst surface structure. For the electro-catalytic reactions that behave like this, the rate (faradaic current density) is the result of the sum of the activity of the different active sites working with very different efficiencies or abilities. In this scenario, different atomic arrangements on the catalyst surface are expected to play different roles in surface-catalyzed reactions. In this article, the functionalities that surface defects (steps) can play in the adsorption and electrocatalytic oxidation of CO on model platinum surfaces are reviewed. Surface defects are indirectly related to the up catalysis as well as to the inhibition of reaction pathways of CO electro-oxidation under very particular conditions; these surface entities are also indirectly related to restrictions for the mobility of adsorbed CO on the (111) terraced surfaces. We analyze the selective activation and deactivation of surface sites by the pH effect, and typical catalytic properties of extended surfaces and shaped-controlled nanoparticles have been discussed thoroughly. We present a model of most active sites involved in the pathways of CO2 formation from the electro-oxidation of adsorbed CO.

Morita–Baylis–Hillman reaction: how do optimal enzyme active sites compare with organocatalysts

Theoretical active site models for the Morita–Baylis–Hillman reaction uncover the importance of cooperativity between the oxyanion hole and the acid–base catalyst in catalyzing multiple steps via minimal reorganization.

Kinetically favorable edge-type iron–cobalt atomic pair sites synthesized via a silica xerogel approach for efficient bifunctional oxygen electrocatalysis

The edge-type Fe–Co atomic pair structure as the main active site model decreases the energy gap and modifies charge transfer, affording superior bifunctional activity for the ORR and OER.

FeFe]‐Hydrogenase models featuring dithiolato‐bridgehead functionality: Preparation, structures, and electrocatalytic proton reduction

AbstractTo further develop the active site models of [FeFe]‐hydrogenases, a new series of diiron model complexes [{(μ‐SCH2)2CHCH2OC(O)C6H4I)}Fe2(CO)6] (I is located at ‐para for 1, ‐meta for 2, and ‐ortho for 3) bearing dithiolato‐bridgehead functionality were successfully prepared by treatments of bridghead‐hydroxyl‐containing parent complex [{(μ‐SCH2)2CHCH2OH)}Fe2(CO)6] (A) and three different iodobenzoic acids (IC6H4CO2H) in the presence of 4‐dimethylaminopyridine (DMAP) as catalyst and dicyclohexylcarbodiimide (DCC) as dehydrating reagent. All these new complexes 1–3 have been fully characterized through elemental analysis, FT‐IR and NMR (1H, 13C) spectroscopies, and X‐ray crystallography. Further electrochemical and electrocatalytic properties of complexes 1–3 are studied and compared in the absence and presence of acetic acid (HOAc) as a proton source by cyclic voltammetry (CV), indicating that they may be considered as the active biomimetic electrocatalysts for proton reduction to hydrogen (H2).

Calculation of Resonance Raman Spectra and Excited State Properties for Blue Copper Protein Model Complexes

Laccase enzymes have been investigated for their potential as eco-friendly biofuel cell cathode catalysts. In these applications, electrons transfer from the electrode directly to the so-called Type 1 (T1) redox site within the enzyme. Over the years, resonance Raman (RR) spectroscopy has been utilized to probe the nature of ligand interactions with the Cu2+ center at the T1 site of the resting, oxidized form of laccases and related blue copper proteins. Spectral interpretation has been guided by parallel studies of small molecule T1 site mimics. Reported herein are results of time-dependent density functional theory (TDDFT) calculations performed on a series of experimentally well-studied T1 site mimics based on Cu2+ thiolate complexes stabilized by either a tridentate tris(pyrazolyl)hydroborate ligand or a bidentate β-diketiminate ligand. Vertical excitation energies were computed for the first 10 doublet states of each complex. The electronic absorption and RR spectra derived show excellent agreement with the experiment. RR spectra were dominated by a strong Cu–S stretching transition near 400 cm–1, which is sensitive to molecular conformation within the thiolate ligand. The results provide a foundation to build from in advancing models of laccase catalytic active sites in support of sustainable technologies.

Microbial Denitrification: Active Site and Reaction Path Models Predict New Isotopic Fingerprints.

The study of isotopic fingerprints in nitrate (δ15N, δ18O, Δ17O) has enabled pivotal insights into the global nitrogen cycle and revealed new knowledge gaps. Measuring populations of isotopic homologs of intact NO3 - ions (isotopologues) shows promise to advance the understanding of nitrogen cycling processes; however, we need new theory and predictions to guide laboratory experiments and field studies. We investigated the hypothesis that the isotopic composition of the residual nitrate pool is controlled by the N-O bond-breaking step in Nar dissimilatory nitrate reductase using molecular models of the enzyme active sites and associated kinetic isotope effects (KIEs). We integrated the molecular model results into reaction path models representing the reduction of nitrate under either closed-system or steady-state conditions. The predicted intrinsic KIE (15ε and 18ε) of the Nar active site matches observed fractionations in both culture and environmental studies. This is what would be expected if the isotopic composition of marine nitrate were controlled by dissimilatory nitrate reduction by Nar. For a closed system, the molecular models predict a pronounced negative 15N-18O clumping anomaly in residual nitrate. This signal could encode information about the amount of nitrate consumed in a closed system and thus constrain initial nitrate concentration and its isotopic composition. Similar clumped isotope anomalies can potentially be used to distinguish whether a system is open or closed to new nitrate addition. These mechanistic predictions can be tested and refined in combination with emerging ESI-Orbitrap measurements.