Habib and Hofman Chemistry Central Journal (2017) 11:35 DOI 10.1186/s13065-017-0263-7 RESEARCH ARTICLE Open Access Efect of molybdenum and tungsten on the reduction of nitrate in nitrate reductase, a DFT study Uzma Habib1* and Matthias Hofman2 Abstract The molybdenum and tungsten active site model complexes, derived from the protein X-ray crystal structure of the frst W-containing nitrate reductase isolated from Pyrobaculum aerophilum, were computed for nitrate reduction at the COSMO-B3LYP/SDDp//B3LYP/Lanl2DZ(p) energy level of density functional theory. The molybdenum containing active site model complex (Mo–Nar) has the largest activation energy (34.4 kcal/mol) for the oxygen atom transfer from the nitrate to the metal center as compared to the tungsten containing active site model complex (W–Nar) (12.0 kcal/mol). Oxidation of the educt complex is close to thermoneutral ( 1.9 kcal/mol) for the Mo active site model complex but strongly exothermic ( 34.7 kcal/mol) for the W containing active− site model complex, however, the M­ VI to ­MIV reduction requires equal amount− of reductive power for both metal complexes, Mo–Nar or W–Nar. Keywords: Nitrate reductase, DFT studies, Molybdenum, Tungsten Background and type of reaction they catalyze, these mononuclear Molybdenum and tungsten are the only 4d (Mo) and 5d MPT containing enzymes have been grouped into three (W) transition metals prefer to be essential for biologi- subfamilies (Fig. 1), xanthine oxidase family, sulfte oxi- cal systems. Mononuclear enzymes containing Mo or dase family, and DMSO (dimethylsulfoxide) reductase W at their active sites generally catalyze oxygen atom family [1]. transfer reactions [1, 2]. Despite the high similarity Nitrate reductases (NRs) play key roles in the frst between the chemical properties of Mo and W, W-con- step of biological nitrogen cycles [7–9] i.e., assimilatory taining enzymes are by far less common. Mo-containing ammonifcation (to incorporate nitrogen into biomol- enzymes are found in almost all forms of life [1], whereas ecules), denitrifcation (to generate energy for cellular W-containing enzymes seem to be popular for organisms function) and dissimilatory ammonifcation (to dissipate such as hyperthermophilic archaea that live in extreme extra energy by respiration). Tey always catalyze the environments [2]. However, W-containing enzymes have reduction of nitrate to nitrite, and have been classifed also been found in organisms that do not need extreme into three groups, assimilatory nitrate reductases (Nas), conditions [3–5], suggesting a more important role for respiratory nitrate reductases (Nar) and periplasmic tungsten [6]. nitrate reductases (Nap). Nas belongs to the sulfte oxi- Mononuclear enzymes contain a cofactor that com- dase family and is located in the cytoplasm [10]. It is the prises metallopterin (MPT) or some of its nucleotide frst enzyme of a reduction sequence for nitrogen incor- variants, each of which is coordinated to Mo or W with poration into the biomass that maintains the bioavailabil- an enedithiolene motif. Based on the active site structure ity of nitrate to plants, algae, fungi, archaea and bacteria [11, 12]. Dissimilatory nitrate reductases, Nar and Nap belong to the DMSO reductase family of mononuclear *Correspondence: [email protected] MPT containing molybdo-enzymes. Tey are linked to 1 Research Center for Modeling and Simulation (RCMS), National University of Science and Technology (NUST), H‑12, Islamabad, Pakistan respiratory electron transport systems and are located in Full list of author information is available at the end of the article the membrane and periplasm, respectively. Tey catalyze © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Habib and Hofman Chemistry Central Journal (2017) 11:35 Page 2 of 12 Fig. 1 Active site composition of subfamilies of mononuclear Mo/W enzymes the frst step of the catabolic, anaerobic respiration path- termed as NarGH where NarG hosts the metal (Mo or way in bacteria and archaea [14]. W) catalytic site. Te metal is coordinated by two metal- Nitrate reduction, catalyzed by membrane bound res- lopterin guanine dinucleotide (bis-MGD) ligands, a car- piratory nitrate reductase (Nar), is an important step of boxyl group of Asp222 and a water molecule. Te NarH the denitrifcation in the anaerobic respiratory pathways component possesses an iron–sulfur (FeS) redox active employed by a diverse group of bacteria and archaea [13]. subunit [19]. Nar was found to contain a Mo cofactor in all microbes NarGH reduces nitrate to nitrite, changing the oxida- from which it was isolated and belongs to the DMSO tion state of metal from +IV to +VI. Two electrons and reductase family [14]. In general, Nar becomes inac- two protons are required for the reductive half reaction, 2− tive by the addition of tungstate (WO4 ) to the growth resulting in the formation of a water molecule and a medium [15], although due to similar chemical proper- nitrite ion (Eq. 1). ties W can replace Mo as the active site metal and can- − + − − NO3 + 2H + 2e ⇋ NO2 + H2O. (1) not only retain but increase its catalytic activity in E. coli TMAO reductase [16], the Desulfovibrio alaskensis for- Te active site of dissimilatory nitrate reductase (Des- mate dehydrogenase [17] and the Rhodobactercapsula- ulfovibrio desulfuricans), in the reduced state contains a tus DMSO reductase [18]. However, recently the nitrate Mo atom bound by two metalopterin dithiolene ligands reductase (Nar) from the hyperthermophilic denitrify- and a cysteinate residue. An experimental study on small ing archaeon Pyrobaculum aerophilum has been shown model complexes demonstrates that nitrate reduction by to retain its activity even at a tungsten rich environment primary (direct) oxo transfer [23] is a feasible reaction [19]. pathway (Fig. 2) [24]. Pyrobaculum aerophilum, a hyperthermophilic Here we present a density functional theory (DFT) archaeon, is naturally exposed to high levels of tung- study on model complexes derived from the protein sten, a heavy metal that is abundant in high temperature X-ray crystal structure of P. aerophilum [19] nitrate environments. Tungsten was reported to stimulate the reductase (Nar). Te purpose of the study was to inves- growth of several mesophilic methanogens and some tigate (i) the efect on the reduction of nitrate when W mesophilic and thermophilic bacteria [14]. Te growth of replaces Mo at the active site, (ii) the energy barriers on P. aerophilum also depends on the presence of tungstate the potential energy surface and (iii) the reason for the in the growth medium which suggests the involvement of activity loss of Nars (respiratory nitrate reductase) in the tungstoenzymes in essential metabolic pathways [20]. presence of W. Pyrobaculum aerophilum is the only hyperthermo- philic archaeon isolated that reduces nitrate via a mem- Computational details brane bound respiratory nitrate reductase (Nar) [20]. All geometries were optimized using Gaussian 09 with Nar purifed from P. aerophilum grown in the absence of the hybrid density functional B3LYP [25] and the LAN- 2− added molybdate (MoO4 ) and with 4.5 µM tungstate L2DZ basis set [26–29] augmented by polarization func- 2− (WO4 ) is a tungsten containing enzyme, which is iden- tions on sulfur atoms (ζ = 0.421) [30]. Te starting nitrate tical to Mo-Nar [21] (previously isolated from P. aerophi- complex geometries for transition state searches were lum), indicating that either metal can serve as the active generated by shortening and lengthening of forming and site ion. Te crystal structure is similar to the previously breaking bonds, respectively. Frequency calculations reported Nar from E. coli [22], a heterodimeric enzyme proved transition states to have exactly one imaginary Habib and Hofman Chemistry Central Journal (2017) 11:35 Page 3 of 12 Fig. 2 Schematic description of the proposed mechansim [1] for the nitrate reduction, where M Mo and Y S–Cys. Also the metalopterin dinu- = = cleotide cofactor is shown frequency with the correct transition vector. Single point nitrogen atoms attached to the beta (β) carbon atoms of energies were computed with the B3LYP functional and His546, Asn52, Tyr220 and Asp222 were kept fxed to their the Stuttgart–Dresden efective core potential basis set crystal structure positions to mimic the steric constraints (SDD) [31, 32] augmented by polarization functions for by the protein matrix. Carbon atom C 7 and the nitrogen all atoms except Mo, W and H (ζ = 0.600, 1.154, 0.864, atom attached to carbon atom C5 were kept fxed for resi- and 0.421 for C, O, N, and S, respectively) [30]. Self- due Gly549. Te MPT ligands were truncated at the pyran consistent reaction feld (SCRF) computations were per- rings and oxygen atoms of these pyran rings were also formed on the optimized geometries to model the protein kept fxed (Fig. 3). surrounding the active site by a conductor like polariz- First, hydrogen atoms were geometry optimized apply- able continuum method (CPCM) [33] as implemented ing one negative overall charge (assuming Mo/W at the in Gaussian 09 [34, 35]. Default Gaussian 03 parameters +VI oxidation state), keeping all heavy-atoms fxed at were used for the evaluation of solute–solvent dispersion their positions. Te resulting geometries served to gener- and repulsion interaction energies [36, 37], and solute ate the diferent starting geometries needed for comput- cavitation energy variations [38]. Te molecular cavity ing the mechanism for nitrate reduction. was specifed using a minimum radius (RMin) of 0.5Ǻ Te starting geometries for the substrate and product and an overlap index (OFac) of 0.8 [39].