Effect of molybdenum and tungsten on the reduction of nitrate in nitrate reductase, a DFT study
© The Author(s) 2017
Received: 10 August 2016
Accepted: 11 April 2017
Published: 26 April 2017
The molybdenum and tungsten active site model complexes, derived from the protein X-ray crystal structure of the first 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 MVI to MIV reduction requires equal amount of reductive power for both metal complexes, Mo–Nar or W–Nar.
Molybdenum and tungsten are the only 4d (Mo) and 5d (W) transition metals prefer to be essential for biological systems. Mononuclear enzymes containing Mo or W at their active sites generally catalyze oxygen atom transfer reactions [1, 2]. Despite the high similarity between the chemical properties of Mo and W, W-containing enzymes are by far less common. Mo-containing enzymes are found in almost all forms of life , whereas W-containing enzymes seem to be popular for organisms such as hyperthermophilic archaea that live in extreme environments . However, W-containing enzymes have also been found in organisms that do not need extreme conditions [3–5], suggesting a more important role for tungsten .
Nitrate reductases (NRs) play key roles in the first step of biological nitrogen cycles [7–9] i.e., assimilatory ammonification (to incorporate nitrogen into biomolecules), denitrification (to generate energy for cellular function) and dissimilatory ammonification (to dissipate extra energy by respiration). They always catalyze the reduction of nitrate to nitrite, and have been classified into three groups, assimilatory nitrate reductases (Nas), respiratory nitrate reductases (Nar) and periplasmic nitrate reductases (Nap). Nas belongs to the sulfite oxidase family and is located in the cytoplasm . It is the first enzyme of a reduction sequence for nitrogen incorporation into the biomass that maintains the bioavailability 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 MPT containing molybdo-enzymes. They are linked to respiratory electron transport systems and are located in the membrane and periplasm, respectively. They catalyze the first step of the catabolic, anaerobic respiration pathway in bacteria and archaea .
Nitrate reduction, catalyzed by membrane bound respiratory nitrate reductase (Nar), is an important step of the denitrification in the anaerobic respiratory pathways employed by a diverse group of bacteria and archaea . Nar was found to contain a Mo cofactor in all microbes from which it was isolated and belongs to the DMSO reductase family . In general, Nar becomes inactive by the addition of tungstate (WO4 2−) to the growth medium , although due to similar chemical properties W can replace Mo as the active site metal and cannot only retain but increase its catalytic activity in E. coli TMAO reductase , the Desulfovibrio alaskensis formate dehydrogenase  and the Rhodobactercapsulatus DMSO reductase . However, recently the nitrate reductase (Nar) from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum has been shown to retain its activity even at a tungsten rich environment .
Pyrobaculum aerophilum, a hyperthermophilic archaeon, is naturally exposed to high levels of tungsten, a heavy metal that is abundant in high temperature environments. Tungsten was reported to stimulate the growth of several mesophilic methanogens and some mesophilic and thermophilic bacteria . The growth of P. aerophilum also depends on the presence of tungstate in the growth medium which suggests the involvement of tungstoenzymes in essential metabolic pathways .
Pyrobaculum aerophilum is the only hyperthermophilic archaeon isolated that reduces nitrate via a membrane bound respiratory nitrate reductase (Nar) . Nar purified from P. aerophilum grown in the absence of added molybdate (MoO4 2−) and with 4.5 µM tungstate (WO4 2−) is a tungsten containing enzyme, which is identical to Mo-Nar  (previously isolated from P. aerophilum), indicating that either metal can serve as the active site ion. The crystal structure is similar to the previously reported Nar from E. coli , a heterodimeric enzyme termed as NarGH where NarG hosts the metal (Mo or W) catalytic site. The metal is coordinated by two metallopterin guanine dinucleotide (bis-MGD) ligands, a carboxyl group of Asp222 and a water molecule. The NarH component possesses an iron–sulfur (FeS) redox active subunit .
Here we present a density functional theory (DFT) study on model complexes derived from the protein X-ray crystal structure of P. aerophilum  nitrate reductase (Nar). The purpose of the study was to investigate (i) the effect on the reduction of nitrate when W replaces Mo at the active site, (ii) the energy barriers on the potential energy surface and (iii) the reason for the activity loss of Nars (respiratory nitrate reductase) in the presence of W.
All geometries were optimized using Gaussian 09 with the hybrid density functional B3LYP  and the LANL2DZ basis set [26–29] augmented by polarization functions on sulfur atoms (ζ = 0.421) . The starting nitrate complex geometries for transition state searches were generated by shortening and lengthening of forming and breaking bonds, respectively. Frequency calculations proved transition states to have exactly one imaginary frequency with the correct transition vector. Single point energies were computed with the B3LYP functional and the Stuttgart–Dresden effective core potential basis set (SDD) [31, 32] augmented by polarization functions for all atoms except Mo, W and H (ζ = 0.600, 1.154, 0.864, and 0.421 for C, O, N, and S, respectively) . Self-consistent reaction field (SCRF) computations were performed on the optimized geometries to model the protein surrounding the active site by a conductor like polarizable continuum method (CPCM)  as implemented in Gaussian 09 [34, 35]. Default Gaussian 03 parameters were used for the evaluation of solute–solvent dispersion and repulsion interaction energies [36, 37], and solute cavitation energy variations . The molecular cavity was specified using a minimum radius (RMin) of 0.5Ǻ and an overlap index (OFac) of 0.8 .
Active site models
First, hydrogen atoms were geometry optimized applying one negative overall charge (assuming Mo/W at the +VI oxidation state), keeping all heavy-atoms fixed at their positions. The resulting geometries served to generate the different starting geometries needed for computing the mechanism for nitrate reduction.
The starting geometries for the substrate and product complexes are generated by slight distortion of M–O and O–NO2 in the optimized transition state geometries, 6a and 6b. Geometries with slightly elongated M–O distance and reduced O–NO2 distance are considered as the starting geometries for the optimization of 5a and 5b educt-substrate complexes whereas reduced M–O distance and elongated O–NO2 distance are considered as the starting geometries for the optimization of 7a and 7b product complexes. The geometry optimizations of these distorted geometries directly lead to complexes, 5a/5b and 7a/7b.
Optimized active site model complexes
The protein X-ray crystal structure of P. aerophilum Nar from the PDB data base (PDB ID: 1R27)  shows that at the active site the metal is coordinated by two metallopterin guanine dinucleotide (bis-MGD) ligands, a carboxyl group of Asp222 and a water molecule . However, the distance of the oxygen atom (Owat) of this coordinated water molecule from the metal center is 1.87 Ǻ which neither falls in the range expected for metal oxide (1.71–1.75 Ǻ) [40, 41], nor for water (2.0–2.3 Ǻ)  ligands. Also, the distance between Owat and oxygen of Asp222 (OAsp) is 1.59 Ǻ, which is only 0.1 Ǻ longer than the typical peroxo O–O− bond length (1.49 Ǻ).
Geometrical features of the optimized model complexes of the reaction mechanism for the molybdenum containing nitrate reductase
O–NO2 − (Ǻ)
Geometrical features of the optimized model complexes of the reaction mechanism for the tungsten containing nitrate reductase
O–NO2 − (Ǻ)
Distorted trigonal prismatic geometries result from geometry optimizations of oxidized model complexes 2a (2b). Optimized data show changes in the S1–S2–S3–S4 dihedral angles from −18.3° to 15.1° (20.2°) and in the M–S bond distances from ~2.455 to ~2.420 Ǻ (~ 2.417 Ǻ) as compared to the protein X-ray crystal structure (Fig. 5; Tables 1, 2). Bond distances between M–OAsp and M–OH are increased from 1.97 to 2.145 Ǻ (2.113 Ǻ) and from 1.874 to 1.990 Ǻ (1.973 Ǻ), respectively. OAsp–OOH bond distance is increased from 1.596 to 2.458 Ǻ (2.439 Ǻ) and the bond angle between OAsp, M and OOH is increased from 49° to 72.8° (73.2°).
Distorted octahedral coordination geometries result from geometry optimizations of oxidized model complexes 3a (3b).Optimized data shows increase in the S1–S2–S3–S4 dihedral angles [from −18.3° to −43.7° (−42.1°)] and in the M–S bond distances [from ~2.455 to ~2.474 Ǻ (~2.461 Ǻ)]. One M–S bond is significantly longer than the other three. However, it is the M–S2 bond (2.537 Ǻ) in the X-ray structure while it is the M–S3 bond [2.591 Ǻ (2.549)] (Fig. 5; Tables 1, 2) in the optimized oxidized model complexes. These sulfur atoms (S2 and S3, respectively) are at the trans position relative to the oxo ligand, a trans-influencing ligand which causes the elongation of the M–S bonds.
Increased bond angles between the OAsp, M and O1 [from 49° to 88° (88°)], and distances between the two oxygen atoms, OAsp–O1 [from 1.596 to 2.684 Ǻ (2.647Ǻ)] are computed in complexes 3a (3b). Slightly elongated M–OAsp distances [from 1.97 to 2.083Ǻ (2.04Ǻ)] and shortened M–O1 distances [from 1.874 to 1.755 Ǻ (1.764Ǻ)] are also observed (Fig. 5; Tables 1, 2).
Optimized reduced complexes 4a/4b
The reaction catalyzed by nitrate reductase is an oxo-transfer reaction, in which an oxygen atom is transferred from nitrate to the reduced metal. As a consequence of the metal reduction from MVI to MIV, the oxo group of the oxidized MVI is lost as hydroxo/water after proton uptake. Optimizations of the reduced active site model complexes 4a (4b) without any additional ligand, i.e. fivefold coordinate metal center give S1–S2–S3–S4 dihedral angles of −0.2° (1.3°), resulting in nearly tetragonal pyramidal geometries. The bond distances between the metal center M and S of the dithiolenes are reduced (Tables 1, 2). The M–OAsp distance is reduced to 2.017 Ǻ (1.980 Ǻ).
Optimized substrate complexes 5a/5b
First, nitrate gets loosely bound in the active site pocket by weak interactions with the active site residues Asn52 and Gly549 resulting in the substrate complexes 5a (5b) (Fig. 5). The computed reaction energies for the substrate complex formation are exothermic, −9.6 kcal/mol (−7.6 kcal/mol) in the gas phase and −4.6 kcal/mol (0.2 kcal/mol) for the polarizable continuum model. There is no significant change in geometrical parameters of the active site relative to the reduced complexes 4a (4b) (Tables 1, 2).
Optimized transition state complexes 6a/6b
Reduction of nitrate is a single step reaction in which the transfer of an oxygen atom proceeds through transition state 6a (6b).The energy barrier computed for 6a, 34.4 kcal/mol in the gas phase and 32.1 kcal/mol in the continuum, is almost three times as large as compared to that of 6b, 12.0 kcal/mol in the gas phase and 11.0 kcal/mol in the continuum. There is also a remarkable difference in the geometries. The Mo containing transition state (6a) has a distorted octahedral geometry with an S1–S2–S3–S4 dihedral angle of 30.5° and Mo–S bond lengths increased from ~2.37 to ~2.45 Ǻ (Table 1). Mo–O and O–NO2 distances are 1.918 and 1.723 Ǻ, respectively. The Mo–OAsp bond distance is elongated to 2.102 Ǻ.
The W containing transition state (6b) on the other hand has a distorted trigonal prismatic geometry where the S1–S2–S3–S4 dihedral angle is 7.6°. The W–S bond lengths are increased from ~2.37 to ~2.45 Ǻ (Table 2). The W–O and O–NO2 bond distances are 1.942 and 1.638Ǻ, respectively i.e., 6b can be considered to be an earlier transition state than 6a. The W–OAsp distance is elongated to 2.079 Ǻ.
In the optimized geometries 6a and 6b, NO3 − is coordinated to the metal at the active center and also forms a hydrogen bond to the Asn55.
Optimized product complexes 7a/7b
The nitrate reduction results in metal oxo product complexes 7a (7b), having distorted octahedral geometries. In the optimized geometries, 7a and 7b, NO2 − is loosely bound in the active site pocket and make hydrogen bonds with the active site residues Asn52 and Gly549. Oxygen atom transfer is computed to be a slightly exothermic step for M=Mo where the product complex (7a) has a relative energy of −7.6 kcal/mol in the gas phase and −1.9 kcal/mol in the continuum. The Mo–O bond distance is reduced to 1.737 Ǻ while the O–NO2 − bond is broken (4.444 Ǻ). The S1–S2–S3–S4 dihedral angle is further increased to 54.5°, the Mo–S bond distances are also increased to ~2.629 Ǻ (Table 1). The Mo–OAsp bond distance is further increased to 2.133 Ǻ.
On the contrary, the W containing product complex (7b) is highly exothermic, with computed relative energies of −43.3 kcal/mol in the gas phase and −34.7 kcal/mol in the continuum. The W–O bond distance is reduced to 1.757 Ǻ while the O−NO2 − bond is broken (5.133 Ǻ). The S1–S2–S3–S4 dihedral angle of the dithiolenes is decreased to −42.4°, whereas the W-S bond distances are increased to ~2.562 Ǻ (Table 2). There is no significant change in the W–OAsp bond distance (2.079 instead of 2.076 Ǻ).
To date, few archaeal Nars have been characterized from P. aerophilum , Haloarcula marimortui [43, 44] and Haloferax mediterranei . These archaeal Nars contain Mo cofactors at their active sites. It is not clear how these microbes maintain their ability to respire with nitrate using Mo-containing Nar in a high temperature environment that is naturally enriched with W but depleted of molybdate (MoO4 2−) . Early attempts to substitute tungsten for molybdenum in molybdo-enzymes failed because the organism was incapable of growing on the tungstate-containing medium . However, the hyperthermophile P. aerophilum is a denitrifying archaeon requiring tungstate (WO4 2−) for growth although it’s Nar is a Mo cofactor containing enzyme . Afshar et al.  demonstrated that the external tungstate concentration affects the denitrification pathway efficiency of this archaeon, resulting in the complete denitrification only at high tungstate concentration.
Recently, Nar purified from P. aerophilum grown in the absence of added molybdate and with 4.5 µM tungstate has been reported  which is a W containing enzyme. P. aerophilum Nar is the first active nitrate reductase that contains a W cofactor. The presence of a W cofactor may be reflective of high concentrations of this metal at high temperatures . As previously described this enzyme can also accommodate Mo as the active site metal .
To compare the properties of Mo and W cofactors containing enzymes, DFT calculations were performed on the active site model complexes derived from the protein X-ray crystal structure of P. aerophilum . The crystal data shows that at the active site the metal is coordinated by bis-MGD ligands, a carboxyl group of Asp222 and an oxo specie. However, there is a controversy about the nature of oxo specie. Based on the optimized data from computed model complexes 1, 2, and 3, this oxo specie is most probably the oxide group.
Computed energies (kcal/mol) relative to the educt–substrate complex for the nitrate reduction
Transition state complex
Oxidized product without nitrite
Reduced product with water
These results are in good agreement with the following experimental findings; (a) the hyperthermophile P. aerophilum is well adapted to a high-tungsten environment and this heavy metal is very important for its anaerobic growth mode on nitrate . (b) In contrast to other mesophilic nitrate reducers, P. aerophilum growth with nitrate is not reduced/stopped at high tungstate concentrations . Similar behaviour have been reported for NAD-dependent glutamate dehydrogenase enzyme in which enzyme isolated form hyperthermophiles shows comparable specific activities to those of enzymes from their mesophilic counterparts .
In conclusion, the computational result shows that the oxo specie attached with the metal at the active site of Nar is probably the oxide group. It is also concluded that the replacement of W with the Mo at the active site impart no effect on the overall reduction of nitrate except the energy barrier for oxygen transfer from nitrate which is low for W containing Nar (W–Nar). The most appropriate justification for this behavior of W–Nar is that P. aerophilum needs to support its growth by nitrate respiration even when the tungsten concentration in the environment is high; the same was concluded experimentally . However, the reason for the activity loss of Nars with the increase in tungstate concentration in the environment needs to be further investigated (Additional file 1).
The author is highly thankful to Graduate Kollege 850 (GK-850) for financial support to complete this work.
The authors declare that they have no competing interests.
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