Enzymatic Reduction of Nitrate to Nitrite: Insight from Density

Dec 16, 2009 - In reaction mechanism iii, the direct coordination of nitrate to Mo is an almost ... Vincent Fourmond , Monique Sabaty , Pascal Arnoux ...
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Organometallics 2010, 29, 436–441 DOI: 10.1021/om9008197

Enzymatic Reduction of Nitrate to Nitrite: Insight from Density Functional Calculations Hujun Xie† and Zexing Cao*,‡ †

Department of Applied Chemistry, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, People’s Republic of China and ‡Department of Chemistry and State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People’s Republic of China Received September 21, 2009

The oxidative half-reaction of oxygen atom transfer from nitrate to the MoIV complex has been investigated by the density functional approach, based on the model cluster [(Me2C2S2)2Mo(MeS2)]derived from the newly identified structure of nitrate reductase. Calculations show that the reduction of nitrate to nitrite can occur through an association of nitrate to the Mo center, followed by rupture of the Mo-O-NO2- bond. In reaction mechanism i, Mo-SCys bond cleavage coupling with the coordination of nitrate to Mo is the rate-determining step with a barrier of 19.8 kcal mol-1. In reaction mechanism iii, the direct coordination of nitrate to Mo is an almost barrier-free process, and the barrier for the rate-determining step of the Mo-O-NO2- bond cleavage is about 11.7 kcal mol-1, significantly lower than those in other plausible mechanisms. Present calculations lend support to the notion that the presence of a disulfide bond in the active site can influence the interconversion of MoIV to MoVI.

1. Introduction Inorganic nitrogen is incorporated into the biosphere by the biological fixation1-6 of atmospheric dinitrogen to NH3 and the denitrification-based removal of nitrates.7-12 Denitrification of nitrate to nitrite is a microbial respiratory process, and it is a key component of the biogeochemical nitrogen cycle involved in the four-step, five-electron reduction of nitrate (NO3-) to dinitrogen (N2) (Scheme 1).13-17 In *To whom correspondence should be addressed. E-mail: zxcao@ xmu.edu.cn. Fax: þ86-592-2183047. (1) Burges, B. K.; Lowe, D. C. Chem. Rev. 1996, 96, 2983. (2) Tanaka, H.; Mori, H.; Seino, H.; Hidai, M.; Mizobe, Y.; Yoshizawa, K. J. Am. Chem. Soc. 2008, 130, 9037. (3) Xie, H. J.; Wu, R. B.; Zhou, Z. H.; Cao, Z. X. J. Phys. Chem. B 2008, 112, 11435. (4) Cao, Z. X.; Zhou, Z. H.; Wan, H. L.; Zhang, Q. E.; Thiel, W. Inorg. Chem. 2003, 42, 6986. (5) Cao, Z. X.; Zhou, Z. H.; Wan, H. L.; Zhang, Q. E. Int. J. Quantum Chem. 2005, 103, 344. (6) Cao, Z. X; Jin, X.; Zhang, Q. J. Theor. Comput. Chem. 2005, 4, 593. (7) Wasser, I. M.; Vries, S.; Karlin, K. D. Chem. Rev. 2002, 102, 1201. (8) Averill, B. A. Chem. Rev. 1996, 96, 2951. (9) Zumft, W. G. Microbiol. Mol. Biol. Rev. 1997, 61, 533. (10) Richardson, D. J.; Watmough, N. J. Curr. Opin. Chem. Biol. 1999, 3, 207. (11) Ferguson, S. J. Antonie Van Leeuwenhoek 1994, 66, 89. (12) Stouthamer, A. H. Antonie Van Leeuwenhoek 1992, 61, 1. (13) Payne, W. J. Denitrification; Wiley-Interscience: New York, 1981. (14) Golterman, H. L., Ed. Denitrification in the Nitrogen Cycle; Plenum Press: New York, 1983. (15) Averill, B. A. Chem. Rev. 1996, 96, 2951. (16) Richardson, D. J.; Watmough, N. J. Curr. Opin. Chem. Biol. 1999, 3, 207. (17) Eady, R. R.; Hasnain, S. S. In Comprehensive Coordination Chemistry II; McCleverty, J. A., Meyer, T. J. Eds.; Elsevier: Amsterdam, 2004; Vol. 8, p 759. pubs.acs.org/Organometallics

Published on Web 12/16/2009

bacteria and archaea, each step of the nitrate reduction is catalyzed by distinct metalloenzymes containing various transition metals (Mo, Fe, Cu), and the metal centers were found to have variable ligand environments, including heme, histidine, or sulfide ligation etc.18-21 Molybdenum-dependent enzymes are ubiquitous in nature and play an important role in biological catalytic reactions.22-24 Nitrate reductase (NAR), as a crucial enzyme in the biological cycle of nitrogen, catalyzes the conversion of nitrate to nitrite, along with the oxidation-state change of molybdenum from þIV to þVI.19,25

NO3 - þ 2e - þ 2Hþ f NO2 - þ H2 O The first crystal structure of periplasmatic nitrate reductase from Desulfovibrio desulfuricans at 1.9 A˚ resolution reveals that the enzyme is composed of four domains. All four domains are involved in cofactor binding, but only one domain is responsible for the binding of an Fe4S4 cluster that serves as an electron pump.26 In this crystal structure, the (18) York, J. T.; Bar-Nahum, I.; Tolman, W. B. Inorg. Chim. Acta 2007, 361, 885. (19) Hofmann, M. J. Biol. Inorg. Chem. 2007, 12, 989. (20) Tocheva, E. I.; Rosell, F. I.; Mauk, A. G.; Murphy, M. E. P. Science 2004, 304, 867. (21) Kurtz, D. M. Dalton Trans. 2007, 37, 4115. (22) Hille, R. Chem. Rev. 1996, 96, 2757. (23) Webster, C. E.; Hall, M. B. J. Am. Chem. Soc. 2001, 123, 5820. (24) Leopoldini, M.; Chiodo, S. G.; Toscano, M.; Russo, N. Chem. Eur. J. 2008, 28, 8674. (25) Leopoldini, M.; Russo, N.; Toscano, M.; Dulak, M.; Wesolowski, T. A. Chem. Eur. J. 2006, 12, 2532. (26) Dias, M. J.; Than, M. E.; Humm, A.; Huber, R.; Bourenkov, G. P.; Bartunik, H. D.; Bursakov, S.; Calvete, J.; Caldeira, J.; Carneiro, C.; Moura, J. J. G.; Moura, I.; Romao, M. J. Structure 1999, 7, 65. r 2009 American Chemical Society

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Scheme 1. Consecutive Four-Step Reduction of Nitrate to Nitrite in Bacterial Denitrification

MoVI is hexacoordinated with a distorted-trigonal-prismatic geometry. The ligands comprise the four sulfur atoms of the dithiolene moieties, another sulfur atom from a side chain Cys140 residue, and a hydroxy or water ligand to complete the coordination sphere of the Mo atom. Recent X-ray structures of nitrate reductase from Desulfovibrio desulfuricans at various resolutions from 1.99 to 2.44 A˚ show that the sixth molybdenum ligand, originally proposed to be an OH-/OH2 ligand, should be assigned as a sulfur atom.27 The cofactor of this enzyme is an organic pterin incorporating a dithiolene moiety, specifically bismolybdopterin guanine dinucleotide (MGD),28 and this structure is very similar to previous nitrate reductase.26 This is the first experimental evidence of a partial S-S bond for the active site of a mononuclear molybdenum enzyme.29-36 Accordingly, the formation and dissociation of a S-S bond may result in a partial intermolecular redox reaction of molybdenum with thiolate, which can influence the interconversion of MoVI to MoIV involved in the catalytic reaction cycle. Previous computational studies by Leopoldini et al.25 suggested that the enzymatic reaction process involves the formation of an [MoIV(SCH3)(NO3)(S2C2Me2)2]2- complex and the consequent N-O bond breaking, leading to NO2and [MoVIO(SCH3)(S2C2Me2)2]- as primary products. The singlet state was predicted to be the ground state for all stationary points on the potential energy surface, and the predicted activation barrier is about 19.3 and 11.6 kcal mol-1 in the gas phase and protein environments, respectively. Hofmann19 performed DFT calculations using the molybdenum and tungsten complexes as models for the active sites of assimilatory or dissimilatory nitrate reductases. Calculations indicate that one water molecule bound to assimilatory NAR models [(Me2C2S2)MoO(SMe)]- can be replaced by nitrate, but a hydroxyl group may not. The nitrate reduction was predicted to be exothermic by about 14.9 kcal mol-1 with the highest barrier of 13.3 kcal mol-1 for the proteinfree models. For dissimilatory NAR model complexes [(Me2C2S2)2Mo(OOCMe)]-, the exchange of an oxo ligand (assimilatory NAR) for a dithiolato ligand (dissimilatory (27) Najmudin, S.; Gonzalez, P. J.; Trincao, J.; Coelho, C.; Mukhopadhyay, A.; Cerqueira, N. M. F. S. A.; Romao, C. C.; Moura, I.; Moura, J. J. G.; Brondino, C. D.; Romao, M. J. J. Biol. Inorg. Chem. 2008, 13, 737. (28) Collison, D.; Garner, C. D.; Juole, J. A. Chem. Soc. Rev. 1996, 21, 25. (29) Stiefel, E. I.; Miller, K. F.; Bruce, A. E.; Corbin, J. L.; Berg, J. M.; Hodgson, K. O. J. Am. Chem. Soc. 1980, 102, 3624. (30) Berg, J. M.; Spira, D. J.; Hodgson, K. O.; Bruce, A. E.; Miller, K. F.; Corbin, J. L.; Stiefel, E. I. Inorg. Chem. 1984, 23, 3412. (31) Laughlin, L. J.; Eagle, A. A.; George, G. N.; Tiekink, E. R.; Young, C. G. Inorg. Chem. 2007, 46, 939. (32) Stiefel, E. I. In Biological Inorganic Chemistry: Structure and Reactivity; Bertini, I., Gray, H. B., Stiefel, E. I., Valentine, J. S., Eds.; University Science Books: Sausalito, CA, 2007; pp 1-30. (33) Young, C. G. J. Inorg. Biochem. 2007, 101, 1562. (34) Jepson, B. J.; Mohan, S.; Clarke, T. A.; Gates, A. J.; Cole, J. A.; Butler, C. S.; Butt, J. N.; Hemmings, A. M.; Richardson, D. J. J. Biol. Chem. 2007, 282, 6425. (35) Frangioni, B.; Arnoux, P.; Sabaty, M.; Pignol, D.; Bertrand, P.; Guigliarelli, B.; Leger, C. J. Am. Chem. Soc. 2004, 126, 1328. (36) Jepson, B. J.; Anderson, L. J.; Rubio, L. M.; Taylor, C. J.; Butler, C. S.; Flores, E.; Herrero, A.; Butt, J. N.; Richardson, D. J. J. Biol. Chem. 2004, 279, 32212.

Figure 1. Optimized geometry of the model cluster for nitrate reductase.

NAR model) lowers the reaction exothermicity and raises the barrier. More recently, Hofmann37 explored possible mechanisms of nitrate reduction using the different models derived from the newly crystal structure, and the effect of the structural environment at the active site on the relative energetics was studied. Despite these important contributions, the new coordination sphere of molybdenum from the newly characterized crystal structure27 might revise the previous redox chemistry of molybdenum to a certain extent, accordingly. On the basis of the newly identified structure of nitrate reductase, extensive density functional calculations on the enzymatic reduction of nitrate to nitrite have been performed, and plausible reaction mechanisms have been explored theoretically.

2. Computational Methods Density functional calculations were performed to study the nitrate binding and activation by the model cluster [(Me2C2S2)2Mo(MeS2)]-. Figure 1 gives the initial structure of the computational model from the newly determined X-ray structure of nitrate reductase (PDB code 2jip),27 in which the Cys140 residue was modeled as a SCH3 group. The generalized gradient approximation PW91 functional (GGA-PW91)38,39 was adopted in the density functional theory (DFT) calculations. The allelectron and double-numerical basis sets plus polarization functions (DNP) were employed in the spin-unrestricted KohnSham (UKS) computation. The reliability of this method has been verified in previous studies on properties of metal-sulfur clusters.3,6,40-44 In calculations, the electronic singlet states were considered throughout, since the singlet states were shown to be the lowest energy states for all stationary points along the nitrate reduction pathway.25 (37) Hofmann, M. J. Biol. Inorg. Chem. 2009, 14, 1023. (38) Perdew, J. P.; Wang, Y. Phys. Rev. B 1992, 45, 13244. (39) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. Rev. Lett. 1996, 77, 3865. (40) Cao, Z. X.; Mo, Y. R. J. Theor. Comput. Chem. 2008, 4, 473. (41) Cao, Z. X.; Zhou, Z. H.; Wan, H. L.; Zhang, Q. E.; Thiel, W. Inorg. Chem. 2003, 42, 6986. (42) Lovell, T.; Liu, T. Q.; Case, D. A.; Noodleman, L. J. Am. Soc. Chem. 2003, 125, 8377. (43) Pelmenschikov, V.; Case, D. A.; Noodleman, L. Inorg. Chem. 2008, 47, 6162. (44) Lukoyanov, D.; Pelmenschikov, V.; Maeser, N.; Laryukhin, M.; Yang, T. C.; Noodleman, L.; Dean, D. R.; Case, D. A.; Seefeldt, L. C.; Hoffman, B. M. Inorg. Chem. 2007, 46, 11437.

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Scheme 2. Three Proposed Different Reaction Mechanisms for Nitrate Reduction by Nitrate Reductase

Table 1. Selected Bond Lengths (A˚) and Angles (deg) of the Optimized Model Complexes for Nitrate Reductase reaction mechanism i param

reactant

TS

product

reaction mechanism iii reactant

TS

rroduct

Distances

Figure 2. Predicted relative energies and geometries of the reaction species involved in reaction mechanism i. To approximately estimate the effect of the protein environment, the single-point energy calculations at the optimized geometries were carried out by using the conductor-like screening model (COSMO)45,46 with a dielectric constant (ε) of 4.0. All calculations in the present work have been implemented in the DMol3 package.47-49

Mo-S Mo-SCys S-SCys Mo-O N-O Mo-S1 Mo-S2 Mo-S3 Mo-S4

2.420

2.456 2.485

2.216 2.207 1.315 2.406 2.389 2.398 2.392

2.110 1.926 1.726 2.485 2.520 2.427 2.387

Mo-S-SCys Mo-O-N O-Mo-S S1-Mo-S2 S3-Mo-S4 S1-Mo-S3 S2-Mo-S4

117.9 136.5 77.7 80.0 81.5 82.0 80.3

117.4 134.0 81.7 77.6 81.6 79.8 79.7

2.032 1.735 2.479 2.476 2.419 2.409

2.503 2.514 2.064 2.349 1.304 2.468 2.441 2.464 2.467

2.594

66.0 127.3 81.5 83.5 79.0 81.6 82.1

68.3 130.5 83.1 80.8 79.2 80.7 81.0

2.104 1.845 1.792 2.463 2.453 2.455 2.461

2.657 2.686 2.040 1.752 2.486 2.482 2.454 2.473

Angles 110.2 77.2 79.4 82.0 77.8 77.5

66.8 82.6 78.1 80.3 78.4 79.9

3. Results and Discussion On the basis of extensively computational studies on the reaction mechanisms of nitrate reductase, three possible reaction mechanisms have been proposed, as shown in Scheme 2. The nitrate reduction can occur either through a direct rupture of the MoO-NO2- bond (reaction mechanisms i and iii) or via bonding of the nitrate ion to a sulfur ligand of molybdenum (reaction mechanism ii). The optimized structure of the model system (Figure 1) in its ground state is found to be square pyramidal with an average Mo-S (dithiolene) distance of 2.372 A˚ and a S-SCys bond length of 2.098 A˚. These bond lengths agree well (45) 799. (46) 9312. (47) (48) (49)

Klamt, A.; Sh€ urmann, G. J. Chem. Soc., Perkin Trans. 2 1993, Andzelm, J.; Kolmel, C.; Klamt, A. J. Chem. Phys. 1995, 103, Delley, B. J. Phys. Chem. 1996, 100, 6107. Delley, B. J. Chem. Phys. 1990, 92, 508. Delley, B. J. Chem. Phys. 2000, 113, 7756.

with the experimental values determined by Najmudin and co-workers,27 while predicted Mo-S and Mo-SCys bond lengths show relatively longer values of 2.396 and 2.658 A˚, respectively. Reaction Mechanism i. As Figure 2 displays, in reaction mechanism i, the Mo-SCys bond breaks first and, following coordination of nitrate to MoIV in the vacant position, leads to a stable intermediate complex. Previous experiments27 and theoretical calculations19,25 suggested that the stable complex from the association of nitrate to molybdenum should be a precursor to the oxidative reaction. In the present calculation, the binding barrier of nitrate in reaction mechanism i is predicted to be 19.8 kcal mol-1. As Figure 2 and Table 1 show, as a consequence of the interaction between Mo atom and nitrate, the geometries of the initial reaction complex (reactant) show slight changes in contrast to the isolated model cluster. The N-O bond of substrate lengthens from 1.265 to 1.315 A˚, and all Mo-S (dithiolene) distances fall in the range from 2.389 to 2.406 A˚.

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Table 2. Selected Charge Populations for the Reaction Species of Nitrate Reductase Mo

S ligand

SCys

S1

S2

S3

S4

Reaction Mechanism i reactant 0.566 TS 0.684 product 0.885

-0.252 -0.219 -0.144

-0.310 -0.359 -0.336 -0.359 -0.379 -0.316 -0.385 -0.377 -0.350 -0.383 -0.201 -0.285 -0.300 -0.330 -0.319

Reaction Mechanism iii reactant 0.532 TS 0.763 product 0.925

-0.307 -0.282 -0.219

-0.139 -0.336 -0.374 -0.277 -0.374 -0.247 -0.405 -0.419 -0.351 -0.377 -0.083 -0.312 -0.346 -0.286 -0.309

The Mo-S and Mo-O bond lengths are predicted to be 2.420 and 2.207 A˚, respectively. The S-SCys bond is about 2.216 A˚, slightly longer than the experimental value of 2.184 A˚. The present results are consistent with those of previous computational studies.19,25 As can be seen in Figure 2, the activation barrier of N-O bond cleavage is about 14.7 and 15.3 kcal mol-1 in the gasphase and protein environments, respectively, slightly lower than those found by Hofmann19 and Russo.27 The N-O distance is predicted to be 1.726 A˚ at the transition state (TS). As Table 1 shows, the Mo-S distance (2.456 A˚) in TS slightly increases in comparison with the reactant, while the S-SCys separation (2.110 A˚) decreases slightly. The ligand arrangements appear to be remarkably distorted with respect to the ideal trigonal-prismatic and octahedral geometries. As shown in Table 2, the charge populations on the Mo atom, S ligand, and SCys atom in the TS state are 0.684, -0.219, and -0.316, respectively. The only imaginary frequency of -476 cm-1 confirms the saddle-point character of the TS state and corresponds to the dissociation of Mo-O-NO2. Once the N-O bond breaks, the oxo MoVI complex is formed with the release of the nitrite product. In the product state, the positive charge of Mo (0.885) increases remarkably related to those in reactant and TS states. The complex exhibits obvious distorted geometries. The Mo-S and MoO bond lengths are calculated to be 2.485 and 1.735 A˚, respectively. The S-SCys bond length is about 2.032 A˚, shorter than those of the reactant and TS. In the gas phase, the reduction of nitrate to nitrite will release an energy of 49.7 kcal mol-1. Reaction Mechanism ii. In reaction mechanism ii, the nitrate is directly bound to the sulfur ligand at first. The binding barrier of nitrate is predicted to be 56.8 kcal mol-1, which is significantly higher than that for reaction mechanism i and previous theoretical results.19,25 Geometry optimization indicates that, once nitrate binds to the sulfur ligand, the N-O bond cleavage takes place spontaneously. Selected key species involved in the reaction pathway are shown in Figure 3. These energetic results show that the binding of nitrate to the sulfur ligand is energetically unfavorable. Obviously, such a high barrier arises from the dissociation of oxygen in NO3-, where the oxygen atoms are saturated by the strongly delocalized π-bonding interactions and thus significant N-O bond breaking is requisite for the O 3 3 3 S coupling. Reaction Mechanism iii. As can be seen in Scheme 2, in the case of reaction mechanism iii, the nitrate directly binds to the Mo center, yielding a complex as an initial reactant, and the S-SCys bond still survive in the formation of the complex. Previous studies showed that the disulfide bond can

Figure 3. Optimized structures of relevant stationary points along reaction mechanism ii.

influence the redox chemistry of molybdenum.37,50-52 The binding barrier of nitrate is found to be 1.7 kcal mol-1, much lower than that in reaction mechanism i and reaction mechanism ii. In the reactive complex, the conformation rearrangement of the pseudodithiolene coordinated to the Mo center is indispensable to facilitate the access of nitrate (Figure 4). As Table 1 shows, all the Mo-S(dithiolene) distances in the reactant are in a range from 2.441 to 2.468 A˚, and the Mo-O, Mo-S, and Mo-SCys bond lengths are predicted to be 2.349, 2.503, and 2.514 A˚, respectively. The S-SCys bond length is about 2.064 A˚, slightly shorter than the experimental value of 2.184 A˚.24 The N-O bond length of the nitrate substrate lengthens from 1.265 to 1.304 A˚ due to the interaction between Mo and nitrate. As Figure 4 displays, the N-O distance is predicted to be 1.792 A˚ in the transition state. In comparison with the reactant, the Mo-S (2.594 A˚) distance in the TS state slightly increases, while the S-SCys distance (2.104 A˚) slightly decreases. It is interesting to note that the Mo-SCys bond breaks in the transition state, and the ligand arrangements appear to be quite distorted with respect to the ideal trigonalprismatic and octahedral geometries. The only imaginary frequency of -330 cm-1 confirms the character of the TS state, and it corresponds to the stretching vibration modes of the Mo-O(nitrate) and O(nitrate)-N(nitrate) bonds. The predicted activation barrier for the N-O bond cleavage is about 11.7 and 12.6 kcal mol-1 in the gas phase and protein environments, respectively. These barriers are obviously lower than those in the reaction mechanisms i and ii, as well as those by Hofmann19 and Russo.27 As Table 2 shows, the charge populations on the Mo atom are 0.763 and 0.925 in the transition state and the product, respectively. Compared to the reactant, there is an intramolecular electron transfer from Mo to the leaving NO2 moiety. The disulfide bond in the reactant may influence the interconversion of MoIV to MoVI, involving both Mo and ligand-based redox chemistry as suggested in previous studies.27 The presence of a partial disulfide bond in the reactant state can facilitate the nitrate binding and the N-O bond cleavage. These results are in good agreement with previous studies.29-33,37 Present calculations show that reaction mechanism iii is favorable energetically. (50) Stiefel, E. I.; Miller, K. F.; Bruce, A. E.; Corbin, J. L.; Berg, J. M.; Hodgson, K. O. J. Am. Chem. Soc. 1980, 102, 3624. (51) Laughlin, L. J.; Eagle, A. A.; George, G. N.; Tiekink, E. R.; Young, C. G. Inorg. Chem. 2007, 46, 939. (52) Young, C. G. J. Inorg. Biochem. 2007, 101, 1562.

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Figure 4. Predicted relative energies of the reaction species involved in reaction mechanism iii.

Figure 5. Optimized geometries of possible intermediates involved in the alternating two-electron and two-proton reduction steps to the ready state in different reaction mechanisms of nitrate reductase. Scheme 3. Sequence of Alternating Two-Proton and TwoElectron Reduction Steps to the Ready State of Nitrate Reductase

The N-O bond cleavage yields the oxo MoVI complex and the nitrite product, and the charge of the Mo center increases remarkably (0.925). The MoVI complex exhibits a distorted octahedral coordination of the Mo atom, in which the Mo-S and Mo-SCys, Mo-O distances are predicted to be 2.657, 2.686, and 1.752 A˚, respectively. The S-SCys bond length is about 2.040 A˚, slightly shorter than that of the reactant and transition states. As Figure 4 shows, the formation of products are exothermic by 54.2 kcal mol-1. Protonation/Electron Reduction Steps. As displayed in Scheme 3, after N-O bond cleavage, the catalytic cycle consists of a sequence of alternating two-electron and twoproton reduction steps, and the release of one water molecule from the model cluster leads to the fully initial state of the active site in the nitrate reductase. Previous studies showed that an Fe4S4 cluster can serve as an electron pump,26 and indefinite water molecules or residues around the model cluster can act as proton donors.25,27 Figure 5 shows the

Figure 6. Relative energies of the intermediates involved in the alternating two-electron and two-proton reduction steps to the ready state for the different reaction mechanisms i-iii of nitrate reductase.

optimized geometries of possible intermediates involved in the alternating two-electron and two-proton reduction reactions in the reaction mechanisms i-iii. In the case of reaction mechanism iii, the formation of stable intermediate Iint1 is exothermic by 11.0 kcal mol-1 as shown in Figure 6. Relative to the product, the Mo-O bond of Iint1 significantly

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lengthens from 1.752 to 2.046 A˚ due to the interaction between Mo and hydroxide. The Mo-S and Mo-SCys bond lengths are predicted to be 2.679 and 2.553 A˚, respectively. The S-SCys bond is about 2.060 A˚, slightly longer than the product value of 2.040 A˚. The intermediate Iint1 evolves to the Iint2 with an exothermicity of 23.5 kcal mol-1. The predicted Mo-S and Mo-SCys bond lengths are 2.576 and 2.447 A˚, respectively. The S-SCys bond length is about 2.086 A˚, and the Mo-O bond further lengthens in contrast to Iint2 with a value of 3.624 A˚, facilitating the release of a water molecule. The intermediate Iint2 may return to the ready state (model cluster) for the next catalytic turnover.

4. Conclusions Density functional calculations have been used to explore the reaction mechanism of nitrate reduction by the Modependent nitrate reductase. On the basis of calculations, the nitrate reduction can occur either through a direct rupture of the Mo-O-NO2- bond (reaction mechanisms i and iii) or via bonding of the nitrate ion to a sulfur ligand of

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molybdenum (reaction mechanism ii). The barriers of the rate-determining step in reaction mechanisms i and ii are predicted to be 19.8 and 56.8 kcal mol-1, respectively. These values are significantly higher than those in reaction mechanism iii and previous theoretical results. In the case of reaction mechanism iii, the nitrate directly binds to the Mo center, and the S-SCys bond still survive in the formation of the complex. The predicted activation barrier of N-O bond cleavage is about 11.7 kcal mol-1, significantly lower than those in other plausible mechanisms. Present calculations indicate that reaction mechanism iii is more favorable energetically. The existence of a disulfide bond can influence the interconversion of MoIV to MoVI, and it plays an important role in nitrate reduction.

Acknowledgment. This work was supported by the National Science Foundation of China (Grant Nos. 20673087, 20733002, and 20873105), the Ministry of Science and Technology (Grant No. 2004CB719902), and Science Fund for Young Scholars of Zhejiang Gongshang University, China (Grant No. 1110XJ200966).