Theoretical Investigation on the Role of the Central Carbon Atom and

Research Article pubs.acs.org/acscatalysis

Theoretical Investigation on the Role of the Central Carbon Atom and Close Protein Environment on the Nitrogen Reduction in Mo Nitrogenase Li Rao,† Xin Xu,‡ and Carlo Adamo*,§,∥ †

Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, China ‡ Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, MOE Laboratory for Computational Physical Science, Department of Chemistry, Fudan University, Shanghai 200433, China § Chimie ParisTech, PSL Research University, CNRS, Institut de Recherche de Chimie Paris (IRCP), F-75005 Paris, France ∥ Institut Universitaire de France, 103 Boulevard Saint Michel, F-75005 Paris, France S Supporting Information *

ABSTRACT: A theoretical study elucidating the mechanism of N2 reduction in Mo nitrogenase was carried out using a QM/QM′ approach based on density functional theory/semiempirical methods. Resting on the consolidated Lowe−Thorneley catalytic cycle, the identified reaction mechanism corresponds to an alternating pathway where the two nitrogen atoms are alternately reduced. Furthermore, this new mechanism provides a clear mechanistic basis to most of the experimental observations, including the noninnocent role played by the carbon atom located in the center of the MoFe cofactor and by the surrounding amino acids (such as α-96ARG, α-195HIS, and α-70VAL). It also provides evidence for the presence of H2 evolution in the global reaction cycle. Our calculations indicate a large flexibility of the cofactor upon hydrogenation and subsequent N2 chemical binding, with the average Fe−C distance increasing of 0.26 Å in going from the E0 to the E4 state, in agreement with experimental evidence. Taken together, these results give new insights into the activity of Mo nitrogenase, clearly considering the most relevant experimental findings. KEYWORDS: Mo nitrogenase, enzymatic catalysis, MoFe cofactor, nitrogen reduction, DFT

1. INTRODUCTION

investigations give more clear indications supporting the presence of a carbon atom.9−11 The structure of the resulting, and now commonly accepted, MoFe cofactor is thus the one shown in Figure 1. The determination of the active-site structure provides a firm framework for the mechanism of N2 reduction, which follows the general reaction

Nitrogen is essential for all living organisms, being one of the main constituents of proteins, nucleic acids, and many other biomolecules. The majority of nitrogen used by organisms is provided by N2 fixation occurring in microorganisms called diazotrophs and catalyzed by enzymes belonging to the nitrogenase family.1 Among various nitrogenase enzymes, the molybdenum−nitrogenase is the most common in nature and has attracted the most attention. Indeed, it was characterized nearly half a century ago,2 and reports concerning experimental investigations are abundant in literature (see, for instance, refs 3 and 4). In particular, the general framework concerning the catalytic activity of Mo nitrogenase is clearly defined. N2 reduction takes place at the iron−molybdenum (MoFe) cofactor where the substrate N2 is adsorbed and reduced by incoming electrons and protons (from Fe protein and solvent, respectively).3 The cofactor structure was identified as [Fe7MoS9-homocitrate] in 1996,5 but subsequent research revealed the existence of an interstitial atom, although these studies did not clearly discriminate between carbon, nitrogen, or oxygen.6 The argument concerning the nature of the interstitial atom and its role lasted for some time,7,8 but recent © 2016 American Chemical Society

N2 + 8e− + 16MgATP + 8H+ → 2NH3 + H 2 + 16MgADP + 16H3PO4

According to this reaction, the MoFe cofactor accepts eight electrons and eight protons through a complex (and not completely understood) interplay with magnesium ATP and its close environment (water molecules).3,12 In order to rationalize the experimental data, the whole mechanism has been divided into nine steps (labeled En, n = 0−8, as shown in Figure 2), characterized by consecutive one-electron reductions and arrival of protons. This scheme is known as the Lowe− Received: November 16, 2015 Revised: January 13, 2016 Published: January 25, 2016 1567

DOI: 10.1021/acscatal.5b02577 ACS Catal. 2016, 6, 1567−1577

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second molecule is not released until the E7 state (after 7e−/ 7H+ added) has been reached.3 Experimental data also point out the active role of the protein environment close to the MoFe cofactor. In particular, several residue mutations around the MoFe cofactor (see Figure 1) are found to affect its catalytic activity and substrate availability: mutations of residue α-96ARG have been reported to decrease the N2 reduction rate, while the α-195HIS→GLN mutation eliminates the N2 reduction ability, probably disrupting the delivery of protons for reduction of the nitrogenous substrates.18,19 The molecular volume of the residue α-70 VAL strongly correlates with the substrate availability, affecting the interaction with the so-called iron waist of the MoFe-cofactor, composed by Fe atoms 2, 3, 6, and 7.19,20 Finally, hydrazine (N2H4) and diazene (N2H2) are both reduced to ammonia by the wild-type nitrogenase,3,17 and the substitution of α-70VAL with smaller α-70ALA increases the rate of their reduction.21 Despite all these indications, it is still not possible to propose a detailed catalytic cycle to date, and only two draf t mechanisms are proposed in literature.22 The first is the socalled distal pathway, wherein the distal nitrogen is protonated first and then liberated as NH319 at the E5 state. In the second mechanism the two nitrogen atoms are reduced alternately, with a late cleavage of the N−N bond (the so-called alternating pathway) and the leaving of the first ammonia at the E7 state.19,22 Some help in the rationalization of all these experimental data was provided by several modeling studies.23−32 Theoretical calculations clearly confirmed that N2 binds to one or two Fe atoms of the MoFe cofactor and indicated the alternating mechanism as the most probable.26,28 In addition, it was stated that the incoming hydrogen prefers sulfur to iron atoms and, in particular, that the S2B atom (see Figure 1 for labeling) could be easily protonated.29 Finally, the rate-determining step of the whole catalytic cycle could have a relatively high barrier, up to 28 kcal/mol, while single reaction steps are found to be endothermic (up to 21 kcal/mol)29 or strongly exothermic (down to −94 kcal/mol).26 These theoretical studies also emphasized the nontrivial problems in modeling the N2 reduction at the MoFe cofactor, where the complexity arises from different features, such as the size or the entangled electronic states on the catalytic system. Therefore, the slow progress in the field is made in a step-bystep way, where each contribution brings a missing piece to the puzzle. Indeed several points still deserve a deeper analysis. For instance, previous theoretical studies only considered six hydrogens in the main reaction channel.25,29 As a consequence, a direct interconnection between H2 release and N2 reduction has not been considered or it was excluded, in disagreement with the LT scheme and the related experimental evidence. Furthermore, the computational models were usually limited to the bare MoFe cofactor (sometimes including the α-275CYS and α-442HIS residues) so that the role of the closest protein residues has not been deeply investigated and rationalized. Finally, some of these studies considered nitrogen as interstitial atom, spectator in the whole catalytic cycle.31,32 Indeed, the substitution of nitrogen with a carbon atom was claimed to affect only slightly the reaction energetics.31 This point is somehow in contrast with recent experimental indications which suggest that not only the N2 molecule binds to an iron atom in trans-position to an interstitial carbon but also its coordination depends on the Fe−C bond properties, thus

Figure 1. MoFe cofactor and surrounding residues. The labeling follows the convention adopted in the PDB crystallographic structure 1M1N (see ref 6).

Figure 2. Scheme of the Lowe−Thorneley catalytic cycle proposed for the N2 reduction. In parentheses are the reported nitrogenous intermediates as indicated by the presented computations.

Thorneley (LT) scheme,13 and it fits well the most relevant experimental observations for the catalytic cycle of Mo nitrogenase. In particular, it has been observed that at least three, and most likely four, electrons (and an equivalent number of protons) must accumulate in the MoFe protein before N2 binding14 can occur. Analogously, the release of one hydrogen molecule is found to accompany the reduction of each N2 molecule. Opinions in the literature are split between a stoichiometric and a nonstoichiometric hypothesis.15 The first indicates that the release of H2 is a consequence of N2 binding and a part of the N2 reduction. However, the explanation of how the nitrogenase could further reduce nitrogen while the hydrogen atoms prefer to be released as H2 rather than attack the nitrogen atoms is still not clear. The nonstoichiometric assumption treats the H2 evolution as a side reaction and rejects its intimate relationship with N2 reduction. This last hypothesis is in poor agreement with other experimental evidence such as the facts that H2 could act as an inhibitor of N2 reduction and that only in the presence of N2 is D2 gas reduced to HD.3 Recently, an E4 intermediate has been trapped, and evidence showed that this intermediate is activated for N2 binding by the accumulation of four protons and four electrons.16,17 Furthermore, it could relax to an E2 intermediate releasing molecular hydrogen. In light of this experimental observation, the stoichiometric mechanism seems to be the most plausible route. The other relevant event along the LT scheme is, of course, the release of ammonia, whose first molecule is released after the addition of at least 5e− and 5 H+ (state E5), while the 1568


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and unrestricted (spin polarized) Kohn−Sham formalism. The structures were optimized using the Los Alamos pseudopotentials and the associated valence basis sets for Fe and Mo, while the 631G(d) basis was adopted for the remaining atoms.39,40 Single-point energies were then evaluated at the optimized structures with the Stuttgart quasi-relativistic pseudopotentials and the 6-311+G(2d,p) basis.41 The same combination of functional and basis sets was used for the high level region in the ONIOM calculation, while the low level of theory applied was the semiempirical method PM6.42 The chosen functional has been shown to provide accurate energy evaluations for organometallic chemistry and metal-containing enzymes (see, for instance, refs 43−46 and Table S1 and related comments in the Supporting Information, SI), and a similar QM/QM′ approach, combining M06-2X and a semiempirical model, was successfully used in enzymatic reaction investigations.46 Furthermore, the selected computational protocol also reproduces with a very good accuracy the main bond distances of the MoFe cofactor in the E0 state, with deviations ranging between 0.04 and 0.14 Å (see Table S2, SI). All calculations were performed using the Gaussian09 program.47 Previous calculations have shown that the MoFe cofactor possesses a complex electronic structure, presenting a number of electronic states close in energies.29 From a technical point of view, these states are characterized by different charge and spin distributions among the different sites of the cofactor. Taking into account the fact that a clear S = 3/2 EPR signal was experimentally observed,48 the total charge of the resting E0 state model could be assigned as +2, 0, −2, etc. Without counter charges, it is not likely for a highly positively charged cofactor to be stably buried in the center of protein since solvation effects would tend to separate the cofactor from the protein. Thus, we can exclude highly charged states and assign a total charge of −2 to the resting state with the consideration of the counter charge from two nearby positively charged ARG residues. Considering also the negative charge of sulfur atom in α-275CYS and the negative charge of the homocitrate, the total charge of the [Fe7S9MoC] cluster is 0, as in the commonly accepted [Fe7S9MoN]0 redox state.25,29,31 The spin multiplicity of the E0 state was determined to be 4, corresponding to a spin state S = 3/2, as suggested by the EPR data. After the influx of electrons, the spin multiplicity of the MoFe cofactor could be decreased or increased, depending on whether the incoming electron pairs a single electron of MoFe cofactor or not. Taking into account that the MoFe cofactor enters an EPR-silent state after the addition of incoming electrons,3 the reduced cofactor are most likely low spin states. Thus, the spin multiplicities of E1, E3, E5, and E7 were set to be 1 (S = 0), while the spin multiplicities of E2, E4, E6 were set to be 2 (S = 1/2). To be more cautious, we also calculated the key E4 state reaction with spin multiplicity 4, and the result is given in the SI, Table S1. The obtained results show that the calculated barrier height is not sensitive to the choice of spin multiplicity. Concerning the spin distribution within the cofactor, different electronic states are possible, implying a choice of the reference state. Among all of the possibilities, the broken-symmetry state, corresponding to a global antiferro coupling between close irons, seems the most reasonable. This approach is usually used for modeling Fe−S clusters in protein,49 and in particular, it was successfully applied to the study of the FeMo cofactor.7,31 The spin densities were monitored, so as to maintain the same electronic state of the MoFe cofactor all along a single reaction path (the En path). An example of the resulting spin distribution in reaction intermediates is given in the SI.

providing evidence for an active role of the interstitial carbon atom.33 Within this complex experimental and theoretical context, we believe that there is room for accurate theoretical investigations aimed at reconciling the discrepancies between experimental and theoretical findings to provide a clear and more substantial picture of N2 fixation. In particular, the present work focuses on the role played by the central carbon atom, the surrounding protein residues, and the H2 evolution in the N2 reduction catalyzed by the MoFe cofactor. On the basis of the DFT results obtained, we propose a new N2 catalytic cycle, taking into account all the above-mentioned features and providing a clear mechanistic basis to most experimental observations, thus offering important insights to rationalize the N2 fixation mechanism.

2. COMPUTATIONAL DETAILS As mentioned above, the modeling of the N2 reduction represents a very difficult and challenging playground for computational approaches, since the MoFe cofactor has a complicated electronic structure29 and a very large size, even for cost-effective computational methods like those based on density functional theory (DFT), when the most important surrounding residues are to be considered. The situation is even more involved when the reaction mechanism investigated is characterized by flat potential energy surfaces. All these points have been clearly stressed in (almost all) previous theoretical studies, and therefore, a particular attention is required in defining a reliable computational approach. In the following, we will elucidate our own computational protocol, giving arguments to support our choices. 2.1. Structural Models. A cluster model was cut off from the crystal structure PDB ID 1M1N,6 and it consists of the [Fe7S9MoC] cluster, the homocitrate group, and residues α-275CYS and α-442HIS. Following an approach well-established in the literature (see, for instance, refs 24, 34, and 35), only the coordinates of the most involved atoms were relaxed, while peripheral units were kept frozen in positions in the X-ray structure. This constrained optimization is necessary to avoid unrealistic movements of the various groups of the active site.35 In particular, mutation experiments indicated that the N2 reduction takes place on the 4Fe−4S surface, involving Fe atoms 2, 3, 6, 7 and S atoms 3B, 5A, 2B, 2A,18 so that the whole 4Fe−4S cluster, the central C atom, and the imidazole of α-195HIS were optimized. Frequency analysis was then carried out to characterize the localized stationary states (minima or first-order saddle points), projecting out the vibrational modes associated with the frozen degrees of freedom. Thermodynamic quantities (enthalpies and Gibbs free energies) were then computed in the usual way at 298 K and 1 atm. Notably, to the best of our knowledge, this is the first time that such energetic quantities are reported for the N2 reduction reaction. The effect of the surrounding protein was then estimated using an ONIOM approach,36 where the high level region consists of the mentioned [Fe7S9MoC] cluster, the homocitrate group, residues α275CYS, α-442HIS, α-195HIS, and α-96ARG, and the NxHy species. The low-level region includes the MoFe cofactor and all of the residues within 8 Å from the MoFe cofactor (about 780 atoms in total). The resulting system was fully optimized, and the Gibbs free energies were obtained by combining the total energies obtained by ONIOM computations with the thermodynamic corrections obtained for the cluster model. Finally, in order to estimate further polarization effects of the surrounding protein environment, single-point PCM calculations on these systems were performed using a low dielectric constant (ε = 4), a value that was used in many previous works.35 The effect of this third layer, treated following the so-called ONIOM-X approach,37 was indeed found to be negligible (maximum variation of 1 kcal/mol on the relative enthalpies). 2.2. Electronic Approach. The calculations on the cluster model were carried out using the M06-2X exchange-correlation functional38

3. RESULTS AND DISCUSSION The N2 reduction at the MoFe cofactor is a multistep reaction, where protons and electrons arrive from the external protein environment, making the choice of the reference energy value nontrivial. Therefore, following a consolidated procedure,26 the reaction path of each En state has been characterized, taking the energy of the reactants as the zero-energy reference. All of the reported hydrogenation energies are computed as the energy difference between the energy of the hydrogenated En state and 1569


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Figure 3. Hydrogenation energies (kcal/mol) from E0 to different possible E1 structures. Data in parentheses are obtained using the cluster model calculations, without the protein environment. The zero-energy reference is taken as the E0 state MoFe cofactor and one free H atom.

Figure 4. Relative Gibbs free energies (kcal/mol) for the reaction path of the E4 state. Data in parentheses are obtained by corresponding cluster model without the protein environment. The zero-energy reference refers to the MoFe cofactor in the E4 state and one free N2 molecule, labeled E4 + N2 in the figure.

place at sulfur atoms and, in particular, at the S2B atom (see Figure 1) as indicated by previous calculations.27 Instead, our calculations suggest that the first hydrogen prefers to bind the central carbon rather than the S2B atom by about 5 kcal/mol, the hydrogenation energies being −72.4 (E0 to E1-CH) and −67.2 kcal/mol (E0 to E1-S2B-H), respectively (see Figure 3). According to the calculated hydrogenation energies, the second hydrogen goes to S2B, while the third one prefers the S2A

the sum of the energy of the En-1 state and a free H atom. A lower hydrogenation energy corresponds, of course, to a more stable species. 3.1. Preliminary Phase: States E0−E3. The first steps, from E0 to E3, can be considered as preliminary to the main catalytic activity, since experiments indicate that these states are not able to bind N2.8,50 The preparatory event is the multiple hydrogenation of the cofactor, which has been proposed to take 1570


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Figure 5. Representation of the optimized structures of the intermediates of the E4 state.

Figure 6. Key interaction distances between surrounding residues and MoFe cofactor: (a) α-195HIS and the first hydrogenation of N2 (E4TS1); (b) α-96ARG and the release of H2 (E4TS2); (c) α-70VAL and the binding of N2 (E4LS2).

atom, rather than S5B (hydrogenation energies of −51.8 and −47.8 kcal/mol, respectively). It is interesting to note that the computed hydrogenation energies of S5A and S2B in the cluster model (i.e., without the protein environment) are −70.5 and −68.3 kcal/mol, respectively. The large decrease of the S5A hydrogenation energy in going from the full to cluster model depends on the

nearby α-96ARG residue which, forming a strong H-bond with the sulfur atom (see Figure 1), impedes the S5A hydrogenation. The hydrogenation of S2A is also unfavorable by 10 kcal/mol as indicated by the difference between cluster and full-model calculations. All these results reveal the non-negligible role played by the surrounding protein residues, which reduce the 1571


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Figure 7. Relative Gibbs free energy (kcal/mol) for the E5 and E6 states. Data in parentheses were obtained by cluster model calculations.

structural rearrangement (simply labeled E4 in Figure 4) is the starting point for the reaction leading to the release of the hydrogen molecule. The energy profile is given in Figure 4, while the structures of all the intermediates are reported in Figure 5. It should be noticed that the fourth incoming hydrogen atom would prefer the S atom instead of the central carbon. This rearrangement (E4CH2 in figure S7) is 21.6 kcal/ mol higher in energy than the stable E4 structure, the starting point for the reaction. As shown in Figure 4, the coordination of one N2 molecule at Fe6 is accompanied by the transfer of one hydrogen atom from S2A to N2, and it is facilitated by the interaction with the central carbon (see Figure 5 for details on structure). Such a coordination activates the N−N bond which is elongated from 1.10 to 1.35 Å. The corresponding barrier height is just 9.8 kcal/mol. Note that such a barrier is 44.7 kcal/mol in the cluster model, thus suggesting that this reaction could not take place in a bare MoFe cofactor. Indeed, the complete absence of N2 reduction activity for the bare FeMo cofactor in the absence of the protein has been experimentally observed.51 The significant variation of the barrier is due to the strong hydrogen bond formed between the N atom and the positively charged α195HIS, as shown in Figure 6a. This is in agreement with the α195HIS mutation experiments which found that the mutation eliminates the N2 reduction ability.18,19 In a similar manner, the less efficient N2 reduction observed in the α-70VAL→ILE mutation experiments has been taken as an indication of N2 binding on the Fe6 atom,19 as also indicated by our

stability of the hydrogenated sulfur atoms and, hence, aid the hydrogenation of the incoming N2 molecule. The possibility of hydrogen atoms bonded to iron, either in a head-on rearrangement or in a bridging mode over two Fe atoms, has been hypothesized.22,28 All efforts to locate one of these structures ended up with the hydrogen moving to the S2B atom or the central carbon during structural optimizations. Nevertheless, the relatively short distance (about 2.2 Å) between the C−H hydrogen atom and the nearby Fe atom found in the computed structures could explain the experimental finding. Finally, the hydrogenation energy of S3B and the second hydrogenation energy of central carbon are significantly higher, being computed to be about −36 and −39 kcal/mol, respectively. Our results suggest that, at the beginning of the E4 state, the MoFe cofactor has received four protons which are located, for the above-mentioned energetic reasons, at the interstitial carbon, S2B, S2A, and S5A, respectively (see Figure 5 for details on structure). 3.2. E4 State: N2 Binding, First Reduction, and Hydrogen Release. At the beginning of the E4 state, the previous hydrogenation of the interstitial carbon has already weakened the carbon−Fe6 bond, leading to a structural rearrangement of the Fe−C bond and to the activation of the Fe atom toward N2 coordination. This flexible iron−carbon interaction and its role in the enzymatic catalysis has been recently hypothesized in the literature, albeit the hydrogenation of carbon has not been directly evidenced.33 The corresponding 1572


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Figure 8. Representation of the optimized structures of the most stable intermediates of the E5−E8 states.

computations. Furthermore, the α-70VAL residue has been found to control the substrate availability in a previous experimental study.19 Our computations indicate that such a control is realized via the interaction between α-70VAL side group and the intermediate species (E4LS2), as shown in Figure 6c. The hydrogenation of S2A, taking place at the E3 state, represents a condition sine qua non for the beginning of the N2 reduction. Nevertheless, such reaction could happen in absence of an H atom on S5A, that is at the E3 state, but it is energetically disfavored. Indeed, the computed barrier is 66.1 kcal/mol, which indicates that the 4Fe-4S cluster is not activated enough to bind an N2 molecule in the E3 state. Overall, these results suggest that the N2 molecule binds to the MoFe cofactor in the E4 state, and this event takes place only after the full hydrogenation of the interstitial carbon and the two sulfurs S2B and S5A. The central carbon is, therefore, not innocent in the N2 binding event, and a rationale is given for the accumulation of four hydrogen atoms in the MoFe cofactor before N2 binds. The formation of E4LS2 (see Figure 4) is the first step of N2 reduction, and it is exothermic by 12.3 kcal/mol. This intermediate, presenting an under-coordinated Fe atom, evolves through a barrier-less reaction to give the CH−N−NH2 intermediate (E4LS3), which is followed by the formation of one hydrogen molecule (E4TS2) and its release. This latter step is the rate-determining step of the whole mechanism, the associated barrier being 20.5 kcal/mol. Nonetheless, E4TS2 is

6.0 kcal/mol lower than the initial reactant E4N2 energy, so that this barrier can be overcome with the help of the excess energy released during the previous steps. The final product of E4 is a C−N−NH2 species (E4LS4), which is 15.1 kcal/mol lower than E4LS3 and 41.6 kcal/mol exothermic with respect to the starting reference (E4+ N2). In such a species the N−N bond is still activated, being about 1.35 Å. Our results on the cluster model indicate that from E4LS3 to E4LS4 the release of H2 is slightly unfavorable by 1.4 kcal/mol, so that it can be argued that the surrounding protein residues also play a role in H2 release. The positively charged α-96ARG residue interacts with the H atom that bonds to the S2A site, which significantly facilitates the H leaving as H2 together with the other H atom on the central carbon (see Figure 6b for a schematic illustration). This is in agreement with the experimental report that α-96ARG mutations could decrease the N2 reduction rate.18 3.3. E5−E8 States: Ammonia Release. Starting from the last product of the E4 state, i.e., E4LS4, there are different sites available for the attack of the incoming hydrogen in the next state, E5, as illustrated in Figure 7. The most stable one leads to the formation of the NH−NH 2 species (E5−NHNH 2 intermediate), whose structure is reported in Figure 8. The further hydrogenation of NH2, which results in an N−NH3 species, is only slightly higher in energies (+0.7 kcal/mol, E5− NNH3 intermediate), while the attack at the central carbon and the S2B site are significantly disfavored (see Figure 7). The small difference in the Gibbs free energies between the E5− 1573


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Figure 9. Relative Gibbs free energy (kcal/mol) for the E7 and E8 states. Data in parentheses were obtained by cluster model calculations.

barrierless release of the first NH3 molecule. The remaining amine moiety is further reduced in the last state of the catalytic cycle (the E8 state) and the second ammonia is then released from the MoFe cofactor (also barrier free), finally recovering the original E0 state. The structures of the most stable intermediates, E7−NH2 and E8−NH3, are reported in Figure 8. It should be pointed out that according to the cluster calculations the formation of the second NH3 is disfavored by 3.8 kcal/mol, as compared with the hydrogenation of sulfur atom. The second NH3 release is facilitated due to the destabilization of hydrogenated S2B by H-bond with α-195HIS and the steric repulsion between α-70VAL and NxHy species. Overall, the characterized reaction path corresponds to the alternating mechanism.22

NHNH2 and the E5−NNH3 intermediates suggests that both reaction channels starting from these intermediates are possible. In particular, the N−N bond cleavage and the release of an NH3 molecule can happen in the E5 state, leaving a bare N atom to be hydrogenated in the following E6−E8 states. This channel corresponds to the distal pathway mentioned in the literature.22,52 However, according to our calculations, the release of NH3 from the E5-NNH3 species is exothermic by 44.0 kcal/mol with an energy barrier of 34.9 kcal/mol (see Figure S9, SI). Therefore, even if it is thermodynamically favored, this side channel is actually kinetically forbidden. Our calculations suggest instead that the mechanism corresponds to an alternating pathway in which the two N atoms are hydrogenated alternatively, with a hydrazine-bound state generated at the E6 state and the first NH3 released during the E7 state (see infra), in agreement with the literature.22,52 It is interesting also to note that the cluster model predicts a greater stability for the E5-SH and E5-CH intermediates (see Figure 7) due to the lack of protein steric effects. In the E6 state, the continued hydrogenation of NH-NH2 species, leading to the formation of hydrazine (NH2−NH2), is preferred over the hydrogen attack to the central carbon (E6-CH, see Figure 7) or to the sulfur atom (E6-SH), the relative energies of these two intermediates being 10.7 and 22.6 kcal/mol, respectively. Also in this case, the protein environment plays a relevant role, since cluster computations indicate that both E6-CH and E6SH species are the most stable. In the E7 state (see Figure 9), the incoming H atom still preferentially bonds to the nitrogen atom, which results in the

4. COMMENTS Beyond the details on the energetics of the stationary points, the presented computational results provide important insights into the overall mechanism of N2 reduction and its interlock with experimental data. As mentioned above, the first four states (from E0 to E3) can be regarded as preparatory events, where up to three protons and three electrons arrive at the MoFe cofactor. No coordination of N2 has been found before the E3 state, in agreement with the LT scheme (see Figure 2). N2 binding happens during, more probably, the E4 state, as suggested experimentally,22 and it is promoted by an elongated Fe−C bond. Indeed, the average Fe−C distance increases of 0.26 Å in going from the E0 to the E4 state, and a similar 1574


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Figure 10. Relative Gibbs free energies (kcal/mol) for the coordination of diazene (a) and hydrazine (b) starting from the E0 state.

Aiso of the S2B hydrogen and one of the hydrogens bound to the central carbon are calculated to be 18.2 and 22.6 MHz, respectively, in good agreement with the experimental data within the limit of the theoretical approach used.55,56 The rate-determining step in the E4 state is the release of a hydrogen molecule, thus also placing this event in the reduction mechanism (see Figure 2). To the best of our knowledge, this is the first theoretical mechanism that provides a consistent explanation for the evolution of H2. The first ammonia molecule is then released in the E7 state, followed by the second one in the E8 state, which is compatible with the experimental indications.19,52 Therefore, we suggest that the N2 reduction takes place with an alternating-type mechanism, iterating from E0 to E8 state with the continuous supply of electrons, protons and N2. Globally, our calculations also point out the noninnocent role of the protein backbone, which facilitates the release of H2, as well as the nitrogen hydrogenations. Our modeling also provides some hints on the role of the residues α-195HIS, α96ARG, and α-70VAL. In particular, the role of α-195HIS in stabilizing the initially activated N2 species clearly emerges from our computations, in agreement with the experimental findings

variation (0.35 Å) has been experimentally found upon the coordination of a propargyl alcohol molecule on the external face of the MoFe cofactor.53 This structural flexibility is the key point in the stabilization of the NxHy intermediates all along the reaction and emphasizes the active role of the central carbon (which is nevertheless strongly buried inside the cofactor22). For example, during the hydrogenation and the subsequent N2 chemical binding, the size of the cofactor increases, as indicated by the S2B−S5A distance, which varies from 6.2 Å in the E0 state to 7.7 Å in the E4LS3 intermediate. Consequent to this breathing motion of the cofactor, the surrounding environment can have a stabilizing (or destabilizing) effect following the decreasing (or increasing) steric interaction or the creation (disruption) of weak hydrogen bonds (see below). These theoretical results strongly support the experimental findings and related hypotheses on the MoFe flexibility.33,53 A further confirmation of the nature of the E4 intermediate comes from the analysis of the isotropic hyperfine coupling constants (Aiso). In particular, experimental EPR investigations have characterized the Aiso of two protons at about 22 and 24 MHz22,54 in the E4 state. These protons were assigned to two different iron atoms.54 We did not find such a structure, but the 1575


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on the disruption of the reaction upon substitution with α195GLN.19 The α-96ARG residue is shown to stabilize the transition state E4TS2 and both effects should facilitate the reaction turnover. The interaction between the α-70VAL side chain group and the species bound to Fe−Mo cofactor (like in E4LS2) confirmed its role as limiting the substrate size. Finally, some considerations on hydrazine and diazene should be done, whose coordination, and subsequent reduction have been characterized in wild-type nitrogenease.21 The E0 state MoFe cofactor could bind one NHNH molecule to form the E4-NHNH species (Figure S8) or bind one NH2NH2 molecule to form the E6−NH2NH2 species (Figure 6). The calculated binding Gibbs free energies are −7.4 kcal/mol and −9.0 kcal/mol, respectively, all reported in Figure 10. The E4− NHNH species will then undergo a hydrogen atom transfer to form the E4LS4 intermediate or will be hydrogenated to E5− NHNH2 by the next incoming hydrogen atom, thus joining the N2 fixation catalytic cycle (see Figure 10). E6−NH2NH2 is, instead, the final product of the E6 state, already an intermediate of the N2 pathway. Our mechanism is thus fully compatible with the experimental hypothesis that diazene and hydrazine join the normal N2-reduction pathway.22,52

ACKNOWLEDGMENTS Dr. Ilaria Ciofini (Chimie ParisTech, Paris) and Dr. Frédéric Labat (Chimie ParisTech, Paris) are gratefully acknowledged for helpful suggestions. This work was granted access to the HPC resources of MesoPSL financed by the Region Ile de France and the project Equip@Meso (reference ANR-10EQPX-29-01) of the Programme Investissements d’Avenir supervised by the Agence Nationale pour la Recherche.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet ay The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.5b02577. Comments on the computational approach and spin atomic populations for selected intermediates; representation of the structures of the reaction intermediates not reported in the text; Cartesian coordinates of all the reaction intermediates (PDF)

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REFERENCES

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5. CONCLUSIONS In this work, we propose a mechanism for N2 reduction in molybdenum−nitrogenase based on the alternating hydrogenation of the two nitrogen atoms. This mechanism fits into the LT scheme, commonly used to place the experimental observations on N 2 reduction. The proposed reaction mechanism explains the relevant role of the α-195HIS residue in stabilizing the first reduction intermediate, while the surrounding residues such as α-70VAL and α-96ARG are also found to affect the catalytic activity via their interactions with the intermediate species. More importantly, the noninnocent role of the central carbon on the catalytic activity is evidenced and explained, as well as the flexibility of the MoFe cofactor. To the best of our knowledge, this is the first theoretical investigation that confirms the stoichiometric hypothesis of H2 evolution hypothesized in the LT scheme. More generally, the overall agreement with the experimental observations and hypotheses gives a solid foundation to the proposed mechanism, which can therefore represent a useful starting point for future experimental investigations of the N2 fixation mechanism.

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Research Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 1576


Research Article

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Theoretical Investigation on the Role of the Central Carbon Atom and

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