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Nitrogenase is the only enzyme that can cleave the strong triple bond in N 2 , making nitrogen available for biological life. There are three isozymes of nitrogenase, differing in the composition of the active site, viz., Mo, V, and Fe-nitrogenase. Recently, the first crystal structure of Fe-nitrogenase was presented. We have performed the first combined quantum mechanical and molecular mechanical (QM/MM) study of Fe-nitrogenase. We show with QM/MM and quantum-refinement calculations that the homocitrate ligand is most likely protonated on the alcohol oxygen in the resting E 0 state. The most stable broken-symmetry (BS) states are the same as for Mo-nitrogenase, i.e., the three Noodleman BS7-type states (with a surplus of β spin on the eighth Fe ion), which maximize the number of nearby antiferromagnetically coupled Fe-Fe pairs. For the E 1 state, we find that protonation of the S2B μ 2 belt sulfide ion is most favorable, 14-117 kJ/mol more stable than structures with a Fe-bound hydride ion (the best has a hydride ion on the Fe2 ion) calculated with four different density-functional theory methods. This is similar to what was found for Mo-nitrogenase, but it does not explain the recent EPR observation that the E 1 state of Fe-nitrogenase should contain a photolyzable hydride ion. For the E 1 state, many BS states are close in energy, and the preferred BS state differs depending on the position of the extra proton and which density functional is used.

Nitrogenase is the only enzyme that can cleave the triple bond in N 2 , making nitrogen available for all lifeforms. Previous computational studies have given widely diverging results regarding the reaction mechanism of the enzyme. For example, some recent studies have suggested that one of the μ 2 -bridging sulfide ligands (S2B) may dissociate from one of the Fe ions when protonated in the doubly reduced and protonated E 2 state, whereas other studies indicated that such half-dissociated states are unfavorable. We have examined how the relative energies of 26 structures of the E 2 state depend on details of combined quantum mechanical and molecular mechanical (QM/MM) calculations. We show that the selection of the broken-symmetry state, the basis set, relativistic effects, the size of the QM system, relaxation of the surroundings, and the conformations of the bound protons may affect the relative energies of the various structures by up to 12, 22, 9, 20, 37, and 33 kJ/mol, respectively. However, they do not change the preferred type of structures. On the other hand, the choice of the DFT functional strongly affects the preferences. The hybrid B3LYP functional strongly prefers doubly protonation of the central carbide ion, but such a structure is not consistent with experimental EPR data. Other functionals suggest structures with a hydride ion, in agreement with the experiments, and show that the ion bridges between Fe2 and Fe6. Moreover, there are two structures of the same type that are degenerate within 1-5 kJ/mol, in agreement with the observation of two EPR signals. However, the pure generalized gradient approximation (GGA) functional TPSS favors structures with a protonated S2B also bridging Fe2 and Fe6, whereas r 2 SCAN (meta-GGA) and TPSSh (hybrid) prefer structures with S2B dissociated from Fe2 (but remaining bound to Fe6). The energy difference between the two types of structure is so small (7-18 kJ/mol) that both types need to be considered in future investigations of the mechanism of nitrogenase.

CO-bound forms of nitrogenase are N 2 -reduction inhibited and likely intermediates in Fischer-Tropsch chemistry. Visible-light photolysis at 7 K was used to interrogate all three known CO-related EPR-active forms as exhibited by the α-H195Q variant of Azotobacter vinelandii nitrogenase MoFe protein. The hi(5)-CO EPR signal converted to the hi-CO EPR signal, which reverted at 10 K. FT-IR monitoring revealed an exquisitely light-sensitive "Hi-2" species with bands at 1932 and 1866 cm -1 that yielded "Hi-1" with bands at 1969 and 1692 cm -1 , which reverted to "Hi-2". The similarities of photochemical behavior and recombination kinetics showed, for the first time, that hi-CO EPR and "Hi-1" IR signals arise from one chemical species. hi(5)-CO EPR and "Hi-2" IR signals are from a second species, and lo-CO EPR and "Lo-2" IR signals, formed after prolonged illumination, are from a third species. Comparing FT-IR data with CO-inhibited MoFe-protein crystal structures allowed assignment of CO-bonding geometries in these species.

The nitrogenase active-site cofactor must accumulate 4e - /4H + (E 4 (4H) state) before N 2 can bind and be reduced. Earlier studies demonstrated that this E 4 (4H) state stores the reducing-equivalents as two hydrides, with the cofactor metal-ion core formally at its resting-state redox level. This led to the understanding that N 2 binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H 2 . The state having acquired 2e - /2H + (E 2 (2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e - /H + (E 1 (H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E 1 (H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E 1 (H) state of Fe-nitrogenase shows an S = 1/2 EPR spectrum with g = [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic intracavity , 450 nm photolysis converts to a new and likewise photoactive S = 1/2 state (denoted E 1 (H)*) with g = [2.009, 1.950, 1.860], which results in a photostationary state, with E 1 (H)* relaxing to E 1 (H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E 1 (H)/E 1 (H)* photointerconversion. These observations indicate that the addition of the first e - /H + to the FeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core is formally one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.

Carbon monoxide (CO) is a well-known inhibitor of nitrogenase activity. Under turnover conditions, CO binds to FeMoco, the active site of Mo nitrogenase. Time-resolved IR measurements suggest an initial terminal CO at 1904 cm -1 that converts to a bridging CO at 1715 cm -1 , and an X-ray structure shows that CO can displace one of the bridging belt sulfides of FeMoco. However, the CO-binding redox state(s) of FeMoco (E n ) and the role of the protein environment in stabilizing specific CO-bound intermediates remain elusive. In this work, we carry out an in-depth analysis of the CO-FeMoco interaction based on quantum chemical calculations addressing different aspects of the electronic structure. (1) The local electronic structure of the Fe-CO bond is studied through diamagnetically substituted FeMoco. (2) A cluster model of FeMoco within a polarizable continuum illustrates how CO binding may affect the spin-coupling between the metal centers. (3) A QM/MM model incorporates the explicit influence of the amino acid residues surrounding FeMoco in the MoFe protein. The QM/MM model predicts both a terminal and a bridging CO in the E 1 redox state. The scaled calculated CO frequencies (1922 and 1716 cm -1 , respectively) are in good agreement with the experimentally observed IR bands supporting CO binding to the E 1 state. Alternatively, an E 2 state QM/MM model, which has the same atomic structure as the CO-bound X-ray structure, features a semi-bridging CO with a scaled calculated frequency (1718 cm -1 ) similar to the bridging CO in the E 1 model.

The nitrogenase enzymes are responsible for all biological nitrogen reduction. How this is accomplished at the atomic level, however, has still not been established. The molybdenum-dependent nitrogenase has been extensively studied and is the most active catalyst for dinitrogen reduction of the nitrogenase enzymes. The vanadium-dependent form, on the other hand, displays different reactivity, being capable of CO and CO 2 reduction to hydrocarbons. Only recently did a crystal structure of the VFe protein of vanadium nitrogenase become available, paving the way for detailed theoretical studies of the iron-vanadium cofactor (FeVco) within the protein matrix. The crystal structure revealed a bridging 4-atom ligand between two Fe atoms, proposed to be either a CO 3 2- or NO 3 - ligand. Using a quantum mechanics/molecular mechanics model of the VFe protein, starting from the 1.35 Å crystal structure, we have systematically explored multiple computational models for FeVco, considering either a CO 3 2- or NO 3 - ligand, three different redox states, and multiple broken-symmetry states. We find that only a [VFe 7 S 8 C(CO 3 )] 2- model for FeVco reproduces the crystal structure of FeVco well, as seen in a comparison of the Fe-Fe and V-Fe distances in the computed models. Furthermore, a broken-symmetry solution with Fe2, Fe3, and Fe5 spin-down (BS7-235) is energetically preferred. The electronic structure of the [VFe 7 S 8 C(CO 3 )] 2- BS7-235 model is compared to our [MoFe 7 S 9 C] - BS7-235 model of FeMoco via localized orbital analysis and is discussed in terms of local oxidation states and different degrees of delocalization. As previously found from Fe X-ray absorption spectroscopy studies, the Fe part of FeVco is reduced compared to FeMoco, and the calculations reveal Fe5 as locally ferrous. This suggests resting-state FeVco to be analogous to an unprotonated E 1 state of FeMoco. Furthermore, V-Fe interactions in FeVco are not as strong compared to Mo-Fe interactions in FeMoco. These clear differences in the electronic structures of otherwise similar cofactors suggest an explanation for distinct differences in reactivity.

We have studied the geometry and electronic structure of the P-cluster in nitrogenase in four oxidation states: P N , P 1+ , P 2+ , and P 3+ . We have employed combined quantum mechanical and molecular mechanical (QM/MM) calculations, using two different density-functional theory methods, TPSS and B3LYP. The calculations confirm that the side chain of Ser-188 is most likely deprotonated in the partly oxidized P 1+ state, thereby forming a bond to Fe6. Likewise, the backbone amide group of Cys-88 is deprotonated in the doubly oxidized P 2+ state, forming a bond to Fe5. The calculations also confirm the two conformations of the P-cluster in the atomic-resolution crystal structure of the enzyme, representing the P N and P 2+ states, but show that the finer differences between the two structures are not fully reflected in the crystal structure, because the coordinates of only two atoms differ between the two conformations. However, the recent crystal structure of the P 1+ state seems to be of lower quality with many dubious Fe-Fe and Fe-S distances. Quantum refinement of this structure indicates that it is a mixture of the P 1+ and P 2+ states but confirms that the side chain of Ser-188 is most likely deprotonated in both states. TPSS gives structures that are appreciably closer to the crystal structures than does B3LYP. In addition, we have studied all 16-48 possible broken-symmetry states of the four oxidation states of the P-cluster with DFT in the one or two observed spin states. For the reduced P N state, we can settle the most likely state from the calculated energies and geometries. However, for the more oxidized states there are large differences in the predictions obtained with the two DFT methods.

The biomimetic diiron complex 4-(N 2 ) 2 , featuring two terminally bound Fe-N 2 centers bridged by two hydrides, serves as a model for two possible states along the pathway by which the enzyme nitrogenase reduces N 2 . One is the Janus intermediate E 4 (4H), which has accumulated 4[e-/H+], stored as two [Fe-H-Fe] bridging hydrides, and is activated to bind and reduce N 2 through reductive elimination (RE) of the hydride ligands as H 2 . The second is a possible RE intermediate. 1 H and 14 N 35 GHz ENDOR measurements confirm that the formally Fe(II)/Fe(I) 4-(N 2 ) 2 complex exhibits a fully delocalized, Robin-Day type-III mixed valency. The two bridging hydrides exhibit a fully rhombic dipolar tensor form, T ≈ [- t, + t, 0]. The rhombic form is reproduced by a simple point-dipole model for dipolar interactions between a bridging hydride and its "anchor" Fe ions, confirming validity of this model and demonstrating that observation of a rhombic form is a convenient diagnostic signature for the identification of such core structures in biological centers such as nitrogenase. Furthermore, interpretation of the 1 H measurements with the anchor model maps the g tensor onto the molecular frame, an important function of these equations for application to nitrogenase. Analysis of the hyperfine and quadrupole coupling to the bound 14 N of N 2 provides a reference for nitrogen-bound nitrogenase intermediates and is of chemical significance, as it gives a quantitative estimate of the amount of charge transferred between Fe and coordinated N, a key element in N 2 activation for reduction.

Early studies in which nitrogenase was freeze-trapped during enzymatic turnover revealed the presence of high-spin ( S = 3 / 2 ) electron paramagnetic resonance (EPR) signals from the active-site FeMo-cofactor (FeMo-co) in electron-reduced intermediates of the MoFe protein. Historically denoted as 1b and 1c, each of the signals is describable as a fictitious spin system, S' = 1 / 2 , with anisotropic g' tensor, 1b with g' = [4.21, 3.76, ?] and 1c with g' = [4.69, ∼3.20, ?]. A clear discrepancy between the magnetic properties of 1b and 1c and the kinetic analysis of their appearance during pre-steady-state turnover left their identities in doubt, however. We subsequently associated 1b with the state having accumulated 2[e - /H + ], denoted as E 2 (2H), and suggested that the reducing equivalents are stored on the catalytic FeMo-co cluster as an iron hydride, likely an [Fe-H-Fe] hydride bridge. Intra-EPR cavity photolysis (450 nm; temperature-independent from 4 to 12 K) of the E 2 (2H)/1b state now corroborates the identification of this state as storing two reducing equivalents as a hydride. Photolysis converts E 2 (2H)/1b to a state with the same EPR spectrum, and thus the same cofactor structure as pre-steady-state turnover 1c, but with a different active-site environment. Upon annealing of the photogenerated state at temperature T = 145 K, it relaxes back to E 2 (2H)/1b. This implies that the 1c signal comes from an E 2 (2H) hydride isomer of E 2 (2H)/1b that stores its two reducing equivalents either as a hydride bridge between a different pair of iron atoms or an Fe-H terminal hydride.

The P-cluster of the nitrogenase MoFe protein is a [ Fe 8 S 7 ] cluster that mediates efficient transfer of electrons to the active site for substrate reduction. Arguably the most complex homometallic FeS cluster found in nature, the biosynthetic mechanism of the P-cluster is of considerable theoretical and synthetic interest to chemists and biochemists alike. Previous studies have revealed a biphasic assembly mechanism of the two P-clusters in the MoFe protein upon incubation with Fe protein and ATP, in which the first P-cluster is formed through fast fusion of a pair of [ Fe 4 S 4 ] + clusters within 5 min and the second P-cluster is formed through slow fusion of the second pair of [ Fe 4 S 4 ] + clusters in a period of 2 h. Here we report a VTVH MCD and EPR spectroscopic study of the biosynthesis of the slow-forming, second P-cluster within the MoFe protein. Our results show that the first major step in the formation of the second P-cluster is the conversion of one of the precursor [ Fe 4 S 4 ] + clusters into the integer spin cluster [ Fe 4 S 3-4 ] α , a process aided by the assembly protein NifZ, whereas the second major biosynthetic step appears to be the formation of a diamagnetic cluster with a possible structure of [ Fe 8 S 7-8 ] β , which is eventually converted into the P-cluster.

Mo-containing nitrogenase is the main enzyme that is able to take N 2 from the air and form ammonia. The active-site cofactor of the enzyme, termed FeMoco, is unique in nature. It has seven Fe and one Mo atoms connected by S bridges, with a C atom in the center of the cofactor. Another unusual feature is that it has a large homocitrate ligand known to be of importance for catalysis. In the present computational study, the role of the homocitrate ligand is investigated. It is found that a large structural change, which makes Mo III five-coordinated, is energetically favorable in the more reduced states. This is of probable importance for the nitrogenase mechanism.

Herein, a systematic study of a series of molecular iron model complexes has been carried out using Fe L 2,3 -edge X-ray absorption (XAS) and X-ray magnetic circular dichroism (XMCD) spectroscopies. This series spans iron complexes of increasing complexity, starting from ferric and ferrous tetrachlorides ([FeCl 4 ] -/2- ), to ferric and ferrous tetrathiolates ([Fe(SR) 4 ] -/2- ), to diferric and mixed-valent iron-sulfur complexes [Fe 2 S 2 R 4 ] 2-/3- . This test set of compounds is used to evaluate the sensitivity of both Fe L 2,3 -edge XAS and XMCD spectroscopy to oxidation state and ligation changes. It is demonstrated that the energy shift and intensity of the L 2,3 -edge XAS spectra depends on both the oxidation state and covalency of the system; however, the quantitative information that can be extracted from these data is limited. On the other hand, analysis of the Fe XMCD shows distinct changes in the intensity at both L 3 and L 2 edges, depending on the oxidation state of the system. It is also demonstrated that the XMCD intensity is modulated by the covalency of the system. For mononuclear systems, the experimental data are correlated with atomic multiplet calculations in order to provide insights into the experimental observations. Finally, XMCD is applied to the tetranuclear heterometal-iron-sulfur clusters [MFe 3 S 4 ] 3+/2+ (M = Mo, V), which serve as structural analogues of the FeMoco and FeVco active sites of nitrogenase. It is demonstrated that the XMCD data can be utilized to obtain information on the oxidation state distribution in complex clusters that is not readily accessible for the Fe L 2,3 -edge XAS data alone. The advantages of XMCD relative to standard K-edge and L 2,3 -edge XAS are highlighted. This study provides an important foundation for future XMCD studies on complex (bio)inorganic systems.

N 2 reduction by nitrogenase involves the accumulation of four reducing equivalents at the active site FeMo-cofactor to form a state with two [Fe-H-Fe] bridging hydrides (denoted E 4 (4H), the Janus intermediate), and we recently demonstrated that the enzyme is activated to cleave the N≡N triple bond by the reductive elimination (re) of H 2 from this state. We are exploring a photochemical approach to obtaining atomic-level details of the re activation process. We have shown that, when E 4 (4H) at cryogenic temperatures is subjected to 450 nm irradiation in an EPR cavity, it cleanly undergoes photoinduced re of H 2 to give a reactive doubly reduced intermediate, denoted E 4 (2H)*, which corresponds to the intermediate that would form if thermal dissociative re loss of H 2 preceded N 2 binding. Experiments reported here establish that photoinduced re primarily occurs in two steps. Photolysis of E 4 (4H) generates an intermediate state that undergoes subsequent photoinduced conversion to [E 4 (2H)* + H 2 ]. The experiments, supported by DFT calculations, indicate that the trapped intermediate is an H 2 complex on the ground adiabatic potential energy suface that connects E 4 (4H) with [E 4 (2H)* + H 2 ]. We suggest that this complex, denoted E 4 (H 2 ; 2H), is a thermally populated intermediate in the catalytically central re of H 2 by E 4 (4H) and that N 2 reacts with this complex to complete the activated conversion of [E 4 (4H) + N 2 ] into [E 4 (2N2H) + H 2 ].

Despite decades of research, the structure-activity relationship of nitrogenase is still not understood. Only recently was the full molecular structure of the FeMo cofactor (FeMoco) revealed, but the charge and metal oxidation states of FeMoco have been controversial. With the recent identification of the interstitial atom as a carbide and the more recent revised oxidation-state assignment of the molybdenum atom as Mo(III), here we revisit the Mössbauer properties of FeMoco. By a detailed error analysis of density functional theory-computed isomer shifts and computing isomer shifts relative to the P-cluster, we find that only the charge of [MoFe 7 S 9 C] 1- fits the experimental data. In view of the recent Mo(III) identification, the charge of [MoFe 7 S 9 C] 1- corresponds to a formal oxidation-state assignment of Mo(III)3Fe(II)4Fe(III), although due to spin delocalization, the physical oxidation state distribution might also be interpreted as Mo(III)1Fe(II)4Fe(2.5)2Fe(III), according to a localized orbital analysis of the M S = 3/2 broken symmetry solution. These results can be reconciled with the recent spatially resolved anomalous dispersion study by Einsle et al. that suggests the Mo(III)3Fe(II)4Fe(III) distribution, if some spin localization (either through interactions with the protein environment or through vibronic coupling) were to take place.

A series of oxidovanadium(V) complexes [VO(L-κ 4 O,N,N,O)(OR)] (1a, R = Et, L = L 1 ; 1b, R = Me, L = L 1 ; 2, R = Me, L = L 2 ; 3, R = Me, L = L 3 ) were synthesized by the σ-bond metathesis reaction between [VO(OR) 3 ] and the linear diaminebis(phenol) derivatives H 2 L (salans) containing different para-substituents on the phenoxo group [CMe 3 CH 2 CMe 2 , L 1 ; Me, L 2 ; Cl, L 3 ]. As shown by X-ray crystallography complexes 1a, 1b, and 2 exhibit cis-α geometry, do have a stereogenic vanadium center, and exist as a racemic mixture of the Δ cis-α and Λ cis-α enantiomers. In solution, as demonstrated by 1 H and 51 V NMR investigations, the structures of complexes 1-3 are consistent with their solid state. The reactions of 1b, 2, and 3 with NH 2 NMePh in n-hexane afforded the oxidovanadium(IV) [VO(L-κ 4 O,N,N,O)] (4, L 1 ; 5, L 2 ; 6, L 3 ) and 1,4-dimethyl-1,4-diphenyl-2-tetrazene (Me 2 Ph 2 N 4 ) (7) as the main products together with a small amount of hydrazido(2-) vanadium(V) [V(L 3 -κ 4 O,N,N,O)(NNMePh)(OMe)] (8). Proposed reaction course for the oxidation of NH 2 NMePh by 1b-3 is discussed. Compounds 4-8 were characterized by chemical and physical techniques including the X-ray crystallography for 4, 7, and 8. The solid-state (powder) electron paramagnetic resonance spectra and magnetic features strongly indicate that complexes 4-6 are formed as a mixture of a mononuclear (S = 1/2) and a dinuclear (S = 1) species.

We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe-H-Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1-8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped "hydride" state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysis at cryogenic temperature. The protocol rests on the hypothesis that an intermediate trapped in the frozen solid can relax toward the resting state only by the release of a stable reduction product from FeMo-co. In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesis implicitly predicts that states that have accumulated an odd number of electrons/protons (n = 1, 3) during turnover under Ar cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozen state. The present experiments confirm this prediction and, thus, the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis. This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and 1c, trapped under high electron flux.

The catalyzed hydrogenations effected at the active site FeMo-co of nitrogenase have been proposed to involve serial supply of the required multiple protons along a proton wire terminating at sulfur atom S3B of FeMo-co. In conjunction with serial electron transfer to FeMo-co, these protons become H atoms, and then are able to migrate from S3B to other Fe and S atoms of FeMo-co, and to transfer to bound substrate and intermediates. This general model, which can account for all reactions of nitrogenase, involves a preparatory stage in which each incoming H atom is required to move from the proton delivery side of S3B to the opposite migration side of S3B. This report examines the mechanism of this reconfiguration of S3B-H, finding four stable configurations in which S3B-H has pyramidal-trigonal coordination, with one elongated Fe-S3B interaction. The transition states and energies for reconfiguration are described. Pseudotetrahedral four coordination and planar-trigonal coordination for S3B-H are less stable than pyramidal-trigonal coordination. Results are presented for FeMo-co with one, two, three, and four H atoms (the E1H1, E2H2, E3H3, and E4H4 Thorneley-Lowe stages), and the general principles are defined, for application in the various chemical mechanisms of nitrogenase.

Heteroligated cluster cores consisting of weak-field iron, strongly basic nitrogen anions, and sulfide are of interest with respect to observed and conjectured environments in the FeMo cofactor of nitrogenase. Selective syntheses have been developed to achieve such environments with tert-butyl-substituted amide and imide core ligands. A number of different routes were employed, including nominal ligand substitution and oxidative addition reactions, as well as novel transformations involving the combination of different cluster precursors. New cluster products include precursor Fe(2)(μ-NH(t)Bu)(2)[N(SiMe(3))(2)](2) (6), Fe(2)(μ-NH(t)Bu)(2)(μ-S)[N(SiMe(3))(2)](2) (7), which has a rare confacial bitetrahedral geometry previously unknown in iron chemistry, [Fe(2)(μ-N(t)Bu)(μ-S)Cl(4)](2-) (2), and cuboidal [Fe(4)(μ(3)-N(t)Bu)(3)(μ(3)-S)Cl(4)](-) (8), [Fe(4)(μ(3)-N(t)Bu)(2)(μ(3)-S)(2)Cl(4)](2-) (9), and [Fe(4)(μ(3)-N(t)Bu)(μ(3)-S)(3)Cl(4)](2-) (10), as well as selenide-substituted derivatives Fe(2)(μ-NH(t)Bu)(2)(μ-Se)[N(SiMe(3))(2)](2) (7-Se) and [Fe(4)(μ(3)-N(t)Bu)(μ(3)-Se)(3)Cl(4)](2-) (10-Se). The imide-sulfide clusters complete the compositional sets [Fe(2)(μ-N(t)Bu)(n)(μ-S)(2-n)Cl(4)](2-) (n = 0-2) and [Fe(4)(μ(3)-N(t)Bu)(n)(μ(3)-S)(4-n)Cl(4)](z) (n = 0-4), represented previously only by the all-imide and all-sulfide core congeners, and they share chemical and physical properties with the parent homoleptic core species. All imide-sulfide cores are compositionally stable and show no evidence of core ligand exchange over days in solution. Beyond structural differences, the impact of mixed core ligation is most evident in redox potentials, which show progressive decreases of -435 (for z = 1-/2-) or -385 mV (for z = 2-/3-) for each replacement of sulfide by the more potent imide donor, and a corresponding effect may be expected for the interstitial heteroligand in the FeMo cofactor. Cluster 10 presents an [Fe(4)NS(3)] core framework virtually isometric with the isostructural [Fe(4)S(3)X] subunit of the FeMo cofactor, thus providing a synthetic structural representation for this portion of the cofactor core.

An extensive series of heterometal-iron-sulfur single cubane-type clusters with core oxidation levels [MFe(3)S(3)Q](3+,2+) (M = Mo, W; Q = S, Se) has been prepared by means of a new method of cluster self-assembly. The procedure utilizes the assembly system [((t)Bu(3)tach)M(VI)S(3)]/FeCl(2)/Na(2)Q/NaSR in acetonitrile/THF and affords product clusters in 30-50% yield. The trisulfido precursor acts as a template, binding Fe(II) under reducing conditions and supplying the MS(3) unit of the product. The system leads to specific incorporation of a μ(3)-chalcogenide from an external source (Na(2)Q) and affords the products [((t)Bu(3)tach)MFe(3)S(3)QL(3)](0/1-) (L = Cl(-), RS(-)), among which are the first MFe(3)S(3)Se clusters prepared. Some 16 clusters have been prepared, 13 of which have been characterized by X-ray structure determinations including the incomplete cubane [((t)Bu(3)tach)MoFe(2)S(3)Cl(2)(μ(2)-SPh)], a possible trapped intermediate in the assembly process. Comparisons of structural and electronic features of clusters differing only in atom Q at one cubane vertex are provided. In comparative pairs of complexes differing only in Q, placement of one selenide atom in the core increases core volumes by about 2% over the Q = S case, sets the order Q = Se > S in Fe-Q bond lengths and Q = S > Se in Fe-Q-Fe bond angles, causes small positive shifts in redox potentials, and has an essentially nil effect on (57)Fe isomer shifts. Iron mean oxidation states and charge distributions are assigned to most clusters from isomer shifts. ((t)Bu(3)tach = 1,3,5-tert-butyl-1,3,5-triazacyclohexane)., (© 2011 American Chemical Society)

A significant limitation in our understanding of the molecular mechanism of biological nitrogen fixation is the uncertain composition of the FeMo-cofactor (FeMo-co) of nitrogenase. In this study we present a systematic, density functional theory-based evaluation of spin-coupling schemes, iron oxidation states, ligand protonation states, and interstitial ligand composition using a wide range of experimental criteria. The employed functionals and basis sets were validated with molecular orbital information from X-ray absorption spectroscopic data of relevant iron-sulfur clusters. Independently from the employed level of theory, the electronic structure with the greatest number of antiferromagnetic interactions corresponds to the lowest energy state for a given charge and oxidation state distribution of the iron ions. The relative spin state energies of resting and oxidized FeMo-co already allowed exclusion of certain iron oxidation state distributions and interstitial ligand compositions. Geometry-optimized FeMo-co structures of several models further eliminated additional states and compositions, while reduction potentials indicated a strong preference for the most likely charge state of FeMo-co. Mössbauer and ENDOR parameter calculations were found to be remarkably dependent on the employed training set, density functional, and basis set. Overall, we found that a more oxidized [Mo(IV)-2Fe(II)-5Fe(III)-9S(2-)-C(4-)] composition with a hydroxyl-protonated homocitrate ligand satisfies all of the available experimental criteria and is thus favored over the currently preferred composition of [Mo(IV)-4Fe(II)-3Fe(III)-9S(2-)-N(3-)] from the literature., (© 2011 American Chemical Society)

The all-ferrous, carbene-capped Fe(4)S(4) cluster, synthesized by Deng and Holm (DH complex), has been studied with density functional theory (DFT). The geometry of the complex was optimized for several electronic configurations. The lowest energy was obtained for the broken-symmetry (BS) configuration derived from the ferromagnetic state by reversing the spin projection of one of the high spin (S(i) = 2) irons. The optimized geometry of the latter configuration contains one unique and three equivalent iron sites, which are both structurally and electronically clearly distinguishable. For example, a distinctive feature of the unique iron site is the diagonal Fe···S distance, which is 0.3 Å longer than for the equivalent irons. The calculated (57)Fe hyperfine parameters show the same 1:3 pattern as observed in the Mössbauer spectra and are in good agreement with experiment. BS analysis of the exchange interactions in the optimized geometry for the 1:3, M(S) = 4, BS configuration confirms the prediction of an earlier study that the unique site is coupled to the three equivalent ones by strong antiferromagnetic exchange (J > 0 in J Σ(j<4)Ŝ(4)·Ŝ(j)) and that the latter are mutually coupled by ferromagnetic exchange (J' < 0 in J' Σ(i

Treatment of [Et(4)N][Tp*WS(3)] (1) (Tp* = hydridotris(3,5-dimethylpyrazol-1-yl)borate) with 2 equiv of AgSCN in MeCN afforded a novel neutral compound [(Tp*WS(2))(2)(μ-S(2))] (2). Reactions of 2 with excess CuX (X = Cl, Br, I) in MeCN and CH(2)Cl(2) or CHCl(3) formed three neutral W/Cu/S clusters [{Tp*W(μ(3)-S)(3)Cu(3)(μ-Cl)}(2)Cu(μ-Cl)(2)(μ(7)-Cl)(MeCN)](2) (3), [{Tp*W(μ(3)-S)(3)Cu(3)}(2)Br(μ-Br)(2)(μ(4)-Br)(MeCN)] (4), and [{Tp*W(μ(3)-S)(3)Cu(3)}(2){Cu(2)(μ-I)(4)(μ(3)-I)(2)}] (5), respectively. On the other hand, treatment of 2 with CuX (X = Cl, Br) in the presence of Et(4)NX (X = Cl, Br) produced two anionic W/Cu/S clusters [Et(4)N][{Tp*W(μ(3)-S)(3)Cu(3)X}(2)(μ-X)(2)(μ(4)-X)] (6: X = Cl; 7 X = Br). Compounds 2-7 were characterized by elemental analysis, IR, UV-vis, (1)H NMR, electrospray ionization (ESI) mass spectra, and single-crystal X-ray crystallography. The dimeric structure of 2 can be viewed as two [Tp*WS(2)] fragments in which two W atoms are connected by one S(2)(2-) dianion. Compounds 3-7 all possess unique halide-bridged double cubanelike frameworks. For 3, two [Tp*W(μ(3)-S)(3)Cu(3)](2+) dications are linked via a μ(7)-Cl(-) bridge, two μ-Cl(-) bridges, and a [Cu(MeCN)(μ-Cl)(2)](+) bridge. For 4, one [Tp*W(μ(3)-S)(3)Cu(3)(MeCN)](2+) dication and one [Tp*W(μ(3)-S)(3)Cu(3)Br](+) cation are linked via a μ(4)-Br(-) and two μ-Br(-) bridges. For 5, the two [Tp*W(μ(3)-S)(3)Cu(3)](2+) dications are bridged by a linear [(μ-I)(2)Cu(μ(3)-I)(2)Cu(μ-I)(2)](4+) species. For 6 and 7, two [Tp*W(μ(3)-S)(3)Cu(3)X](+) cations are linked by a μ(4)-X(-) and two μ-X(-) bridges (X = Cl, Br). In addition, the third-order nonlinear optical (NLO) properties of 2-7 in MeCN/CH(2)Cl(2) were investigated by using femtosecond degenerate four-wave mixing (DFWM) technique.

The iron-molybdenum cofactor (FeMo-co), which is the catalytic center for the enzymatic conversion of N(2) to NH(3), has the composition [NFe(7)MoS(9)(homocitrate)], and, with a cluster of eight transition metal atoms and nine sulfur atoms, has a complex delocalized electronic structure. The electronic dimensions of FeMo-co and of each of its derivatives appear as sets of electronic states lying close in energy. These electronic dimensions naturally partner the geometrical changes and the reactivity patterns during the catalytic cycle, and also connect with spectroscopic investigations of the mechanism. This paper describes straightforward computational procedures for the determination and management of the low-lying electronic states of FeMo-co and of its coordinated intermediates and transition states during density functional simulations of steps in the catalytic mechanism. General principles for the distribution of electron spin density over all atoms are presented, using several proposed intermediates as examples. A tough general irony arises in the distribution of spin density over FeMo-co and its derivatives: the less interesting atoms get the spin, and the most interesting atoms do not.

A series of V(III) complexes of varying coordination number (5, 6, and 7) all containing the PS3 ligand (PS3 = trianion of tris(2-thiophenyl)phosphine and its derivatives with other phenyl substituents) has been prepared and structurally characterized. The complexes have general formula [V(PS3)L(n)](0,-), where n = 1 (from L = Cl(-), 1-Me-Im, N(3)(-)), 2 (from L = 2,2'-bpy; counting each N of the bidentate ligand), and 3 (from L = 1-Me-Im, N(2)H(4)). The complexes have also been investigated by direct current (DC) magnetic susceptibility and high-frequency and -field electron paramagnetic resonance (HFEPR). HFEPR, supported by magnetometry, has provided accurate spin Hamiltonian parameters that describe the S = 1 spin ground state of the complexes. Of particular interest are the zero-field splitting (zfs) parameters which, together with structural data, are the empirical starting point for detailed computational studies. The computational methods included density functional theory (DFT), which was only marginally successful, and more advanced ab initio methods (CASSCF and SORCI). The zfs in these complexes is relatively small in magnitude (|D| approximately 1 cm(-1)) and is the result of multiple, often counteracting, spin-orbit coupling (SOC) and spin-spin coupling (SSC) contributions. The specific origin of each of these contributions is described in detail. The results indicate the level of electronic structure calculation possible for transition metal complexes even with multiple unpaired electrons and highly covalent, heavier atom donor ligands.

A C-H bond of Cp*(2)Co was found to be cleaved by a [Fe(8)S(7)] cluster model of the nitrogenase P-cluster. This is the first example of C-H bond activation mediated by a biologically relevant Fe/S cluster. The reaction mechanism probably consists of electron transfer from Cp*(2)Co to the [Fe(8)S(7)] cluster and subsequent proton abstraction by the reduced form of the cluster.

It is well established that the cysteinate-coordinated [Fe(4)S(4)] cluster of the iron protein of nitrogenase from Azotobacter vinelandii (Av2) can attain the all-ferrous core oxidation state. Mössbauer and electron paramagnetic resonance (EPR) studies have shown that the all-ferrous cluster has a ground-state spin S = 4 and an effective 3:1 site symmetry in the spin structure and (57)Fe quadrupole interactions. Recently, Deng and Holm reported the synthesis of [Fe(4)S(4)(Pr(i)(2)NHCMe(2))(4)],(2) (1; Pr(i)(2)NHCMe(2) = 1,3-diisopropyl-4,5-dimethylimidazol-2-ylidene) and showed that the all-ferrous carbene-coordinated cluster is amenable to physicochemical studies. Mössbauer and EPR studies of 1, reported here, reveal that the electronic structure of this complex is strikingly similar to that of the protein-bound cluster, suggesting that the ground-state spin and the 3:1 site ratio are consequences of spontaneous distortions of the cluster core. To gain insight into the origin of the peculiar ground state of the all-ferrous clusters in 1 and Av2, we have studied a theoretical model that is based on a Heisenberg-Dirac-van Vleck Hamiltonian whose exchange-coupling constants are a function of the Fe-Fe distances. By combining the exchange energies with the elastic deformation energies in the harmonic approximation, we obtain for a T(2) distortion a minimum with spin S = 4 and a C(3v) core structure in which one iron is unique and three irons are equivalent. This minimum has all of the spectroscopic and structural characteristics of the all-ferrous clusters of 1 and Av2. Our analysis maps the unique spectroscopic iron site to a specific site in the X-ray structure of the [Fe(4)S(4)](0) core both in complex 1 and in Av2.

Broken symmetry density functional theory (BS-DFT) has been used to address the hyperfine parameters of the single atom ligand X, proposed to be coordinated by six iron ions in the center of the paramagnetic FeMo-cofactor (FeMoco) of nitrogenase. Using the X = N alternative, we recently found that any hyperfine signal from X would be small (calculated A(iso)(X = (14)N) = 0.3 MHz) due to both structural and electronic symmetry properties of the [Mo-7Fe-9S- X] FeMoco core in its resting S = 3/2 state. Here, we extend our BS-DFT approach to the 2e(-) reduced S = 1/2 FeMoco state. Alternative substrates coordinated to this FeMoco state effectively perturb the electronic and/or structural symmetry properties of the cofactor. Using an example of an allyl alcohol (H2C=CH-CH2-OH) product ligand, we consider three different binding modes at single iron site and three different BS-DFT spin state structures and show that this binding would enhance the key hyperfine signal A(iso)(X) by at least 1 order of magnitude (3.8 < or = A(iso)(X = (14)N) < or = 14.7 MHz), and this result should not depend strongly on the exact identity of X (nitrogen, carbon, or oxygen). The interstitial atom, when the nucleus has a nonzero magnetic moment, should therefore be observable by ESR methods for some ligand-bound FeMoco states. In addition, our results illustrate structural details and likely spin-coupling patterns for models for early intermediates in the catalytic cycle.

Both vanadium and molybdenum cofactor clusters are found in nitrogenase. In biomimetic research, many fewer heterometal MFe3S4 cubane-type clusters have been synthesized with M = V than with M = Mo because of the well-established structural relationship of the latter to the molybdenum coordination unit in the enzyme. In this work, a series of single cubane and edge-bridged double cubane clusters containing the cores [VFe3(mu3-S)4]2+ and [V2Fe6(mu3-S)6(mu4-S)2]2+ have been prepared by ligand substitution of the phosphine clusters [(Tp)VFe3S4(PEt3)3]1+ and [(Tp)2V2Fe6S8(PEt3)4]. The single cubanes [(Tp)VFe3S4L3]2- and double cubanes [(Tp)2V2Fe6S8L4]4- (L= F-, N3-, CN-, PhS-) are shown by X-ray structures to have trigonal symmetry and centrosymmetry, respectively. Single cubanes form the three-member electron transfer series [(Tp)VFe3S4L3]3-,2-,1-. The ligand dependence of redox potentials and electron distribution in cluster cores as sensed by 57Fe isomer shifts (delta) have been determined. Comparison of these results with those previously determined for the analogous molybdenum clusters (Pesavento, Berlinguette, and Holm Inorg. Chem. 2007, 46, 510) allows detection of the influence of heterometal M on the properties. At constant M and variable L, redox potentials are lowest for pi-donor ligands and largest for cyanide and relate approximately with decreasing ferrous character in clusters with constant charge z = 2-. At constant L and z and variable M, EV > E(Mo) and delta(av)V < delta(av)Mo, demonstrating that M = Mo clusters are more readily oxidized and suggesting a qualitative relation between lower potentials (greater ease of oxidation) and ferrous character.

Six of the seven iron atoms in the iron-molybdenum cofactor of nitrogenase display an unusual geometry, which is distorted from the tetrahedral geometry that is most common in iron-sulfur clusters. This distortion pulls the iron along one C3 axis of the tetrahedron toward a trigonal pyramid. The trigonal pyramidal coordination geometry is rare in four-coordinate transition metal complexes. In order to document this geometry in a systematic fashion in iron(II) chemistry, we have synthesized a range of four-coordinate iron(II) complexes that vary from tetrahedral to trigonal pyramidal. Continuous shape measures are used for a quantitative comparison of the stereochemistry of the Fe atoms in the iron-molybdenum cofactor with those of the presently and previously reported model complexes, as well as with those in polynuclear iron-sulfur compounds. This understanding of the iron coordination geometry is expected to assist in the design of synthetic analogues for intermediates in the nitrogenase catalytic cycle.

We report the synthesis, spectroscopy, and structural characterization of iron-alkyne and -alkene complexes of the type L(Me)Fe(ligand) [L(Me) = bulky beta-diketiminate, ligand = HCCPh, EtCCEt, CH2CHPh, EtCHCHEt, HCC(p-C6H4OCH3), HCC(p-C6H4CF3)]. The neutral ligand exchanges rapidly at room temperature, and the equilibrium constants have been measured or estimated. The binding affinity toward the low-coordinate Fe follows the trend HCCPh > EtCCEt > CH2CHPh > EtCHCHEt approximately PPh3 > benzene >> N2. This trend is consistent with a model in which pi back-bonding from the formally Fe(I) center is the dominant interaction in determining the relative binding affinities. In nitrogenase, alkynes are reduced while alkenes are unreactive, and this work suggests that the different binding affinities to low-coordinate Fe might explain the differential activity of the enzyme toward these two substrates.

Our explorations of the reactivity of Fe/Mo/S clusters of some relevance to the FeMoco nitrogenase have led to new double-fused cubane clusters with the Mo2Fe6S8 core as derivatives of the known (Cl4-cat)2Mo2Fe6S8(PPr3)6 (I) fused double cubane. The new clusters have been obtained by substitution reactions of the PPr3 ligands with Cl-, BH4-, and N3-. By careful control of the conditions of these reactions, the clusters [(Cl4-cat)(PPr3)MoFe3S4(BH4)2]2(Bu4N)4 (II), [(Cl4-cat)(PPr3)MoFe3S4(PPr3)(BH4)]2(Bu4N)2 (III), [(Cl4-cat)(PPr3)MoFe3S4(N3)2]2(Bu4N)4 (IV), [(Cl4-cat)(PPr3)MoFe3S4(PPr3)(N3)]2(Bu4N)2 (V), and [(Cl4-cat)(PPr3)MoFe3S4Cl2]2(Et4N)4 (VI) have been obtained and structurally characterized. A study of their electrochemistry shows that the reduction potentials for the derivatives of I are shifted to more positive values than those of I, suggesting a stabilization of the reduced clusters by the anionic ligands BH4- and N3-. Using 1H NMR spectroscopy, we have explored the lability of the BH4- ligand in II in coordinating solvents and its hydridic character, which is apparent in its reactivity toward proton sources such as MeOH or PhOH.

Members of the cluster set [(Tp)2Mo2Fe6S8L4]z contain the core unit M2Fe6(mu3-S)6(mu4-S)2 in which two MoFe3S4 cubanes are coupled by two Fe-(mu4-S) interactions to form a centrosymmetric edge-bridged double cubane cluster. Some of these clusters are synthetic precursors to [(Tp)2Mo2Fe6S9L2]3-, which possess the same core topology as the P(N) cluster of nitrogenase. In this work, the existence of a three-member electron-transfer series of single cubanes [(Tp)MoFe3S4L3](z) (z = 3-, 2-, 1-) and a four-member series of double cubanes [(Tp)2Mo2Fe6S8L4]z (z = 4-, 3-, 2-, 1-) with L = F-, Cl-, N3, PhS- is demonstrated by electrochemical methods, cluster synthesis, and X-ray structure determinations. The potential of the [4-/3-] couple is extremely low (<-1.5 V vs SCE in acetonitrile) such that the 4- state cannot be maintained in solution under normal anaerobic conditions. The chloride double cubane redox series was examined in detail. The members [(Tp)2Mo2Fe6S8Cl4]4-,3-,2- were isolated and structurally characterized. The redox series includes the reversible steps [4-/3-] and [3-/2-]. Under oxidizing conditions, [(Tp)2Mo2Fe6S8Cl4]2- cleaves with the formation of single cubane [(Tp)MoFe3S4Cl3]1-. The quasireversible [2-/1-] couple is observed at more positive potentials than those of the single cubane redox step. Structure comparison of nine double cubanes suggests that significant dimensional changes pursuant to redox reactions are mainly confined to the Fe2(mu4-S)2 bridge rhomb. The synthesis and structure of [(Tp)2Mo2Fe6S9F2.H2O]3-, a new topological analogue of the P(N) cluster of nitrogenase, is described. (Tp = hydrotris(pyrazolyl)borate(1-)).

The catalytic cycle of acetylene reduction at the FeMo cofactor of nitrogenase has been investigated on the basis of density functional theory. C2H2 binds to the same site as N2, but it binds to a less reduced state of the cofactor. In a manner similar to that of N2 binding, one of the sulfur bridges opens during acetylene binding. The model explains the strong noncompetitive inhibition of N2 reduction by C2H2 and the weak competitive inhibition of C2H2 reduction by N2. Our proposed mechanism is consistent with experimentally observed stereoselectivity and the ability of C2H2 to suppress H2 production by nitrogenase.

The synthesis and structure of the first Mo/Fe/S/BH(4) cluster is reported. Reaction of (Cl(4)-cat)(2)Mo(2)Fe(6)S(8)(PPr(3))(6) with 4 equiv of Bu(4)NBH(4) results in the formation of [(Cl(4)-cat)(PPr(3))MoFe(3)S(4)(BH(4))(2)](2)(Bu(4)N)(4) (Cl(4)-cat = tetrachloro-catecholate) which has been fully characterized. X-ray structural determination of this double-fused cubane reveals four BH(4)(-) ligands bound to four Fe atoms in a bidentate fashion. A synopsis of the solution characterization as well as the reactivity of this cluster is also presented.

Density functional theory calculations were carried out on the structurally characterized [(Cl(4)-cat)Mo(py)Fe(3)S(3) (CO)(4)(P(n)Pr(3))(3)], A, and (Cl(4)-cat)Mo(py)Fe(3)S(3)(CO)(6)(PEt(3))(2), B, and also on A(2)(-) and B(2+) clusters. The Fe-Fe distances in these molecules depend on the total number of valence electrons (60 e(-) in A and B(2)(+) and 62 e(-) in A(2)(-) and B) and undergo great structural changes upon addition or removal of electrons. The changes are consistent with known electron-counting rules in organometallic chemistry. The weak nature of the Fe-Fe bonding interactions in these clusters is apparent in the very similar energies of states with widely different Fe-Fe distances.

Edge-bridged Mo-Fe-S double cubanes are versatile precursors for the synthesis of other clusters of the same nuclearity. Thus, the double cubane [(Tp)(2)Mo(2)Fe(6)S(8)(PEt(3))(4)] sustains terminal ligand substitution with retention of the Mo(2)Fe(6)(micro(3)-S)(6)(micro(4)-S)(2) core structure and rearrangement to the Mo(2)Fe(6)(micro(2)-S)(2)(micro(3)-S)(6)(micro(6)-S) topology of the nitrogenase P(N) cluster upon reaction with certain nucleophiles. Four distinct processes for the conversion of double cubanes to P(N)-type clusters are documented, affording the products [(Tp)(2)Mo(2)Fe(6)S(9)(SR)(2)](3)(-), [(Tp)(2)Mo(2)Fe(6)S(8)(OMe)(3)](3)(-), and [(Tp)(2)Mo(2)Fe(6)S(7)(OMe)(4)](2)(-). In the latter clusters, two methoxides are terminal ligands and one or two are micro(2)-bridging ligands. The reverse transformation of a P(N)-type cluster to an edge-bridged double cubane has been demonstrated by the reaction of [(Tp)(2)Mo(2)Fe(6)S(8)(OMe)(3)](3)(-) with Me(3)SiX to afford [(Tp)(2)Mo(2)Fe(6)S(8)X(4)](2)(-) (X = Cl(-), Br(-)). Edge-bridged double cubanes have been obtained in the oxidation states [Mo(2)Fe(6)S(8)](2+,3+,4+). The stable oxidation state of P(N)-type clusters is [Mo(2)Fe(6)S(9)](+). Structures of five double cubanes and four P(N)-type clusters are reported. The P(N)-type clusters are synthetic representations of the biologically unique topology of the native P(N) cluster. Best-fit superpositions of the native and synthetic cluster cores gives weighted rms deviations in atom positions of 0.20-0.38 A. This study and an earlier investigation (Zhang, Y.; Holm, R. H. J. Am. Chem. Soc. 2003, 125, 3910-3920) provide a comprehensive account of the synthesis of structural analogues of the native P(N) cluster and provide the basis for continuing investigation of the synthesis of weak-field Mo-Fe-S clusters related to nitrogenase. (Tp = tris(pyrazolyl)hydroborate(1-).)

The geometries and stabilities of the FeFe cofactor at different oxidation states and its complexes with N(2) have been determined by density functional calculations. These calculations support an EPR-inactive resting state of the FeFe cofactor with four Fe(2+) and four Fe(3+) sites (4Fe(2+)4Fe(3+)). FeFeco(mu(6)-N(2)) with a central dinitrogen ligand is predicted to be the most stable complex of the FeFe cofactor with N(2). It is easily formed by penetration of N(2) into the trigonal Fe(6) prism of the FeFe cofactor with an approximate barrier of 4 kcal mol(-1). The present DFT results suggest that an FeFeco(mu(6)-N(2)) entity is a plausible intermediate in dinitrogen fixation by nitrogenase. CO is calculated to bind even more strongly than N(2) to the FeFe cofactor so that CO may inhibit the reduction of nitrogen by Fe-only nitrogenase.

The influence of the interstitial atom, X, discovered in a recent crystallographic study of the MoFe protein of nitrogenase, on the electric hyperfine interactions of (57)Fe has been investigated with density functional theory. A semiempirical theory for the isomer shift, delta, is formulated and applied to the cofactor. The values of delta for the relevant redox states of the cofactor are predicted to be higher in the presence of X than in its absence. The analysis strongly suggests a [Mo(4+)4Fe(2+)3Fe(3+)] oxidation state for the S = 3/2 state M(N). Among C(4-), N(3-), and O(2-), oxide is found to be the least likely candidate for X. The analysis suggests that X should be present in the cofactor states M(OX) and M(R) as well as in the alternative nitrogenases. The calculations of the electric field gradients (EFGs) indicate that the small values for DeltaE(Q) in M(N) result from an extensive cancellation between valence and ligand contributions. X emerges from the analysis of the hyperfine interactions as an ionically bonded species. Its major effect is on the asymmetry parameters for the EFGs at the six equatorial sites, Fe(Eq). A spin-coupling scheme is proposed for the state [Mo(4+)4Fe(2+)3Fe(3+)] that is consistent with the measured (57)Fe A-tensors and DeltaE(Q) values for M(N) and identifies the unique site exhibiting the small A value with the terminal Fe site, Fe(T). The optimized structure of a cofactor model has been calculated for several oxidation states. The study reveals a contraction in the average Fe-Fe distance upon increasing the number of electrons stored in the cluster, in accord with extended X-ray absorption fine structure studies. The reliability of the adopted methodology for predicting redox-structural correlations is tested for cuboidal [4Fe-4S] clusters. The calculations reveal a systematic increase in the S...S sulfide distances, in quantitative agreement with the available data. These trends are rationalized by a simple electrostatic model.

A synthetic method affording a topological analogue of the electron-transfer P-cluster of nitrogenase (Fe(8)S(7)(mu(2)-S(Cys))(2)) in the P(N) state has been devised, based in part on our previous development of cubane-type VFe(3)S(4) clusters (Hauser, C.; Bill, E.; Holm, R. H. Inorg. Chem. 2002, 41, 1615-1624). The cluster [(Tp)VFe(3)S(4)Cl(3)](2-) (1) is converted to [(Tp)VFe(3)S(4)(PR(3))(3)](1+) (R = Et (2), Bu (3)) by reaction with R(3)P. The phosphine ligands are readily substituted, leading to [(Tp)VFe(3)S(4)(SR)(3)](2-) (R = Ph (4), H (5)). Reduction of 2 or 3 with cobaltocene produces the edge-bridged double cubanes [(Tp)(2)V(2)Fe(6)S(8)(PR(3))(4)] (R = Et (6), Bu (7)), which are readily converted to [(Tp)(2)V(2)Fe(6)S(8)(SPh)(4)](4-) (8). The structures of clusters 3-5 and 8 were proven crystallographically. Cluster 8 has the double-cubane structure previously shown for 6, in which two cubane units are bridged by two Fe-(mu(4)-S) bonds. (57)Fe isomer shifts are consistent with the formulation [VFe(2.33+)(3)S(4)](2+) for the single cubanes and the all-ferrous description 2[VFe(2+)(3)S(4)](1+) for the double cubanes. Reaction of 6 with 4 equiv of (Et(4)N)(HS) in acetonitrile results in the insertion of sulfide with concomitant structural rearrangement and the formation of [(Tp)(2)V(2)Fe(6)S(9)(SH)(2)](4-) (10), obtained in ca. 50% yield as the Et(4)N(+) salt. The cluster has C(2) symmetry, with two all-ferrous VFe(3)S(4) fragments bridged by a common mu(6)-S atom and two mu(2)-S atoms that simulate the bridging atoms in the two Fe-(mu(2)-S(Cys))-Fe bridges of the P(N) cluster. The bridge pattern V(2)Fe(6)(mu(2)-S)(2)(mu(3)-S)(6)(mu(6)-S) and cluster shape match those of the native cluster. A best-fit superposition of the cores of 10 and the P(N) cluster affords a weighted rms deviation in atom positions of 0.33 A. Cluster 10 and [(Tp)(2)Mo(2)Fe(6)S(9)(SH)(2)](3-), prepared by a related route (Zhang, Y.; Holm, R. H. J. Am. Chem. Soc. 2003, 125, 3910-3920), demonstrate that the topology of the P(N) cluster can be achieved in molecular form in the absence of protein structure (Tp = tris(pyrazolyl)hydroborate).

The unifying view that molybdenum is the essential component in nitrogenase has changed over the past few years with the discovery of a vanadium-containing nitrogenase and an iron-only nitrogenase. The principal question that has arisen for the alternative nitrogenases concerns the structures of their corresponding cofactors and their metal-ion valence assignments and whether there are significant differences with that of the more widely known molybdenum-iron cofactor (FeMoco). Spin-polarized broken-symmetry (BS) density functional theory (DFT) calculations are used to assess which of the two possible metal-ion valence assignments (4Fe(2+)4Fe(3+) or 6Fe(2+)2Fe(3+)) for the iron-only cofactor (FeFeco) best represents the resting state. For the 6Fe(2+)2Fe(3+) oxidation state, the spin coupling pattern for several spin state alignments compatible with S = 0 were generated and assessed by energy criteria. The most likely BS spin state is composed of a 4Fe cluster with spin S(a) = (7)/(2) antiferromagnetically coupled to a 4Fe' cluster with spin S(b) = (7)/(2). This state has the lowest DFT energy for the isolated FeFeco cluster and displays calculated Mössbauer isomer shifts consistent with experiment. Although the S = 0 resting state of FeFeco has recently been proposed to have metal-ion valencies of 4Fe(2+)4Fe(3+) (derived from experimental Mössbauer isomer shifts), our isomer shift calculations for the 4Fe(2+)4Fe(3+) oxidation state are in poorer agreement with experiment. Using the Mo(4+)6Fe(2+)Fe(3+) oxidation level of the cofactor as a starting point, the structural consequences of replacement of molybdenum (Mo(4+)) with vanadium (V(3+)) or iron (Fe(3+)) in the cofactor have been investigated. The size of the cofactor cluster shows a dependency on the nature of the heterometal and increases in the order FeMoco < FeVco < FeFeco.

The cofactor (M-center) of the MoFe protein of nitrogenase, a MoFe(7)S(9):homocitrate cluster, contains six Fe sites with a (distorted) trigonal sulfido coordination. These sites exhibit unusually small quadrupole splittings, Delta E(Q) approximately 0.7 mm/s, and isomer shifts, delta approximately 0.41 mm/s. Mössbauer and ENDOR studies have provided the magnetic hyperfine tensors of all iron sites in the S = 3/2 state M(N). To assess the intrinsic zero-field splittings and hyperfine parameters of the cofactor sites, we have studied with Mössbauer spectroscopy two salts of the three-coordinated Fe(II) thiolate complex [Fe(SR)(3)](-) (R = C(6)H(2)-2,4,6-tBu(3)). One of the salts, [Ph(4)P][Fe(SR)(3)] x 2MeCN x C(7)H(8), 1, has a planar geometry with idealized C(3h) symmetry. This S = 2 complex has an axial zero-field splitting with D = +10.2 cm(-1). The magnetic hyperfine tensor components A(x) = A(y) = -7.5 MHz and A(z) = -29.5 MHz reflect an orbital ground state with d(z(2)) symmetry. A(iso) = (A(x) +A(y) +A(z))/3 = -14.9 MHz, which includes the contact interaction (kappa P = -21.9 MHz) and an orbital contribution (+7 MHz), which is substantially smaller than A(iso) approximately -22 MHz of the tetrahedral Fe(II)(S-R)(4) sites of both rubredoxin and [PPh(4)](2)[Fe(II)(SPh)(4)]. The largest component of the electric field gradient (EFG) tensor is negative, as expected for a d(z(2)) orbital. However, Delta E(Q) = -0.83 mm/s, which is smaller than expected for a high-spin ferrous site. This reduction can be attributed to a ligand contribution, which in planar complexes provides a large positive EFG component perpendicular to the ligand plane. The isomer shift of 1, delta = 0.56 mm/s, approaches the delta-values reported for the six trigonal cofactor sites. The parameters of 1 and their importance for the cofactor cluster of nitrogenase are discussed.

A new series of cubane-type [VFe(3)S(4)](z)() clusters (z = 1+, 2+, 3+) has been prepared as possible precursor species for clusters related to those present in vanadium-containing nitrogenase. Treatment of [(HBpz(3))VFe(3)S(4)Cl(3)](2)(-) (2, z = 2+), protected from further reaction at the vanadium site by the tris(pyrazolyl)hydroborate ligand, with ferrocenium ion affords the oxidized cluster [(HBpz(3))VFe(3)S(4)Cl(3)](1)(-) (3, z = 3+). Reaction of 2 with Et(3)P results in chloride substitution to give [(HBpz(3))VFe(3)S(4)(PEt(3))(3)](1+) (4, z = 2+). Reaction of 4 with cobaltocene reduced the cluster with formation of the edge-bridged double-cubane [(HBpz(3))(2)V(2)Fe(6)S(8)(PEt(3))(4)] (5, z = 1+, 1+), which with excess chloride underwent ligand substitution to afford [(HBpz(3))(2)V(2)Fe(6)S(8)Cl(4)](4)(-) (6, z = 1+, 1+). X-ray structures of (Me(4)N)[3], [4](PF(6)), 5, and (Et(4)N)(4)[6] x 2MeCN are described. Cluster 5 is isostructural with previously reported [(Cl(4)cat)(2)(Et(3)P)(2)Mo(2)Fe(6)S(8)(PEt(3))(4)] and contains two VFe(3)S(4) cubanes connected across edges by a Fe(2)S(2) rhomb in which the bridging Fe-S distances are shorter than intracubane Fe-S distances. Mössbauer (2-5), magnetic (2-5), and EPR (2, 4) data are reported and demonstrate an S = 3/2 ground state for 2 and 4 and a diamagnetic ground state for 3. Analysis of (57)Fe isomer shifts based on an empirical correlation between shift and oxidation state and appropriate reference shifts results in two conclusions. (i) The oxidation 2 --> 3 + e(-) results in a change in electron density localized largely or completely on the Fe(3) subcluster and associated sulfur atoms. (ii) The most appropriate charge distributions are [V(3+)Fe(3+)Fe(2+)(2)S(4)](2+) (Fe(2.33+)) for 1, 2, and 4 and [V(3+)Fe(3+)(2)Fe(2+)S(4)](3+) (Fe(2.67+)) for 3 and [V(2)Fe(6)S(8)(SEt)(9)](3+). Conclusion i applies to every MFe(3)S(4) cubane-type cluster thus far examined in different redox states at parity of cluster ligation. The formalistic charge distributions are regarded as the best current approximations to electron distributions in these delocalized species. The isomer shifts require that iron atoms are mixed-valence in each cluster.

The Mo-site and its ligand environment of the FeMo-cofactor (FeMo-co) were studied using the hybrid density functional method B3LYP. The structure and stability of the model complex (S-ligand)3(N-ligand)Mo[(S)-OCH(CH3)C(O)O-] along with its various protonated and reduced/oxidized forms were calculated. Several hypotheses were tested: (i) ligand environment of the Mo-site, (ii) monodentate vs bidentate coordination of the Mo-bound homocitrate ligand, (iii) substrate coordination to the Mo center, and (iv) Mo-His interaction. It was found that the decoordination of one of the homocitrate (lactate in the model) "legs", the bidentate-->monodentate rearrangement, does not occur spontaneously upon either single/double protonation or one-electron reduction. However, it could occur only upon substrate coordination to the Mo-center of the single-protonated forms of the complex. It was shown that one-electron reduction, single-protonation, and substrate coordination facilitate the bidentate<-->monodentate rearrangement of the homocitrate (lactate) ligand of FeMo-co. It was demonstrated that the smallest acceptable model of His ligand in FeMo-co is methylimidazolate (MeIm-). Our studies suggest that the epsilon-N of the FeMo-co-bound His residue is not protonated, and as a consequence the cluster is tightly bound to the protein matrix via a strong Mo-N delta bond.

Transformations of the edge-bridged double cubane cluster [(Cl4cat)2(Et3P)2Mo2Fe6S8(PEt3)4] (1) under reducing conditions have been investigated as synthetic approaches to the clusters of nitrogenase. Cluster 1 is a versatile precursor to different Mo-Fe-S cluster types. The reaction system 1/K(C14H10) in THF yields the reduced cluster [(Cl4cat)2(Et3P)2Mo2Fe6S8(PEt3)4]1- (2), which as its crystalline Et4N+ salt retains the edge-bridged structure of 1. X-ray structural and Mössbauer spectroscopic results indicate an unsymmetrical electron distribution with localized [MoFe3S4]2+,1+ cubane-type units. The system 1/2K(C14H10)/2HS- in THF/acetonitrile affords [(Cl4cat)4(Et3P)4Mo4Fe12S20K3(DMF)]5- (3), whose structure was determined as the Ph3PMe+ salt. The cluster consists of two isostructural Mo2Fe6S9 fragments connected by two mu 2-S bridges. Three potassium ions are bound between the two fragments. In each fragment, the iron atoms are present in tetrahedral FeS4 and the molybdenum atoms in octahedral MoO2PS3 coordination units, and two MoFe3(mu 3-S)3 cuboidal units are bridged by a common mu 6-S atom. The fragments have idealized mirror symmetry and are isostructural with two of the fragments present in the previously reported high-nuclearity cluster [(Cl4cat)6(Et3P)6Mo6Fe20S30]8- (4) (Osterloh, F.; Sanakis, Y.; Staples, R. J.; Münck, E.; Holm, R. H. Angew. Chem., Int. Ed. Engl. 1999, 38, 2066). On the basis of overall shape, atom connectivities, and metric features, the Mo2Fe6S9 fragment is a topological analogue of the P-cluster of nitrogenase in the PN (reduced) state. A third cluster type, formed as a minor byproduct in the reaction system leading to 2, was crystallographically identified as [(Cl4cat)2(Et3P)2Mo2Fe6S8(PEt3)4]4-, whose core is made up of two MoFe3(mu 3-S)3 cuboidal units bridged by two mu 2-S atoms and connected by a direct Fe-Fe bond. Full structural details and the redox properties of 2 and 3 are reported.