Nitrogen reduction by the Fe sites of synthetic [Mo3S4Fe] cubes.

Nitrogen (N2) fixation by nature, which is a crucial process for the supply of bio-available forms of nitrogen, is performed by nitrogenase. This enzyme uses a unique transition-metal-sulfur-carbon cluster as its active-site co-factor ([(R-homocitrate)MoFe7S9C], FeMoco)1,2, and the sulfur-surrounded iron (Fe) atoms have been postulated to capture and reduce N2 (refs. 3-6). Although there are a few examples of synthetic counterparts of the FeMoco, metal-sulfur cluster, which have shown binding of N2 (refs. 7-9), the reduction of N2 by any synthetic metal-sulfur cluster or by the extracted form of FeMoco10 has remained elusive, despite nearly 50 years of research. Here we show that the Fe atoms in our synthetic [Mo3S4Fe] cubes11,12 can capture a N2 molecule and catalyse N2 silylation to form N(SiMe3)3 under treatment with excess sodium and trimethylsilyl chloride. These results exemplify the catalytic silylation of N2 by a synthetic metal-sulfur cluster and demonstrate the N2-reduction capability of Fe atoms in a sulfur-rich environment, which is reminiscent of the ability of FeMoco to bind and activate N2.

Incorporation of an Asymmetric Mo-Fe-S Cluster as an Artificial Cofactor into Nitrogenase.

Nitrogenase employs a sophisticated electron transfer system and a Mo-Fe-S-C cofactor, designated the M-cluster [(cit)MoFe7 S9 C]), to reduce atmospheric N2 to bioaccessible NH3 . Previously, we have shown that the cofactor-free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe-S cluster [Fe6 S9 (SEt)2 ]4- . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe5 S9 (SH)]3- as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold. The resultant semi-artificial enzyme catalytically reduces C2 H2 to C2 H4 , and CN- into short-chain hydrocarbons, yet it is clearly distinct in activity from its [Fe6 S9 (SEt)2 ]4- -reconstituted counterpart, pointing to the possibility to employ molecular design and cluster synthesis strategies to further develop semi-artificial or artificial systems with desired catalytic activities.

Metal-Sulfur Compounds in N2 Reduction and Nitrogenase-Related Chemistry.

Transition metal-sulfur (M-S) compounds are an indispensable means for biological systems to convert N2 into NH3 (biological N2 fixation), and these may have emerged by chemical evolution from a prebiotic N2 fixation system. With a main focus on synthetic species, this article provides a comprehensive review of the chemistry of M-S compounds related to the conversion of N2 and the structures/functions of the nitrogenase cofactors. Three classes of M-S compounds are highlighted here: multinuclear M-S clusters structurally or functionally relevant to the nitrogenase cofactors, mono- and dinuclear transition metal complexes supported by sulfur-containing ligands in N2 and N2Hx (x = 2, 4) chemistry, and metal sulfide-based solid materials employed in the reduction of N2. Fair assessments on these classes of compounds revealed that our understanding is still limited in N2 reduction and related substrate reductions. Our aims of this review are to compile a collection of studies performed at atomic to mesoscopic scales and to present potential opportunities for elucidating the roles of metal and sulfur atoms in the biological N2 fixation that might be helpful for the development of functional materials.

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4 Se4 ] Cluster.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe4 S4 ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe4 Se4 ] cluster that is used to substitute the [Fe4 S4 ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4 Se4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4 Se4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe4 Se4 ]0 ) in the presence of dithionite, where its [Fe4 S4 ] counterpart in AvNifH exists solely in the reduced state ([Fe4 S4 ]1+ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe4 Se4 ] and [Fe4 S4 ] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen-substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.

Characterization of a Mo-Nitrogenase Variant Containing a Citrate-Substituted Cofactor.

Nitrogenase converts N2 to NH3 , and CO to hydrocarbons, at its cofactor site. Herein, we report a biochemical and spectroscopic characterization of a Mo-nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDKCit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDKCit reveals a shift of CO reduction from H2 evolution toward hydrocarbon formation and an opposite shift of N2 reduction from NH3 formation toward H2 evolution. Consistent with a shift in the Mo K-edge energy of NifDKCit relative to that of its wild-type counterpart, EPR analysis demonstrates a broadening of the line-shape and a decrease in the intensity of the cofactor-originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution.

An EPR and VTVH MCD spectroscopic investigation of the nitrogenase assembly protein NifB.

NifB, a radical SAM enzyme, catalyzes the biosynthesis of the L cluster (Fe8S9C), a structural homolog and precursor to the nitrogenase active-site M cluster ([MoFe7S9C·R-homocitrate]). Sequence analysis shows that NifB contains the CxxCxxxC motif that is typically associated with the radical SAM cluster ([Fe4S4]SAM) involved in the binding of S-adenosylmethionine (SAM). In addition, NifB houses two transient [Fe4S4] clusters (K cluster) that can be fused into an 8Fe L cluster concomitant with the incorporation of an interstitial carbide ion, which is achieved through radical SAM chemistry initiated at the [Fe4S4]SAM cluster upon its interaction with SAM. Here, we report a VTVH MCD/EPR spectroscopic study of the L cluster biosynthesis on NifB, which focuses on the initial interaction of SAM with [Fe4S4]SAM in a variant NifB protein (MaNifBSAM) containing only the [Fe4S4]SAM cluster and no K cluster. Titration of MaNifBSAM with SAM reveals that [Fe4S4]SAM exists in two forms, labeled [Formula: see text] and [Formula: see text]. It is proposed that these forms are involved in the synthesis of the L cluster. Of the two cluster types, only [Formula: see text] initially interacts with SAM, resulting in the generation of Z, an S = ½ paramagnetic [Fe4S4]SAM/SAM complex.

X-Ray Crystallographic Analysis of NifB with a Full Complement of Clusters: Structural Insights into the Radical SAM-Dependent Carbide Insertion During Nitrogenase Cofactor Assembly.

NifB is an essential radical SAM enzyme required for the assembly of an 8Fe core of the nitrogenase cofactor. Herein, we report the X-ray crystal structures of Methanobacterium thermoautotrophicum NifB without (apo MtNifB) and with (holo MtNifB) a full complement of three [Fe4 S4 ] clusters. Both apo and holo MtNifB contain a partial TIM barrel core, but unlike apo MtNifB, holo MtNifB is fully assembled and competent in cofactor biosynthesis. The radical SAM (RS)-cluster is coordinated by three Cys, and the adjacent K1- and K2-clusters, representing the precursor to an 8Fe cofactor core, are each coordinated by one His and two Cys. Prediction of substrate channels, combined with in silico docking of SAM in holo MtNifB, suggests the binding of SAM between the RS- and K2-clusters and putative paths for entry of SAM and exit of products of SAM cleavage, thereby providing important mechanistic insights into the radical SAM-dependent carbide insertion concomitant with cofactor core formation.

Electron Paramagnetic Resonance and Magnetic Circular Dichroism Spectra of the Nitrogenase M Cluster Precursor Suggest Sulfur Migration upon Oxidation: A Proposal for Substrate and Inhibitor Binding.

The active site of the nitrogen-fixing enzyme Mo-nitrogenase is the M cluster ([MoFe7 S9 C⋅R-homocitrate]), also known as the FeMo cofactor or FeMoco. The biosynthesis of this highly complex metallocluster involves a series of proteins. Among them, NifB, a radical-SAM enzyme, is instrumental in the assembly of the L cluster ([Fe8 S9 C]), a precursor and all-iron core of the M cluster. In the absence of sulfite, NifB assembles a precursor form of the L cluster called the L* cluster ([Fe8 S8 C]), which lacks the final ninth sulfur. EPR and MCD spectroscopies are used to probe the electronic structures of the paramagnetic, oxidized forms of both the L and L* clusters, labeled LOx and [L*]Ox . This study shows that both LOx and [L*]Ox have nearly identical EPR and MCD spectra, thus suggesting that the two clusters have identical structures upon oxidation; in other words, a sulfur migrates away from LOx following oxidation, thereby rendering the cluster identical to [L*]Ox . It is proposed that a similar migration could occur to the M cluster upon oxidation, and that this is an instrumental part of both M cluster formation and nitrogenase substrate/inhibitor binding.

Electrochemical Characterization of Isolated Nitrogenase Cofactors from Azotobacter vinelandii.

The nitrogenase cofactors are structurally and functionally unique in biological chemistry. Despite a substantial amount of spectroscopic characterization of protein-bound and isolated nitrogenase cofactors, electrochemical characterization of these cofactors and their related species is far from complete. Herein we present voltammetric studies of three isolated nitrogenase cofactor species: the iron-molybdenum cofactor (M-cluster), iron-vanadium cofactor (V-cluster), and a homologue to the iron-iron cofactor (L-cluster). We observe two reductive events in the redox profiles of all three cofactors. Of the three, the V-cluster is the most reducing. The reduction potentials of the isolated cofactors are significantly more negative than previously measured values within the molybdenum-iron and vanadium-iron proteins. The outcome of this study provides insight into the importance of the heterometal identity, the overall ligation of the cluster, and the impact of the protein scaffolds on the overall electronic structures of the cofactors.

A V-Nitrogenase Variant Containing a Citrate-Substituted Cofactor.

Nitrogenases catalyze the ambient reduction of N2 and CO at its cofactor site. Herein we present a biochemical and spectroscopic characterization of an Azotobacter vinelandii V nitrogenase variant expressing a citrate-substituted cofactor. Designated VnfDGKCit , the catalytic component of this V nitrogenase variant has an αß2 (δ) subunit composition and carries an 8Fe P* cluster and a citrate-substituted V cluster analogue in the αß dimer, as well as a 4Fe cluster in the "orphaned" ß-subunit. Interestingly, when normalized based on the amount of cofactor, VnfDGKCit shows a shift of N2 reduction from H2 evolution toward NH3 formation and an opposite shift of CO reduction from hydrocarbon formation toward H2 evolution. These observations point to a role of the organic ligand in proton delivery during catalysis and imply the use of different reaction sites/mechanisms by nitrogenase for different substrate reductions. Moreover, the increased NH3 /H2 ratio upon citrate substitution suggests the possibility to modify the organic ligand for improved ammonia synthesis in the future.

Spectroscopic Characterization of an Eight-Iron Nitrogenase Cofactor Precursor that Lacks the "9th Sulfur".

Nitrogenases catalyze the reduction of N2 to NH4+ at its cofactor site. Designated the M-cluster, this [MoFe7 S9 C(R-homocitrate)] cofactor is synthesized via the transformation of a [Fe4 S4 ] cluster pair into an [Fe8 S9 C] precursor (designated the L-cluster) prior to insertion of Mo and homocitrate. We report the characterization of an eight-iron cofactor precursor (designated the L*-cluster), which is proposed to have the composition [Fe8 S8 C] and lack the "9th sulfur" in the belt region of the L-cluster. Our X-ray absorption and electron spin echo envelope modulation (ESEEM) analyses strongly suggest that the L*-cluster represents a structural homologue to the l-cluster except for the missing belt sulfur. The absence of a belt sulfur from the L*-cluster may prove beneficial for labeling the catalytically important belt region, which could in turn facilitate investigations into the reaction mechanism of nitrogenases.

A Comparative Analysis of the CO-Reducing Activities of MoFe Proteins Containing Mo- and V-Nitrogenase Cofactors.

The Mo and V nitrogenases are structurally homologous yet catalytically distinct in their abilities to reduce CO to hydrocarbons. Here we report a comparative analysis of the CO-reducing activities of the Mo- and V-nitrogenase cofactors (i.e., the M and V clusters) upon insertion of the respective cofactor into the same, cofactor-deficient MoFe protein scaffold. Our data reveal a combined contribution from the protein environment and cofactor properties to the reactivity of nitrogenase toward CO, thus laying a foundation for further mechanistic investigation of the enzymatic CO reduction, while suggesting the potential of targeting both the protein scaffold and the cofactor species for nitrogenase-based applications in the future.

Reduction of C1 Substrates to Hydrocarbons by the Homometallic Precursor and Synthetic Mimic of the Nitrogenase Cofactor.

Solvent-extracted nitrogenase cofactors can reduce C1 substrates (CN-, CO and CO2) to hydrocarbons in reactions driven by a strong reductant, SmI2 (E0' = -1.55 V vs SCE). Here we show that a synthetic [Et4N]4[Fe6S9(SEt)2] cluster (designated the Fe6RHH-cluster), which mimics the homometallic [Fe8S9C] core of the nitrogenase cofactor (designated the L-cluster), is capable of conversion of C1 substrates into hydrocarbons in the same reactions. Comparison of the yields and product profiles between these homometallic clusters and their heterometallic counterparts points to possible roles of the heterometal, interstitial carbide and belt sulfur-bridged iron atoms in catalysis. More importantly, the observation that a "simplified", homometallic cofactor mimic can perform Fischer-Tropsch-like hydrocarbon synthesis suggests future biotechnological adaptability of nitrogenase-based biomimetic compounds for recycling C1 substrates into useful chemical and fuel products.

Synthetic Analogues of Nitrogenase Metallocofactors: Challenges and Developments.

Nitrogenase is the only known biological system capable of reducing N2 to NH3 , which is a critical component of bioavailable nitrogen fixation. Since the discovery of discrete iron-sulfur metalloclusters within the nitrogenase MoFe protein, synthetic inorganic chemists have sought to reproduce the structural features of these clusters in order to understand how they facilitate the binding, activation and hydrogenation of N2 . Through the decades following the initial identification of these clusters, significant progress has been made to synthetically replicate certain compositional and functional aspects of the biogenic clusters. Although much work remains to generate synthetic iron-sulfur clusters that can reduce N2 to NH3 , the insights borne from past and recent developments are discussed in this concept article.

Interconversion between [Fe4S4] and [Fe2S2] Clusters Bearing Amide Ligands.

Structural conversion of [Fe4S4] clusters into [Fe2S2] clusters has been suggested to be a fundamental process for some O2-sensing proteins. While the formation of [Fe2S2] clusters from synthetic [Fe4S4] clusters has been unprecedented, an all-ferric [Fe4S4](4+) cluster Fe4S4{N(SiMe3)2}4 (1) was found to split in the presence of pyridines, giving [Fe2S2] clusters Fe2S2{N(SiMe3)2}2(L)2 (2, L = pyridines). The structural conversion between [Fe4S4] and [Fe2S2] clusters appeared to be reversible, and the thermodynamic parameters for the equilibrium reactions between 1 + L and 2 were determined. Assembly of two [Fe2S2] clusters was also induced by chemical reductions of Fe2S2{N(SiMe3)2}2(Py)2 (Py = pyridine), and the resultant [Fe4S4] clusters [1](-) and [1](2-) were found to split into two [Fe2S2] clusters by oxidation with [Cp2Fe](+) in the presence of pyridine.

Structure and Reactivity of an Asymmetric Synthetic Mimic of Nitrogenase Cofactor.

The Mo nitrogenase catalyzes the ambient reduction of N2 to NH3 at its M-cluster site. A complex metallocofactor with a core composition of [MoFe7 S9 C], the M-cluster, can be extracted from the protein scaffold and used to facilitate the catalytic reduction of CN- , CO, and CO2 into hydrocarbons in the isolated state. Herein, we report the synthesis, structure, and reactivity of an asymmetric M-cluster analogue with a core composition of [MoFe5 S9 ]. This analogue, referred to as the Mo-cluster, is the first synthetic example of an M-cluster mimic with Fe and Mo positioned at opposite ends of the cluster. Moreover, the ability of the Mo-cluster to reduce C1 substrates to hydrocarbons suggests the feasibility of developing nitrogenase-based biomimetic approaches to recycle C1  waste into fuel products.

Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme.

Nitrogenase catalyzes substrate reduction at its cofactor center ([(Cit)MoFe7S9C](n-); designated M-cluster). Here, we report the formation of an artificial, nitrogenase-mimicking enzyme upon insertion of a synthetic model complex ([Fe6S9(SEt)2](4-); designated Fe6(RHH)) into the catalytic component of nitrogenase (designated NifDK(apo)). Two Fe6(RHH) clusters were inserted into NifDK(apo), rendering the conformation of the resultant protein (designated NifDK(Fe)) similar to the one upon insertion of native M-clusters. NifDK(Fe) can work together with the reductase component of nitrogenase to reduce C2H2 in an ATP-dependent reaction. It can also act as an enzyme on its own in the presence of Eu(II)DTPA, displaying a strong activity in C2H2 reduction while demonstrating an ability to reduce CN(-) to C1-C3 hydrocarbons in an ATP-independent manner. The successful outcome of this work provides the proof of concept and underlying principles for continued search of novel enzymatic activities based on this approach.

A convenient route to synthetic analogues of the oxidized form of high-potential iron-sulfur proteins.

An amide-bound [Fe4S4](3+) cluster, [Fe4S4{N(SiMe3)2}4](-) (1), was found to serve as a convenient precursor for synthetic analogues of the oxidized form of high-potential iron-sulfur proteins. Treatment of 1 with 4 equiv of bulky thiols led to replacement of the amide ligands with thiolates, giving rise to a series of [Fe4S4(SR)4](-) clusters (R = Dmp (2a), Tbt (2b), Eind (2c), Dxp (2d), Dpp (2e); Dmp = 2,6-di(mesityl)phenyl, Tbt = 2,4,6-tris[bis(trimethylsilyl)methyl]phenyl, Eind = 1,1,3,3,5,5,7,7-octaethyl-s-hydrindacen-4-yl, Dxp = 2,6-di(m-xylyl)phenyl, Dpp = 2,6-diphenylphenyl). These clusters were characterized by the mass spectrum, the EPR spectrum, and X-ray crystallography. The redox potentials of the [Fe4S4](3+/2+) couple, -0.82 V (2a), -0.86 V (2b), -0.84 V (2c), -0.74 V (2d), and -0.63 V (2e) vs Ag/Ag(+) in THF, are significantly more negative than that of [Fe4S4(SPh)4](-/2-) (-0.21 V).

Synthetic analogues of [Fe4S4(Cys)3(His)] in hydrogenases and [Fe4S4(Cys)4] in HiPIP derived from all-ferric [Fe4S4{N(SiMe3)2}4].

The all-ferric [Fe(4)S(4)](4+) cluster [Fe(4)S(4){N(SiMe(3))(2)}(4)] 1 and its one-electron reduced form [1](-) serve as convenient precursors for the synthesis of 31-site differentiated [Fe(4)S(4)] clusters and high-potential iron-sulfur protein (HiPIP) model clusters. The reaction of 1 with four equivalents (equiv) of the bulky thiol HSDmp (Dmp = 2,6-(mesityl)(2)C(6)H(3), mesityl = 2,4,6-Me(3)C(6)H(2)) followed by treatment with tetrahydrofuran (THF) resulted in the isolation of [Fe(4)S(4)(SDmp)(3)(THF)(3)] 2. Cluster 2 contains an octahedral iron atom with three THF ligands, and its Fe(S)(3)(O)(3) coordination environment is relevant to that in the active site of substrate-bound aconitase. An analogous reaction of [1](-) with four equiv of HSDmp gave [Fe(4)S(4)(SDmp)(4)](-) 3, which models the oxidized form of HiPIP. The THF ligands in 2 can be replaced by tetramethyl-imidazole (Me(4)Im) to give [Fe(4)S(4)(SDmp)(3)(Me(4)Im)] 4 modeling the [Fe(4)S(4)(Cys)(3)(His)] cluster in hydrogenases, and its one-electron reduced form [4](-) was synthesized from the reaction of 3 with Me(4)Im. The reversible redox couple between 3 and [3](-) was observed at E(1/2) = -820 mV vs. Ag/Ag(+), and the corresponding reversible couple for 4 and [4](-) is positively shifted by +440 mV. The cyclic voltammogram of 3 also exhibited a reversible oxidation couple, which indicates generation of the all-ferric [Fe(4)S(4)](4+) cluster, [Fe(4)S(4)(SDmp)(4)].

Non-centrosymmetric coordination polymer with a highly hindered octahedral copper center bridged by mandelate.

A novel chiral coordination polymer, [Cu(C(6)H(5)CH(OH)COO)(µ-C(6)H(5)CH(OH)COO)] (1-L and 1-D), was synthesized through a reaction of copper acetate with L-mandelic acid at room temperature. Although previously reported copper mandelate prepared by hydrothermal reaction was a centrosymmetric coordination polymer because of the racemization of mandelic acid, the current coordination polymer shows noncentrosymmetry and a completely different structure from that previously reported. The X-ray crystallography for 1-L revealed that the copper center of the compound showed a highly distorted octahedral structure bridged by a chiral mandelate ligand in the unusual coordination mode to construct a one-dimensional (1D) zigzag chain structure. These 1D chains interdigitated each other to give a layered structure as a result of the formation of multiple aromatic interactions and hydrogen bonds between hydroxyl and carboxylate moieties at mandelate ligands. The coordination polymer 1-L belongs to the noncentrosymmetric space group of C2 to show piezoelectric properties and second harmonic generation (SHG) activity.