Evaluation of the Catalytic Relevance of the CO‐Bound States of V‐Nitrogenase

Binding and activation of CO by nitrogenase is a topic of interest because CO is isoelectronic to N₂, the physiological substrate of this enzyme. The catalytic relevance of one‐ and multi‐CO‐bound states (the lo‐CO and hi‐CO states) of V‐nitrogenase to C−C coupling and N₂ reduction was examined. Enzymatic and spectroscopic studies demonstrate that the multiple CO moieties in the hi‐CO state cannot be coupled as they are, suggesting that C−C coupling requires further activation and/or reduction of the bound CO entity. Moreover, these studies reveal an interesting correlation between decreased activity of N₂ reduction and increased population of the lo‐CO state, pointing to the catalytic relevance of the belt Fe atoms that are bridged by the single CO moiety in the lo‐CO state. Together, these results provide a useful framework for gaining insights into the nitrogenase‐catalyzed reaction via further exploration of the utility of the lo‐CO conformation of V‐nitrogenase.     

Enzymatic and catalytic reduction of dinitrogen to ammonia: Density functional theory characterization of alternative molybdenum active sites

We used density functional calculations to model dinitrogen reduction by a FeMo cofactor containing a central nitrogen atom and by a Mo‐based catalyst. Plausible intermediates, reaction pathways, and relative energetics in the enzymatic and catalytic reduction of N₂ to ammonia at a single Mo center are explored. Calculations indicate that the binding of N₂ to the Mo atom and the subsequent multiple proton–electron transfer to dinitrogen and its protonated species involved in the conversion of N₂ are feasible energetically. In the reduction of N₂ the Mo atom experiences a cycled oxidation state from Mo(IV) to Mo(VI) by nitrogenase and from Mo(III) to Mo(VI) by the molybdenum catalyst, respectively, tuning the gradual reduction of N₂. Such a wide range of oxidation states exhibited by the Mo center is crucial for the gradual reduction process via successive proton–electron transfer. Present results suggest that the Mo atom in the N‐centered FeMo cofactor is a likely alternative active site for dinitrogen binding and reduction under mild conditions once there is an empty site available at the Mo site. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Mutual Effects of Substrates and Inhibitors in Reactions Catalyzed by Isolated Iron–Molybdenum Cofactor of Nitrogenase

The inhibiting effects of CO and N₂ on the ability of the nitrogenase iron–molybdenum cofactor (FeMoco) to catalyze acetylene reduction outside the protein were studied to obtain data on the mechanism of substrate reduction at the active center of the enzyme nitrogenase. It was found that CO and N₂ reacted with FeMoco that was separated from the enzyme and reduced by zinc amalgam (E = –0.84 V relative to a normal hydrogen electrode (NHE)) (I) or europium amalgam (E = –1.4 V relative to NHE) (II). In system I, CO reversibly inhibited the reaction of acetylene reduction to ethylene with K _i = 0.05 atm CO. In system II, CO inhibited the formation of the two products of C₂H₂ reduction in different manners: the mixed-type or competitive inhibition was found for ethylene formation with K _i = 0.003 atm CO and the incomplete competitive inhibition was found for ethane formation with K _i = 0.006 atm CO. The fraction of C₂H₆ in the reaction products was greater than 50% at a CO pressure of 0.05 atm because of the stronger inhibiting effect of CO on the formation of C₂H₄. The change in the product specificity of acetylene-reduction centers under influence of CO was explained by some stabilization of the intermediate complex [FeMoco · C₂H₂] upon the simultaneous coordination of CO to the catalytic cluster. Because of this, the fraction value of ethane as a multielectron reduction product increased. The experimental results suggest that several active sites at the FeMoco cluster reduced outside the protein can be simultaneously occupied by substrates and (or) inhibitors. The inhibition of both ethane and ethylene formation by molecular nitrogen in system II is competitive with K _i = 0.5 atm N₂ for either product. That is, N₂ and C₂H₂ as ligands compete for the same coordination site at the reduced FeMoco cluster. The inhibiting effects of CO and N₂ on the catalytic behaviors of both isolated FeMoco and that in the enzyme were compared.     

Mutual Effects of Substrates and Inhibitors in Reactions Catalyzed by the Nitrogenase Iron–Molybdenum Cofactor outside the Enzyme

The inhibiting effects of CO and N₂ on the ability of the nitrogenase iron–molybdenum cofactor (FeMoco) to catalyze acetylene reduction outside the protein were studied to obtain data on the mechanism of substrate reduction at the active center of the enzyme nitrogenase. It was found that CO and N₂ reacted with FeMoco that was separated from the enzyme and reduced by zinc amalgam (E = –0.84 V with reference to a normal hydrogen electrode (NHE)) (I) or europium amalgam (E = –1.4 V with reference to NHE) (II). In system I, CO reversibly inhibited the reaction of acetylene reduction to ethylene with K _i = 0.05 atm CO. In system II, CO inhibited the formation of the two products of C₂H₂ reduction in different manners: the mixed-type or competitive inhibition of ethylene formation with K _i = 0.003 atm CO and the incomplete competitive inhibition of ethane formation with K _i = 0.006 atm CO. The fraction of C₂H₆ in the reaction products was higher than 50% at a CO pressure of 0.05 atm because of the stronger inhibiting effect of CO on the formation of C₂H₄. A change in the product specificity of acetylene-reduction centers under exposure to CO was explained by some stabilization of the intermediate complex [FeMoco · C₂H₂] upon the simultaneous coordination of CO to the catalytic cluster. Because of this, the fraction of the many-electron reduction product (ethane) increased. The experimental results suggest that several active sites in the FeMoco cluster reduced outside the protein can be simultaneously occupied by substrates and (or) inhibitors. The inhibition of both ethane and ethylene formation by molecular nitrogen in system II is competitive with K _i = 0.5 atm N₂ for either product. That is, N₂ and C₂H₂ as ligands compete for the same coordination site in the reduced FeMoco cluster. The inhibiting effects of CO and N₂ on the catalytic behaviors of FeMoco outside the protein and as an enzyme constituent were compared.     

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^IIDTPA, 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.     

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli. Biochemical and spectroscopic analyses demonstrate the integrity of the heterologously expressed NifEN in composition and functionality and, additionally, the ability of an engineered NifEN variant to mimic NifDK in retaining the matured cofactor at an analogous cofactor‐binding site. This is an important step toward piecing together a viable pathway for the heterologous expression of nitrogenase and identifying variants for the mechanistic investigation of this enzyme.     

Elucidating the Coordination Chemistry and Mechanism of Biological Nitrogen Fixation

How does the enzyme nitrogenase reduce the inert molecule N₂ to NH₃ under ambient conditions that are so different from the energy‐expensive conditions of the best industrial practices? This review focuses on recent theoretical investigations of the catalytic site, the iron–molybdenum cofactor FeMo‐co, and the way in which it is hydrogenated by protons and electrons and then binds N₂. Density functional calculations provide reaction profiles and activation energies for possible mechanistic steps. This establishes a conceptual framework and the principles for the coordination chemistry of FeMo‐co that are essential to the chemical mechanism of catalysis. The model advanced herein explains relevant experimental data.     

Redox transformations of polynuclear molybdenum alkoxides and their interaction with nitrogenase substrates: experimental and theoretical study

The experimental and theoretical study of the electronic structure and IR spectra of the CO-containing molybdenum(0) alkoxide complexes of different nuclearity was carried out. The binding energy of the dinitrogen ligand was calculated for the tetranuclear K₄[Mo(OR)(CO)₃]₄ complexes catalyzing dinitrogen reduction. The theoretical study of structural changes for the 20-electron reduction of the catalytic cluster of the octanuclear [Mg₂Mo₈O₂₂(MeO)₆(MeOH)₄]^2? complex was performed. The interaction of the reduced cluster with the nitrogenase substrate was considered. Probable coordination modes of N₂, C₂H₂, and CO were analyzed, as well as the protonation reactions of the acetylene complexes, giving rise to two- and four-electron reduction products. The results of quantum chemical calculations are in good agreement with the experimental regularities observed for the catalytic reduction of the substrates in the presence of the Mo-Mg cluster.     

Differential Reduction of CO2 by Molybdenum and Vanadium Nitrogenases

The molybdenum and vanadium nitrogenases are two homologous enzymes with distinct structural and catalytic features. Previously, it was demonstrated that the V nitrogenase was nearly 700 times more active than its Mo counterpart in reducing CO to hydrocarbons. Herein, a similar discrepancy between the two nitrogenases in the reduction of CO₂ is reported, with the V nitrogenase being capable of reducing CO₂ to CO, CD₄, C₂D₄, and C₂D₆, and its Mo counterpart only capable of reducing CO₂ to CO. Furthermore, it is shown that the V nitrogenase may direct the formation of CD₄ in part via CO₂‐derived CO, but that it does not catalyze the formation of C₂D₄ and C₂D₆ along this route. The exciting observation of a V nitrogenase‐catalyzed C? C coupling with CO₂ as the origin of the building blocks adds another interesting reaction to the catalytic repertoire of this unique enzyme system. The differential activities of the V and Mo nitrogenases in CO₂ reduction provide an important framework for systematic investigations of this reaction in the future.     

Selective and Efficient Cycloisomerization of Alkynols Catalyzed by a New Ruthenium Complex with a Tetradentate Nitrogen–Phosphorus Mixed Ligand

The new ruthenium complex [Ru(N₃P)(OAc)][BPh₄] ( 4 ), in which N₃P is the N,P mixed tetradentate ligand N,N‐bis[(pyridin‐2‐yl)methyl]‐[2‐(diphenylphosphino)phenyl]methanamine was synthesized. The complex was found to be catalytically active for the endo cycloisomerization of alkynols. The catalytic reactions can be used to synthesize five‐, six‐, and seven‐membered endo‐cyclic enol ethers in good to excellent yields. A catalytic cycle involving a vinylidene intermediate was proposed for the catalytic reactions. Treatment of complex 4 with PhC?CH and H₂O gave the alkyl complex [Ru(CH₂Ph)(CO)(N₃P)][BPh₄] ( 30 ), which supports the assumption that the catalytic reactions involve addition of a hydroxyl group to the C?C bond of vinylidene ligands.     

An organometallic approach to the synthesis of high nuclearity Mo-Fe-S clusters as potential models for the iron-molybdenum cofactor of nitrogenase

Reaction of the [Fe₂S₂(CO)₆]^2– dianion with molybdenum reagents produces a number of high-nuclearity Mo-Fe-S carbonyl clusters with Fe/Mo ratios 5, as well as a variety of new Fe-S carbonyl clusters. The former are particularly relevant as models or precursors to models for the iron-molybdenum cofactor [FeMo-cofactor] of nitrogenase. General strategies for the synthesis of FeMo-cofactor models are briefly reviewed, and the structures of clusters produced in the [Fe₂S₂(CO)₆]^2–/Mo systems examined to date are described.     

NHC‐Containing Manganese(I) Electrocatalysts for the Two‐Electron Reduction of CO2

The synthesis and characterization of the first catalytic manganese N‐heterocyclic carbene complexes are reported: MnBr(N‐methyl‐N′‐2‐pyridylbenzimidazol‐2‐ylidine)(CO)₃ and MnBr(N‐methyl‐N′‐2‐pyridylimidazol‐2‐ylidine)(CO)₃. Both new species mediate the reduction of CO₂ to CO following two‐electron reduction of the Mn^I center, as observed with preparative scale electrolysis and verified with ¹³CO₂. The two‐electron reduction of these species occurs at a single potential, rather than in two sequential steps separated by hundreds of millivolts, as is the case for previously reported MnBr(2,2′‐bipyridine)(CO)₃. Catalytic current enhancement is observed at voltages similar to MnBr(2,2′‐bipyridine)(CO)₃.     

N2 activation by iron-sulfur complexes

Inspired by the determination of the structure of the nitrogenase enzyme cofactor by Rees et al., the binding of an N₂ molecule to some model iron-sulfur compounds was investigated usingab initio calculations. Side-on and end-on coordination to one two and four iron centers were investigated. In most cases, the N₂ is loosely bound and retains its internal triple bond, but a few examples are found where the N₂ is “activated” and has a longer N-N bond length.     

Human mitochondrial amidoxime reducing component (mARC): An electrochemical method for identifying new substrates and inhibitors

As recently as 2006 the mitochondrial amidoxime reducing component (mARC) was identified as the fourth and last Mo enzyme present in humans. Its physiological role remains unknown. mARC is capable of reducing a variety of N-hydroxylated compounds such as amidoximes to their corresponding amidine and there is considerable interest in this enzyme from a pharmaceutical perspective. mARC is a target for N-hydroxylated pro-drugs that may be reductively activated intracellularly to release potent drugs such as cationic amidinium ions, which exhibit a broad spectrum of activity as antithrombotics and against various bacteria and parasites. In the quest for a rapid screen of new mARC substrates and inhibitors we present an electrochemical method which utilizes the natural electron partner of mARC, cytochrome b₅, coupled to an electrochemical electrode. Mediated electron transfer from the electrode via cytochrome b₅ to mARC results in a catalytic current in the presence of substrate.     

On the coupled oxidation-reduction mechanism of molecular nitrogen fixation

A new mechanism for the catalytic reduction of N₂ was proposed. According to the mechanism, reduction is preceded by the oxidation step with the formation of N₂O. The mechanism allows the participation of weaker reducing agents than those in purely reductive processes. Probable individual steps are considered, in particular, the oxygen atom transfer from the superoxide radical anion O₂ ^–· in a cyclic complex containing the N₂ molecule in the coordination sphere of a metal. The proposed mechanism can explain N₂ reduction involving recently discovered nitrogenase in which O₂ ^–· acts as an electron donor and N₂ reduction in purely chemical systems including the air nitrogen and relatively weak reducing agents.     

Dinitrogen Fixation: Rationalizing Strategies Utilizing Molecular Complexes

Dinitrogen (N₂) is the most abundant gas in Earth's atmosphere, but its inertness hinders its use as a nitrogen source in the biosphere and in industry. Efficient catalysts are hence required to ov. ercome the high kinetic barriers associated to N₂ transformation. In that respect, molecular complexes have demonstrated strong potential to mediate N₂ functionalization reactions under mild conditions while providing a straightforward understanding of the reaction mechanisms. This Review emphasizes the strategies for N₂ reduction and functionalization using molecular transition metal and actinide complexes according to their proposed reaction mechanisms, distinguishing complexes inducing cleavage of the N≡N bond before (dissociative mechanism) or concomitantly with functionalization (associative mechanism). We present here the main examples of stoichiometric and catalytic N₂ functionalization reactions following these strategies.     

Bioelectrochemical Haber–Bosch Process: An Ammonia‐Producing H2/N2 Fuel Cell

Nitrogenases are the only enzymes known to reduce molecular nitrogen (N₂) to ammonia (NH₃). By using methyl viologen (N ,N ′‐dimethyl‐4,4′‐bipyridinium) to shuttle electrons to nitrogenase, N₂ reduction to NH₃ can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode that utilizes the enzyme hydrogenase to oxidize molecular hydrogen (H₂) results in an enzymatic fuel cell (EFC) that is able to produce NH₃ from H₂ and N₂ while simultaneously producing an electrical current. To demonstrate this, a charge of 60 mC was passed across H₂ /N₂ EFCs, which resulted in the formation of 286 nmol NH₃ mg⁻¹ MoFe protein, corresponding to a Faradaic efficiency of 26.4 %.     

Magnetically separable g‐C3N4 hybrid nanocomposite: Highly efficient and eco‐friendly recyclable catalyst for one‐pot synthesis of α‐aminonitriles

A magnetically separable graphitic carbon nitride nanocomposite (Fe₃O₄/g‐C₃N₄) as a catalyst for the three‐component condensation reactions of carbonyl compounds, amines and trimethylsilylcyanide was thoroughly investigated. The reaction of these three components was found to be efficient, economical and green and took place in the presence of a catalytic amount of the magnetically separable catalyst to yield the corresponding α‐aminonitriles in good to excellent yields. The prepared nanocomposite was characterized using scanning electron microscopy and energy‐dispersive X‐ray and Fourier transform infrared spectroscopies. The nanocomposite was also found to be reusable could be recovered easily and reused several times without distinct deterioration in its catalytic activity.     

Activation of Small Molecules by Cluster Complexes

The results of theoretical, experimental investigations on activation of small molecules on their coordination to cluster complexes of heavy transition metals with weak- and strong-field ligands are presented. Homogeneous catalytic redox reactions of the CO, N₂, H₂O molecules and the N₃ ^- molecular anion in the presence of cluster complexes of low-valent molybdenum and rhenium are studies. The reaction mechanism is established. Three modifications of the homogeneous cluster catalysis of redox reactions of small molecules are described.     

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 [MoFe₇S₉C], 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 [MoFe₅S₉]. 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.