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 NifDK^Cit, the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDK^Cit 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 NifDK^Cit 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.     

Widening the Product Profile of Carbon Dioxide Reduction by Vanadium Nitrogenase

Two reaction systems based on vanadium nitrogenase were previously shown to reduce CO₂ to hydrocarbons: 1) an enzyme‐based system that used both components of V nitrogenase for ATP‐dependent reduction of CO₂ to ≤C₂ hydrocarbons; and 2) a cofactor‐based system that employed SmI₂ to supply electrons to the isolated V cluster for an ATP‐independent reduction of CO₂ to ≤C₃ hydrocarbons. Here, we report ATP‐independent reduction of CO₂ to hydrocarbons by a reaction system comprising Eu^II DTPA and the VFe protein of V nitrogenase. Combining features of both enzyme‐ and cofactor‐based systems, this system exhibits improved C?C coupling and a broader product profile of ≤C₄ hydrocarbons. The C?C coupling does not employ CO₂‐derived CO, and it is significantly enhanced in D₂O. These observations afford initial insights into the characteristics of this unique reaction and provide a potential template for future design of catalysts to recycle the greenhouse gas CO₂ into useful products.     

The Conversion of Carbon Monoxide and Carbon Dioxide by Nitrogenases

Nitrogenases are the only known family of enzymes that catalyze the reduction of molecular nitrogen (N₂) to ammonia (NH₃). The N₂ reduction drives biological nitrogen fixation and the global nitrogen cycle. Besides the conversion of N₂, nitrogenases catalyze a whole range of other reductions, including the reduction of the small gaseous substrates carbon monoxide (CO) and carbon dioxide (CO₂) to hydrocarbons. However, it remains an open question whether these ‘side reactivities’ play a role under environmental conditions. Nonetheless, these reactivities and particularly the formation of hydrocarbons have spurred the interest in nitrogenases for biotechnological applications. There are three different isozymes of nitrogenase: the molybdenum and the alternative vanadium and iron-only nitrogenase. The isozymes differ in their metal content, structure, and substrate-dependent activity, despite their homology. This minireview focuses on the conversion of CO and CO₂ to methane and higher hydrocarbons and aims to specify the differences in activity between the three nitrogenase isozymes.     

Density functional theory studies of mechanistic aspects of the SCR reaction on vanadium oxide catalysts

Density functional theory (DFT) calculations were carried out on a vanadium oxide cluster containing four vanadium atoms to probe the mechanism of the selective catalytic reduction (SCR) of NO with ammonia. The interaction of ammonia with Brønsted acid sites on this V₄-cluster leads to the formation of NH₄ species bonded to two vanadyl (VO) groups, with a bonding energy of −110 kJ/mol. This adsorbed NH₄ species reacts with NO in a series of steps to form an adsorbed NH₂NO species, which subsequently undergoes decomposition to form N₂, H₂O, and a reduced vanadium oxide cluster (V₄H). The latter reaction occurs via a series of hydrogen-transfer steps by a “push–pull” mechanism with adjacent VO and VOH groups on the vanadium oxide cluster. The rate limiting process in this conversion of NO and NH₃ to give N₂, H₂O, and V₄H involves the reaction of an adsorbed NH₃NHO adduct to form NH₂NO species. The transition state of this step may be stabilized through hydrogen bonding with surrounding vanadia and/or titania moieties.     

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.     

A molybdenum-centred model for nitrogenase catalysis

Density functional theory has been used to relate the intrinsic N₂ binding affinities of the Fe and Mo sites of the iron–molybdenum cofactor of nitrogenase (FeMoco) to those of known N₂ complexes. The results indicate that initial N₂ binding to FeMoco is reversible, and that Mo is the preferred site. A mechanism for N₂ reduction is proposed in which a partially reduced MoNNH₂ species undergoes cleavage across a MoFeS₂ face of FeMoco.     

Effect of the reductant nature on the catalytic removal of N2O on Fe-zeolite-β catalysts

The catalytic reduction of N₂O by H₂, NH₃, CO, propene and n-decane, in the presence of O₂, has been studied on two Fe-zeolite-β (Fe-BEA) catalysts. In the following experimental conditions (GHSV=35 000 h⁻¹, 2000 ppm N₂O, 3% O₂), CO starts to reduce N₂O from 473 K and is the most efficient reductant in the low temperature domain. The absence of strong adsorption and the ability to reduce Fe^III oxo-cations can be put forward to explain this behaviour. At medium temperature range n-decane become very reactive to reduce N₂O. Finally, in the absence of NH₃ slip, this reductant can be considered as an excellent candidate owing to the harmless of its reduction products.     

Effect of reductants in N2O reduction over Fe-MFI catalysts

We investigated the effect of reductants over ion-exchanged Fe-MFI catalysts (Fe-MFI) based on the catalytic performance in N₂O reduction in the presence and absence of an oxygen atmosphere. In the case of N₂O reduction with hydrocarbons (CH₄, C₂H₆, and C₃H₆) in the presence of excess oxygen, the order of N₂O contribution was as follows: CH₄ > C₂H₆ > C₃H₆. This indicates that CH₄ is a more efficient reductant than C₂H₆ and C₃H₆. The TOFs of N₂O decomposition and the N₂O reduction by various reductants (H₂, CO, CH₄) in the absence of oxygen increased with increasing Fe/Al ratio (Fe/Al⩾0.15), wheras the TOFs were lower and constant in the range of Fe/Al⩽0.10. Temperature-programmed reduction with hydrogen (H₂-TPR) showed that the catalysts with a higher Fe/Al ratio were reduced more easily than those with a lower Fe/Al ratio. Temperature-programmed desorption of O₂ (O₂-TPD) showed that oxygen was desorbed at lower temperatures over the catalysts with a higher Fe/Al ratio. As the result of extended X-ray absorption fine structure (EXAFS) analysis, only mononuclear Fe species were observed over Fe(0.10)-MFI after treatment with N₂O or O₂. On the other hand, binuclear Fe species and mononuclear Fe species were observed over Fe(0.40)-MFI after treatment with N₂O or H₂. More reducible Fe species, which gave lower-temperature O₂ desorption, can be due to Fe binuclear species. Since the N₂O reduction with reductants proceeds via a redox mechanism, the reducible binuclear Fe species can exhibit higher activity. Furthermore, CH₄ can be oxidized by N₂O more easily than can H₂ and CO, although it is generally known that the reactivity of methane is very low.     

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

Isoprenoid biosynthesis is an important area for anti‐infective drug development. One isoprenoid target is (E)‐1‐hydroxy‐2‐methyl‐but‐2‐enyl 4‐diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H⁺/2e^? reduction. IspH contains a 4 Fe?4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe?4 S clusters by η¹ coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H₂O, O₂) binding. Based on these ideas, we used in silico methods to find drug‐like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a K_i value of ≈500 nm against Pseudomonas aeruginosa IspH.     

Cyclic Lean Reduction of NO by CO in Excess H2O on Pt–Rh/Ba/Al2O3: Elucidating Mechanistic Features and Catalyst Performance

This study provides insight into the mechanistic and performance features of the cyclic reduction of NO_x by CO in the presence and absence of excess water on a Pt–Rh/Ba/Al₂O₃ NO_x storage and reduction catalyst. At low temperatures (150–200 °C), CO is ineffective in reducing NO_x due to self-inhibition while at temperatures exceeding 200 °C, CO effectively reduces NO_x to main product N₂ (selectivity >70 %) and byproduct N₂O. The addition of H₂O at these temperatures has a significant promoting effect on NO_x conversion while leading to a slight drop in the CO conversion, indicating a more efficient and selective lean reduction process. The appearance of NH₃ as a product is attributed either to isocyanate (NCO) hydrolysis and/or reduction of NO_x by H₂ formed by the water gas shift chemistry. After the switch from the rich to lean phase, second maxima are observed in the N₂O and CO₂ concentrations versus time, in addition to the maxima observed during the rich phase. These and other product evolution trends provide evidence for the involvement of NCOs as important intermediates, formed during the CO reduction of NO on the precious metal components, followed by their spillover to the storage component. The reversible storage of the NCOs on the Al₂O₃ and BaO and their reactivity appears to be an important pathway during cyclic operation on Pt–Rh/Ba/Al₂O₃ catalyst. In the absence of water the NCOs are not completely reacted away during the rich phase, which leads to their reaction with NO and O₂ upon switching to the subsequent lean phase, as evidenced by the evolution of N₂, N₂O and CO₂. In contrast, negligible product evolution is observed during the lean phase in the presence of water. This is consistent with a rapid hydrolysis of NCOs to NH₃, which results in a deeper regeneration of the catalyst due in part to the reaction of the NH₃ with stored NO_x. The data reveal more efficient utilization of CO for reducing NO_x in the presence of water which further underscores the NCO mechanism. Phenomenological pathways based on the data are proposed that describes the cyclic reduction of NO_x by CO under dry and wet conditions.     

Reduction of NO2 stored in a commercial lean NO x trap at low temperatures

Isothermal storage of NO₂ and subsequent reduction with different reducing agents (H₂, CO or H₂ + CO) in a lean NO _x trap catalyst was tested by Temperature Programmed Desorption (TPD) and Temperature Programmed Reduction (TPR) experiments at temperatures representative of automotive “cold-start” conditions (<200 °C) using a commercial NO _x trap catalyst. Results from the TPR experiments revealed that no reduction of stored NO₂ to N₂ was observed at 100–180 °C, and at 200 °C 10% reduction only of NO₂ to N₂ was measured. A special affinity of H₂ to form NH₃ was observed during the reduction of stored NO₂. The formation of NH₃ increases with increasing amount of stored NO₂ and decreases with increasing storage temperature. Direct relation exists between the amount of adsorbed and/or stored NO₂ and the formation of H₂O and NH₃.     

Exploring Electron/Proton Transfer and Conformational Changes in the Nitrogenase MoFe Protein and FeMo-cofactor Through Cryoreduction/EPR Measurements

We combine cryoreduction/annealing/EPR measurements of nitrogenase MoFe protein with results of earlier investigations to provide a detailed view of the electron/proton transfer events and conformational changes that occur during early stages of [e⁻/H⁺] accumulation by the MoFe protein. This includes reduction of: 1) the non-catalytic state of the iron-molybdenum cofactor (FeMo-co) active site that is generated by chemical oxidation of the resting-state cofactor (S=3/2) within resting MoFe (E₀); and 2) the catalytic state that has accumulated n=1 [e⁻/H⁺] above the resting-state level, denoted E₁(1H) (S≥1) in the Lowe-Thorneley kinetic scheme. FeMo-co does not undergo a major change of conformation during reduction of oxidized FeMo-co. In contrast, FeMo-co undergoes substantial conformational changes during the reduction of E₀ to E₁(1H), and of E₁(1H) to E₂(2H) (S=3/2). The experimental results further suggest that the E₁(1H)→E₂(2H) step involves coupled delivery of a proton and an electron (PCET) to FeMo-co of E₁(H) to generate a nonequilibrium S= form E₂(2H)*. This subsequently undergoes conformational relaxation and attendant change in the FeMo-co spin state, to generate the equilibrium E₂(2H) (S=3/2) state. Unexpectedly, these experiments also reveal conformational coupling between FeMo-co and the P cluster, and between the Fe protein binding and FeMo-co, which might play a role in gated electron transfer from reduced Fe protein to FeMo-co.     

Decomposition of ammonia with iron and calcium catalysts supported on coal chars

Decomposition of NH₃ to N₂ with Fe and Ca catalysts supported on brown coal chars has been studied with a cylindrical quartz reactor from a viewpoint of hot gas cleanup. The catalyst is prepared by pyrolyzing a brown coal with Fe or Ca ions added. In the decomposition of 2000 ppm NH₃ diluted with He at 750 °C and at a space velocity of 45,000 l/h, 2-6 wt% Fe catalysts are more active than not only 6 wt% Ca catalyst but also 8 wt% Fe catalyst loaded on a commercial activated carbon. The transmission electron microscope observations show that fine iron particles with the sizes of 20-50 nm account for the higher catalytic performances. When reaction temperature is increased to 850 °C, all of Fe and Ca catalysts on the chars achieve complete decomposition of NH₃. The co-feeding of H₂ with 2000 ppm NH₃ improves the performance of the 2% Fe catalyst at 750 °C, but contrarily the coexistence of syngas (CO/H₂=2) deactivates it remarkably, whereas the addition of CO₂ to syngas restores the catalytic activity of the Fe to the original state without syngas. The powder X-ray diffraction and temperature programmed desorption measurements strongly suggest that the Fe and Ca catalysts promote NH₃ decomposition through cycle mechanisms involving the formation of N-containing intermediate species and the subsequent decomposition to N₂.     

Preparation and characterization of three types of dinuclear iron(III) complexes with H2salbn,N,N′-disalicylidene-1,4-diaminobutane

By the reaction of the tetradentate Schiff base ligand H₂salbn, N,N^′-disalicylidene-1,4-diaminobutane with Fe^IIICl₃·6H₂O in the presence of Et₃N in MeOH a dinuclear iron(III) complex, [Fe^III(salbn)(μ-OMe)]₂ (1), has been obtained, whereas in EtOH, dinuclear complexes, [Fe^III(μ-salbn)]₂(μ-O) (2) and [Fe^III(salbn)]₂(μ-salbn) (3), are obtained. The structure of the complex 1 consists of two Fe(III) centers with one tetradentate salbn ligand (N₂O₂) which are bridged by two methoxo groups to yield a planar Fe₂O₂ core. On the other hand, in the complex 2, each of the two Fe(III) ions has a five-coordinate structure in which both salbn ligands act as a bridging didentate ligand and one oxygen atom bridges two Fe(III) ions to form a μ-oxo structure. The structure of the complex 3, which was obtained by accompanying with complex 2, consists of two six-coordinate Fe(III) centers in which each Fe(III) ion is coordinated by a tetradentate salbn ligand (N₂O₂) and one bridging salbn ligand (NO).     

Ammonia loss from surface applied urea-acid products

Ammonia (NH₃) losses from soils occur only under alkaline conditions; therefore, adequate acidification could prevent NH₃ loss. In acid soils this alkaline condition will exist only as a micro-environment around the decomposing CO(NH₂)₂ granule. The objective of this experiment was to examine the degree of NH₃ loss reduction that occurs when acids are placed with surface applied CO(NH₂)₂. Phosphoric acid, H₂SO₄, HCl and HNO₃ were used with surface applied CO(NH₂)₂ in a laboratory experiment to examine resultant NH₃ loss under very extreme NH₃ loss conditions. Calcium and magnesium chloride salts were added to urea:phosphoric acid to compare the relative effectiveness of acid and Ca + Mg salts for control of NH₃ loss.Little depression of NH₃-N loss was found from CO(NH₂)₂ containing H₃PO₄ and H₂SO₄ when the sand contained free CaCO₃. However, when CO(NH₂)₂:H₃PO₄ (UP) mixtures were applied as 17-19-0 on neutral and acid sands, NH₃ losses were reduced. Molar ratios less than 1:1 (28-12-0, 35-7-0) resulted in NH₃ losses similar to those from CO(NH₂)₂ alone even in acid soils. The 110 g N m^–2 as 17-19-0 reduced relative NH₃-N loss and pH in acidified and neutral soils more effectively than 11 g N m^–2. Ammonia losses are determined by chemical reactions occurring under the individual CO(NH₂)₂ granules; therefore, the use of the high 110 g N m^–2 rates in this research. The 17-19-0 reduced soil pH and retarded the rate of CO(NH₂)₂ hydrolysis with consequent reduction in NH₃ loss. Ammonia loss was reduced only slightly at 11 g N m^–2 from 17-19-0 even in acid soils. Ammonia loss was reduced from 70 to 30% of applied N by applications of HNO₃ and HCl with the CO(NH₂)₂. The HNO₃ and HCl react with CaCO₃ in a calcareous soil to produce CaCl₂ and Ca(NO₃)₂ which are known to reduce NH₃ loss from surface applied CO(NH₂)₂. However, a dry product of HNO₃ · CO(NH₂)₂ is explosive and can not be used as a general fertilizer.Calcium chloride or MgCl₂ combined with CO(NH₂)₂:H₃PO₄ reduced NH₃ loss more at 110 g N m^–2 than at 11 g N m^–2. Calcium chloride reduced NH₃ loss more effectively than MgCl₂. The CaCl₂ and MgCl₂ salts were more effective than H₂SO₄ or H₃PO₄ in reducing NH₃ losses except when (e.g., 17-19-0) mixtures were added to neutral or acidic sands.Contribution from Texas Agric. Exp. Stn., Texas A & M University, College Station, TX 77843.     

The origin of15NH3 produced from the reaction of14NH3 and15NO over vanadia-based SCR catalysts

The effects of H₂O and the vanadia content of the catalyst on the formation of¹⁵NH₃ during the reaction of¹⁵NO and¹⁴NH₃ in the absence of O₂ over V₂O₅-based catalysts have been determined by mass spectrometry and Fourier transform infrared spectroscopy. At 450°C, the contribution of¹⁵NH₃ to the total nitrogen-containing products remains constant at about 20% for water concentrations from 0 to 1.6%. The vanadia content also has little effect on the proportion of¹⁵NH₃ produced. Combination reactions producing¹⁴N¹⁵N and¹⁴N¹⁵NO consume surface oxygen species and oxygen mass balances indicate that the amount of¹⁵NH₃ formed is determined by the extent of these combination reactions. Small concentrations of O₂ (<300 ppm) were sufficient to prevent the formation of¹⁵NH₃. The reduction of NO by H₂ was also studied. Negligible amounts of NH₃ were formed under dry feed conditions, whereas, in the presence of 1.6% H₂O,¹⁵NH₃ represents about one third of the products. A mechanism involving reaction of an adsorbed N_s species with H₂O is used to account for these experimental observations.     

Selective catalytic reduction of NOx by NH3 on Fe/HBEA zeolite catalysts in oxygen-rich exhaust

This paper deals with the systematic study of Fe/HBEA zeolites for the selective catalytic reduction (SCR) of NO_x by NH₃ in diesel exhaust. The catalysts are prepared by incipient wetness impregnation of H-BEA zeolite (Si/Al = 12.5). The SCR examinations performed under stationary conditions show that the pattern with a Fe load of 0.25 wt.% (0.25Fe/HBEA) reveals pronounced performance. The turnover frequency at 200 °C indicates superior SCR activity of 0.25Fe/HBEA (8.5 × 10⁻³ s⁻¹) as compared to commercial Fe-exchanged BEA (0.99 × 10⁻³ s⁻¹) and V₂O₅/WO₃/TiO₂ (1.0 × 10⁻³ s⁻¹). Based upon powder X-ray diffraction (PXRD), temperature programmed reduction by H₂ (HTPR), diffuse reflectance UV–vis spectroscopy (DRUV–VIS) and catalytic data it is concluded that the pronounced performance of 0.25Fe/HBEA is substantiated by its high proportion of isolated Fe oxo sites. Furthermore, isotopic studies show that no association mechanism of NH₃ takes place on 0.25Fe/HBEA, i.e. N₂ is mainly formed from NO and NH₃.The evaluation of 0.25Fe/HBEA under more practical conditions shows that H₂O decreases the SCR performance, while CO and CO₂ do not affect the activity. Contrary, SCR is markedly accelerated in presence of NO₂ referring to fast SCR. Moreover, hydrothermal treatment at 550 °C does not change SCR drastically, whereas a clear decline is observed after 800 °C aging.     

[Fe]-Hydrogenase,Cofactor Biosynthesis and Engineering

[Fe]-hydrogenase catalyzes the heterolytic cleavage of H₂ and reversible hydride transfer to methenyl-tetrahydromethanopterin. The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of this enzyme, in which mononuclear Fe(II) is ligated with a pyridinol and two CO ligands. The pyridinol ligand fixes the iron by an acyl carbon and a pyridinol nitrogen. Biosynthetic proteins for this cofactor are encoded in the hmd co-occurring (hcg) genes. The function of HcgB, HcgC, HcgD, HcgE, and HcgF was studied by using structure-to-function analysis, which is based on the crystal structure of the proteins and subsequent enzyme assays. Recently, we reported the catalytic properties of HcgA and HcgG, novel radical S-adenosyl methionine enzymes, by using an in vitro biosynthesis assay. Here, we review the properties of [Fe]-hydrogenase and the FeGP cofactor, and the biosynthesis of the FeGP cofactor. Finally, we discuss the expected engineering of [Fe]-hydrogenase and the FeGP cofactor.     

The Copper (II) Complexes of Phenyl-2 Pyridyl Ketone and the Triple Complexes Obtained by Adding Ethylenediamine

Copper salts of low pH (1.8–2.5) combine with phenyl-2-pyridyl ketone to form a ketone complex [(Py–CO–C₆H₅)₂Cu]²⁺. An electrically uncharged complex is obtained at pH > 9 [(Py–C(OH)(C₆H₅)O^?)₂Cu]⁰, liberating two protons from two molecules of the ligand. The stability constant of this complex is β = 16.05. By mixing the copper salts (except the halide) containing this ligand with ethylenediamine, a charged triple complex is obtained at pH < 7 [Py–C(OH)(C₆H₅) O–Cu–NH₂C₂H₄NH₃⁺]²⁺. At pH > 9.5, an uncharged triple complex is obtained: {[(Py–C(OH)(C₆H₅)O^?)₂Cu}₂ · NH₂C₂H₄NH₂}⁰. The copper halide salts produce only an uncharged triple complex; the halide ions are coordinated with the copper atoms. All of these complexes in their solid state, are bi- or polynuclear. As a result, they are magnetically subnormal.