MoFe Protein Research Articles

The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters

The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α2β2 heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N2-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity–based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.

Energy Transduction in Nitrogenase.

Nitrogenase is a complicated two-component enzyme system that uses ATP binding and hydrolysis energy to achieve one of the most difficult chemical reactions in nature, the reduction of N2 to NH3. One component of the Mo-based nitrogenase system, Fe protein, delivers electrons one at a time to the second component, the catalytic MoFe protein. This process occurs through a series of synchronized events collectively called the "Fe protein cycle". Elucidating details of the events associated with this cycle has constituted an important challenge in understanding the nitrogenase mechanism. Electron delivery is a multistep process involving three metal clusters with intra- and interprotein events. It is proposed that the first electron transfer event is a gated intraprotein transfer of one electron from the MoFe protein P-cluster to the FeMo cofactor. Measurement of the effect of osmotic pressure on the rate of this electron transfer process revealed that it is gated by protein conformational changes. This first electron transfer is activated by binding of the Fe protein containing two bound ATP molecules. The mechanism of how this protein-protein association triggers electron transfer remains unknown. The second electron transfer event is proposed to be a rapid interprotein "backfill" with transfer of one electron from the reduced Fe protein 4Fe-4S cluster to the oxidized P-cluster. In this way, electron delivery can be viewed as a case of "deficit spending". Such a deficit-spending electron transfer process can be envisioned as a way to achieve one-direction electron flow, limiting the potential for back electron flow. Hydrolysis of two ATP molecules associated with the Fe protein occurs after the electron transfer and therefore is not used to directly drive the electron transfer. Rather, ATP hydrolysis is proposed to contribute to relaxation of the "activated" conformational state associated with the ATP form of the complex, with the free energy from ATP hydrolysis being used to pay back energy associated with component protein association and electron transfer. Release of inorganic phosphate (Pi) and protein-protein dissociation follow electron transfer and ATP hydrolysis. The rate-limiting step for the Fe protein cycle is not dissociation of the two proteins, as previously believed, but rather is release of Pi after ATP hydrolysis, which is then followed by rapid protein-protein complex dissociation. Nitrogenase is composed of two catalytic halves that do not function independently but rather exhibit anticooperative nuclear motion in which electron transfer in one-half of the complex partially inhibits electron transfer and ATP hydrolysis in the other half. Calculations indicated the existence of anticooperative interactions across the entire nitrogenase complex, suggesting a mechanism for the control of events on opposite ends of this large complex. The mechanistic necessity for this anticooperative process remains unknown. This Account presents a working model for how all of these processes work together in the nitrogenase "machine" to transduce the energy from ATP binding and hydrolysis to drive N2 reduction.

Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.

Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

(Invited) Nitrogenase Electrochemistry for Ammonia Production

The Haber-Bosch process, responsible for producing NH3 from H2 and N2, ranks as one of the most important discoveries in the history of chemistry. Its industrial application in synthetic fertilizers contributed to the continuously increasing population and their quality of life. The Haber-Bosch process, however, is a very energy-intensive process with high temperature (500 °C) and pressure (20 MPa) required for efficient NH3 production. Over 1 % of the world’s energy sources were consumed in order to produce ~140 megatons of NH3 in 2015. Additionally, 3 % of global CO2 emissions are due to the Haber-Bosch related technology. A renewable strategy for ammonia production would therefore be valuable. In nature, the ability to reduce N2 to NH3 is limited to a group of bacteria and archaea classified as diazotrophs, all of which share an enzyme called nitrogenase. Using methylviologen (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 which 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, 60 mC of charge 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 %. Importantly, this EFC produces NH3 and electrical energy in a carbon-neutral manner. A protective FeSII protein, which can reversibly lock nitrogenase into a multicomponent protective complex upon exposure to low concentrations of O2, was incorporated into a nitrogenase bioelectrosynthetic cell whereby NH3 was produced using air as a substrate. This marks a significant step forward in overcoming the crippling limitation of nitrogenase’s sensitivity toward O2.

A VTVH MCD and EPR Spectroscopic Study of the Maturation of the "Second" Nitrogenase P-Cluster.

The P-cluster of the nitrogenase MoFe protein is a [ Fe8 S7] 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 [ Fe4 S4]+ clusters within 5 min and the second P-cluster is formed through slow fusion of the second pair of [ Fe4 S4]+ 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 [ Fe4 S4]+ clusters into the integer spin cluster [ Fe4 S3-4]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 [ Fe8 S7-8]β, which is eventually converted into the P-cluster.

Hydride Conformers of the Nitrogenase FeMo-cofactor Two-Electron Reduced State E2(2H), Assigned Using Cryogenic Intra Electron Paramagnetic Resonance Cavity Photolysis.

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 E2(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 E2(2H)/1b state now corroborates the identification of this state as storing two reducing equivalents as a hydride. Photolysis converts E2(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 E2(2H)/1b. This implies that the 1c signal comes from an E2(2H) hydride isomer of E2(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.

Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein.

ABSTRACTThe Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.

Cluster-Dependent Charge-Transfer Dynamics in Iron-Sulfur Proteins.

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.

Application of affinity purification methods for analysis of the nitrogenase system from Azotobacter vinelandii.

Nitrogenases are complex two-component metalloenzymes that catalyze biological nitrogen fixation. Three different nitrogenase types are found in the model nitrogen-fixing microbe Azotobacter vinelandii. In the case of the Mo-dependent enzyme, the two catalytic partners are referred to as the Fe protein and MoFe protein. In addition to genes encoding the catalytic components, there are a total of 68 other gene products known to be variously involved in producing, activating, protecting, sustaining, and regulating formation of the Mo-dependent nitrogenase. In order to support experiments designed to gain insight into the catalytic mechanism and assembly of nitrogenase, four different affinity-based purification protocols have been developed. These include an improved Co2+-based Immobilized Metal Affinity Chromatography (IMAC) method for the purification of MoFe protein, a newly developed StrepTactin Affinity Chromatography (STAC) method for the purification of MoFe protein and its assembly intermediates, a combined IMAC and STAC method for isolation of highly pure MoFe protein, and a STAC-based bait-prey method for isolation of complexes variously involved in the maturation process.

Structural characterization of the nitrogenase molybdenum-iron protein with the substrate acetylene trapped near the active site

The biological reduction of dinitrogen (N2) to ammonia is catalyzed by the complex metalloenzyme nitrogenase. Structures of the nitrogenase component proteins, Iron (Fe) protein and Molybdenum‑iron (MoFe) protein, and the stabilized complexes these component proteins, have been determined, providing a foundation for a number of fundamental aspects of the complicated catalytic mechanism. The reduction of dinitrogen to ammonia is a complex process that involves the binding of N2 followed by reduction with multiple electrons and protons. Electron transfer into nitrogenase is typically constrained to the unique electron donor, the Fe protein. These constraints have prevented structural characterization of the active site with bound substrate. Recently it has been realized that selected amino acid substitutions in the environment of the active site metal cluster (Iron‑molybdenum cofactor, FeMo-co) allow substrates to persist even in the resting state. Reported here is a 1.70Å crystal structure of a nitrogenase MoFe protein α-96Arg➔Gln variant with the alternative substrate acetylene trapped in a channel in close proximity to FeMo-co. Complementary theoretical calculations support the validity of the acetylene interaction at this site and is also consistent with more favorable interactions in the variant MoFe protein compared to the native MoFe protein. This work represents the first structural evidence of a substrate trapped in the nitrogenase MoFe protein and is consistent with earlier assignments of proposed substrate pathways and substrate binding sites deduced from biochemical, spectroscopic, and theoretical studies.

Electrocatalytic CO2 reduction catalyzed by nitrogenase MoFe and FeFe proteins

Nitrogenases catalyze biological dinitrogen (N2) reduction to ammonia (NH3), and also reduce a number of non-physiological substrates, including carbon dioxide (CO2) to formate (HCOO−) and methane (CH4). Three versions of nitrogenase are known (Mo-, V-, and Fe-nitrogenase), each showing different reactivities towards various substrates. Normally, electrons for substrate reduction are delivered by the Fe protein component of nitrogenase, with energy coming from the hydrolysis of 2 ATP to 2 ADP+2 Pi for each electron transferred. Recently, it has been demonstrated that energy and electrons can be delivered from an electrode to the catalytic nitrogenase MoFe-protein without the need for Fe protein or ATP hydrolysis. Here, it is demonstrated that both the MoFe- and FeFe-protein can be immobilized as a polymer layer on an electrode and that electron transfer mediated by cobaltocene can drive CO2 reduction to formate in this system. It was also found that the FeFe-protein diverts a greater percentage of electrons to CO2 reduction versus proton reduction compared to the MoFe-protein. Quantification of electron flow to products exhibited Faradaic efficiencies of CO2 conversion to formate of 9% for MoFe protein and 32% for FeFe-protein, with the remaining electrons going to proton reduction to make H2.

QM/MM Study of the Nitrogenase MoFe Protein Resting State: Broken-Symmetry States, Protonation States, and QM Region Convergence in the FeMoco Active Site.

Nitrogenase is one of the most fascinating enzymes in nature, being responsible for all biological nitrogen reduction. Despite decades of research, it is among the enzymes in bioinorganic chemistry whose mechanism is the most poorly understood. The MoFe protein of nitrogenase contains an iron-molybdenum-sulfur cluster, FeMoco, where N2 reduction takes place. The resting state of FeMoco has been characterized by crystallography, multiple spectroscopic techniques, and theory (broken-symmetry density functional theory), and all heavy atoms are now characterized. The cofactor charge, however, has been controversial, the electronic structure has proved enigmatic, and little is known about the mechanism. While many computational studies have been performed on FeMoco, few have taken the protein environment properly into account. In this study, we put forward QM/MM models of the MoFe protein from Azotobacter vinelandii, centered on FeMoco. By a detailed analysis of the FeMoco geometry and comparison to the atomic resolution crystal structure, we conclude that only the [MoFe7S9C]1- charge is a possible resting state charge. Further, we find that of the three lowest energy broken-symmetry solutions of FeMoco, the BS7-235 spin isomer (where 235 refers to Fe atoms that are "spin-down") is the only one that can be reconciled with experiment. This is revealed by a comparison of the metal-metal distances in the experimental crystal structure, a rare case of spin-coupling phenomena being visible through the molecular structure. This could be interpreted as the enzyme deliberately stabilizing a specific electronic state of the cofactor, possibly for tuning specific reactivity on specific metal atoms. Finally, we show that the alkoxide group on the Mo-bound homocitrate must be protonated under resting state conditions, the presence of which has implications regarding the nature of FeMoco redox states as well as for potential substrate reduction mechanisms.

Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects.

Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H2. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H2. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, was found to change linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate-limiting step of H2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the S-H+ bond breaks and H+ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.

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.

Reversible Protonated Resting State of the Nitrogenase Active Site.

Protonated states of the nitrogenase active site are mechanistically significant since substrate reduction is invariably accompanied by proton uptake. We report the low pH characterization by X-ray crystallography and EPR spectroscopy of the nitrogenase molybdenum iron (MoFe) proteins from two phylogenetically distinct nitrogenases (Azotobacter vinelandii, Av, and Clostridium pasteurianum, Cp) at pHs between 4.5 and 8. X-ray data at pHs of 4.5–6 reveal the repositioning of side chains along one side of the FeMo-cofactor, and the corresponding EPR data shows a new S = 3/2 spin system with spectral features similar to a state previously observed during catalytic turnover. The structural changes suggest that FeMo-cofactor belt sulfurs S3A or S5A are potential protonation sites. Notably, the observed structural and electronic low pH changes are correlated and reversible. The detailed structural rearrangements differ between the two MoFe proteins, which may reflect differences in potential protonation sites at the active site among nitrogenase species. These observations emphasize the benefits of investigating multiple nitrogenase species. Our experimental data suggest that reversible protonation of the resting state is likely occurring, and we term this state “E0H+”, following the Lowe–Thorneley naming scheme.

Bio-Inspired Artificial Electron Transfer in Bioelectrocatalysis

Biological systems have complex electron transport pathways that can be used as an inspiration in designing electrodes for bioelectrocatalysis. This presentation will detail the use of microbial biology as an inspiration for designing electrodes capable of hetergeneous reduction to produce ammonia. In this work, we have studied the use of nitrogenase for bioelectrocatalysis, because this MoFe protein is capable of electrosynthesis of ammonia. Although this protein has been studied by biologists in solution or in cells for decades, it has never been effectively wired to an electrode for heterogeneous electrosynthesis. In this work, we detail strategies for electron transport betweent he MoFe protein and the electrode, as well as strategies for immobilization of the enzyme. We will demonstrate bioelectrocatalysis, as well as electrosynthesis assays of the production of ammonia.

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

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

AbstractNitrogenases 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 %.

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 %.