Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra.

Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E0. Two of these species were preliminary assigned to hydrogenated E2 states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of 33S and 77Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.

What triggers the coupling of proton transfer and electron transfer at the active site of nitrogenase?

In converting N2 to NH3 the enzyme nitrogenase utilises 8 electrons and 8 protons in the complete catalytic cycle. The source of the electrons is an Fe4S4 reductase protein (Fe-protein) which temporarily docks with the MoFe-protein that contains the catalytic active cofactor, FeMo-co, and an electron transfer cluster called the P cluster. The overall mechanism involves 8 repetitions of a cycle in which reduced Fe-protein docks with the MoFe-protein, one electron transfers to the P-cluster, and then to FeMo-co, followed by dissociation of the two proteins and re-reduction of the Fe-protein. Protons are supplied serially to FeMo-co by a Grotthuss proton translocation mechanism from the protein surface along a conserved chain of water molecules (a proton wire) that terminates near S atoms of the FeMo-co cluster [CFe7S9Mo(homocitrate)] where the multiple steps of the chemical conversions are effected. It is assumed that the chemical mechanisms use proton-coupled electron-transfer (PCET) and that H atoms (e- + H+) are involved in each of the hydrogenation steps. However there is neither evidence for, or mechanism proposed, for this coupling. Here I report calculations of cluster charge distribution upon electron addition, revealing that the added negative charge is on the S atoms of FeMo-co, which thereby become more basic, and able to trigger proton transfer from H3O+ waiting at the near end of the proton wire. This mechanism is supported by calculations of the dynamics of the proton transfer step, in which the barrier is reduced by ca. 3.5 kcal mol-1 and the product stabilised by ca. 7 kcal mol-1 upon electron addition. H tunneling is probable in this step. In nitrogenase it is electron transfer that triggers proton transfer.

Structural Insights and Mechanistic Understanding of Iron-Molybdenum Cofactor Biosynthesis by NifB in Nitrogenase Assembly Process.

NifB, a radical S-adenosylmethionine (SAM) enzyme, is pivotal in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), commonly referred to as the M-cluster. This cofactor, located within the active site of nitrogenase, is essential for the conversion of dinitrogen (N2) to NH3. Recognized as the most intricate metallocluster in nature, FeMo-co biosynthesis involves multiple proteins and a sequence of steps. Of particular significance, NifB directs the fusion of two [Fe4S4] clusters to assemble the 8Fe core, while also incorporating an interstitial carbide. Although NifB has been extensively studied, its molecular mechanisms remain elusive. In this review, we explore recent structural analyses of NifB and provide a comprehensive overview of the established catalytic mechanisms. We propose prospective directions for future research, emphasizing the relevance to biochemistry, agriculture, and environmental science. The goal of this review is to lay a solid foundation for future endeavors aimed at elucidating the atomic details of FeMo-co biosynthesis.

Low-temperature trapping of N2 reduction reaction intermediates in nitrogenase MoFe protein-CdS quantum dot complexes.

The biological reduction of N2 to ammonia requires the ATP-dependent, sequential delivery of electrons from the Fe protein to the MoFe protein of nitrogenase. It has been demonstrated that CdS nanocrystals can replace the Fe protein to deliver photoexcited electrons to the MoFe protein. Herein, light-activated electron delivery within the CdS:MoFe protein complex was achieved in the frozen state, revealing that all the electron paramagnetic resonance (EPR) active E-state intermediates in the catalytic cycle can be trapped and characterized by EPR spectroscopy. Prior to illumination, the CdS:MoFe protein complex EPR spectrum was composed of a S = 3/2 rhombic signal (g = 4.33, 3.63, and 2.01) consistent with the FeMo-cofactor in the resting state, E0. Illumination for sequential 1-h periods at 233 K under 1 atm of N2 led to a cumulative attenuation of E0 by 75%. This coincided with the appearance of S = 3/2 and S = 1/2 signals assigned to two-electron (E2) and four-electron (E4) reduced states of the FeMo-cofactor, together with additional S = 1/2 signals consistent with the formation of E6 and E8 states. Simulations of EPR spectra allowed quantification of the different E-state populations, along with mapping of these populations onto the Lowe-Thorneley kinetic scheme. The outcome of this work demonstrates that the photochemical delivery of electrons to the MoFe protein can be used to populate all of the EPR active E-state intermediates of the nitrogenase MoFe protein cycle.

High Affinity Electrostatic Interactions Support the Formation of CdS Quantum Dot:Nitrogenase MoFe Protein Complexes.

Nitrogenase MoFe protein can be coupled with CdS nanocrystals (NCs) to enable photocatalytic N2 reduction. The nature of interactions that support complex formation is of paramount importance in intermolecular electron transfer that supports catalysis. In this work we have employed microscale thermophoresis to examine binding interactions between 3-mercaptopropionate capped CdS quantum dots (QDs) and MoFe protein over a range of QD diameters (3.4-4.3 nm). The results indicate that the interactions are largely electrostatic, with the strength of interactions similar to that observed for the physiological electron donor. In addition, the strength of interactions is sensitive to the QD diameter, and the binding interactions are significantly stronger for QDs with smaller diameters. The ability to quantitatively assess NC protein interactions in biohybrid systems supports strategies for understanding properties and reaction parameters that are important for obtaining optimal rates of catalysis in biohybrid systems.

N2 binding to the E0-E4 states of nitrogenase.

Nitrogenase is the only enzyme that can convert N2 into NH3. The reaction requires the addition of eight electrons and protons to the enzyme and the mechanism is normally described by nine states, E0-E8, differing in the number of added electrons. Experimentally, it is known that three or four electrons need to be added before the enzyme can bind N2. We have used combined quantum mechanical and molecular mechanics methods to study the binding of N2 to the E0-E4 states of nitrogenase, using four different density functional theory (DFT) methods. We test many different structures for the E2-E4 states and study binding both to the Fe2 and Fe6 ions of the active-site FeMo cluster. Unfortunately, the results depend quite strongly on the DFT methods. The TPSS method gives the strongest bonding and prefers N2 binding to Fe6. It is the only method that reproduces the experimental observation of unfavourable binding to the E0-E2 states and favourable binding to E3 and E4. The other three methods give weaker binding, preferably to Fe2. B3LYP strongly favours structures with the central carbide ion triply protonated. The other three methods suggest that states with the S2B ligand dissociated from either Fe2 or Fe6 are competitive for the E2-E4 states. Moreover, such structures with two hydride ions both bridging Fe2 and Fe6 are the best models for E4 and also for the N2-bound E3 and E4 states. However, for E4, other structures are often close in energy, e.g. structures with one of the hydride ions bridging instead Fe3 and Fe7. Finally, we find no support for the suggestion that reductive elimination of H2 from the two bridging hydride ions in the E4 state would enhance the binding of N2.

Statistical analyses of the oxidized P-clusters in MoFe proteins using the bond-valence method: towards their electron transfer in nitrogenases.

26 well selected oxidized P-clusters (P2+) from the crystallographic data deposited in the Protein Data Bank have been analysed statistically by the bond-valence sum method with weighting schemes for MoFe proteins at different resolutions. Interestingly, the oxidation states of P2+ clusters correspond to Fe23+Fe62+ with high electron delocalization, showing the same oxidation states as the resting states of P-clusters (PN) in nitrogenases. The previously uncertain reduction of P2+ to PN clusters by two electrons was assigned as a double protonation of P2+, in which decoordination of the serine residue and the peptide chain of cysteine take place, in MoFe proteins. This is further supported by the obviously shorter α-alkoxy C-O bond (average of 1.398 Å) in P2+ clusters and longer α-hydroxy C-O bond (average of 1.422 Å) in PN clusters, while no change is observed in the electronic structures of Fe8S7 Fe atoms in P-clusters. Spatially, the calculations show that Fe3 and Fe6, the most oxidized and most reduced Fe atoms, have the shortest distances of 9.329 Šfrom the homocitrate in the FeMo cofactor and 14.947 Šfrom the [Fe4S4] cluster, respectively, and may well function as important electron-transport sites.

Electrochemical experiments define potentials associated with binding of substrates and inhibitors to nitrogenase MoFe protein.

Nitrogenases catalyse the 6-electron reduction of dinitrogen to ammonia, passing through a series of redox and protonation levels during catalytic substrate reduction. The molybdenum-iron nitrogenase is the most well-studied, but redox potentials associated with proton-coupled transformations between the redox levels of the catalytic MoFe protein have proved difficult to pin down, in part due to a complex electron-transfer pathway from the partner Fe protein, linked to ATP-hydrolysis. Here, we apply electrochemical control to the MoFe protein of Azotobacter vinelandii nitrogenase, using europium(III/II)-ligand couples as low potential redox mediators. We combine insight from the electrochemical current response with data from gas chromatography and in situ infrared spectroscopy, in order to define potentials for the binding of a series of inhibitors (carbon monoxide, methyl isocyanide) to the metallo-catalytic site of the MoFe protein, and the onset of catalytic transformation of alternative substrates (protons and acetylene) by the enzyme. Thus, we associate potentials with the redox levels for inhibition and catalysis by nitrogenase, with relevance to the elusive mechanism of biological nitrogen fixation.

A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H2 formation.

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E4(4H), which has accumulated 4[e-/H+] as two bridging hydrides, Fe2-H-Fe6 and Fe3-H-Fe7, and protons bound to two sulfurs. E4(4H) is poised to bind/reduce N2 as driven by mechanistically-coupled H2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H2 as the enzyme relaxes to state E2(2H), containing 2[e-/H+] as a hydride and sulfur-bound proton; accumulation of E4(4H) in α-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e-/H+] to the E0 state of the WT MoFe protein and to both α-V70I conformations generating E2(2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e-/H+] generates E4(4H) with Fe2-H-Fe6 as the second hydride. E4(4H) in WT enzyme and a minority α-V70I E4(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2-H-Fe6 followed by slower HP of Fe3-H-Fe7, which leads to transient accumulation of E2(2H) containing Fe3-H-Fe7. In the dominant α-V70I E4(4H) conformation, HP of Fe2-H-Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3-H-Fe7 occurs first and the resulting E2(2H) contains Fe2-H-Fe6. It is this HP suppression in E4(4H) that enables α-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in α-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme.

The Fe Protein Cycle Associated with Nitrogenase Catalysis Requires the Hydrolysis of Two ATP for Each Single Electron Transfer Event.

A central feature of the current understanding of dinitrogen (N2) reduction by the enzyme nitrogenase is the proposed coupling of the hydrolysis of two ATP, forming two ADP and two Pi, to the transfer of one electron from the Fe protein component to the MoFe protein component, where substrates are reduced. A redox-active [4Fe-4S] cluster associated with the Fe protein is the agent of electron delivery, and it is well known to have a capacity to cycle between a one-electron-reduced [4Fe-4S]1+ state and an oxidized [4Fe-4S]2+ state. Recently, however, it has been shown that certain reducing agents can be used to further reduce the Fe protein [4Fe-4S] cluster to a super-reduced, all-ferrous [4Fe-4S]0 state that can be either diamagnetic (S = 0) or paramagnetic (S = 4). It has been proposed that the super-reduced state might fundamentally alter the existing model for nitrogenase energy utilization by the transfer of two electrons per Fe protein cycle linked to hydrolysis of only two ATP molecules. Here, we measure the number of ATP consumed for each electron transfer under steady-state catalysis while the Fe protein cluster is in the [4Fe-4S]1+ state and when it is in the [4Fe-4S]0 state. Both oxidation states of the Fe protein are found to operate by hydrolyzing two ATP for each single-electron transfer event. Thus, regardless of its initial redox state, the Fe protein transfers only one electron at a time to the MoFe protein in a process that requires the hydrolysis of two ATP.

Connecting the geometric and electronic structures of the nitrogenase iron-molybdenum cofactor through site-selective 57Fe labelling.

Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the 57Fe-based spectroscopic data-although rich with information-combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.

Structural consequences of turnover-induced homocitrate loss in nitrogenase.

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein - a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA - that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site. We further find a population of the ∆NifV variant complexed to an endogenous protein identified through structural and proteomic approaches as the uncharacterized protein NafT. Recognition by endogenous NafT demonstrates the physiological relevance of the HCA-compromised form, perhaps for cofactor insertion or repair. Our results point towards a dynamic active site in which HCA plays a role in enabling nitrogenase catalysis by facilitating activation of the FeMo-cofactor from a relatively stable form to a state capable of reducing dinitrogen under ambient conditions.

The binding of reducible N2 in the reaction domain of nitrogenase.

The binding of N2 to FeMo-co, the catalytic site of the enzyme nitrogenase, is central to the conversion to NH3, but also has a separate role in promoting the N2-dependent HD reaction (D2 + 2H+ + 2e- → 2HD). The protein surrounding FeMo-co contains a clear channel for ingress of N2, directly towards the exo-coordination position of Fe2, a position which is outside the catalytic reaction domain. This led to the hypothesis [I. Dance, Dalton Trans., 2022, 51, 12717] of 'promotional' N2 bound at exo-Fe2, and a second 'reducible' N2 bound in the reaction domain, specifically the endo-coordination position of Fe2 or Fe6. The range of possibilities for the binding of reducible N2 in the presence of bound promotional N2 is described here, using density functional simulations with a 486 atom model of the active site and surrounding protein. The pathway for ingress of the second N2 through protein, past the first N2 at exo-Fe2, and tumbling into the binding domain between Fe2 and Fe6, is described. The calculations explore 24 structures involving 6 different forms of hydrogenated FeMo-co, including structures with S2BH unhooked from Fe2 but tethered to Fe6. The calculations use the most probable electronic states. End-on (η1) binding of N2 at the endo position of either Fe2 or Fe6 is almost invariably exothermic, with binding potential energies ranging up to -18 kcal mol-1. Many structures have binding energies in the range -6 to -14 kcal mol-1. The relevant entropic penalty for N2 binding from a diffusible position within the protein is estimated to be 4 kcal mol-1, and so the binding free energies for reducible N2 are suitably negative. N2 binding at endo-Fe2 is stronger than at endo-Fe6 in three of the six structure categories. In many cases the reaction domain containing reducible N2 is expanded. These results inform computational simulation of the subsequent steps in which surrounding H atoms transfer to reducible N2.

The HD Reaction of Nitrogenase: a Detailed Mechanism.

Nitrogenase is the enzyme that converts N2 to NH3 under ambient conditions. The chemical mechanism of this catalysis at the active site FeMo-co [Fe7 S9 CMo(homocitrate)] is unknown. An obligatory co-product is H2 , while exogenous H2 is a competitive inhibitor. Isotopic substitution using exogenous D2 revealed the N2 -dependent reaction D2 +2H+ +2e- →2HD (the 'HD reaction'), together with a collection of additional experimental characteristics and requirements. This paper describes a detailed mechanism for the HD reaction, developed and elaborated using density functional simulations with a 486-atom model of the active site and surrounding protein. First D2 binds at one Fe atom (endo-Fe6 coordination position), where it is flanked by H-Fe6 (exo position) and H-Fe2 (endo position). Then there is synchronous transfer of these two H atoms to bound D2 , forming one HD bound to Fe2 and a second HD bound to Fe6. These two HD dissociate sequentially. The final phase is recovery of the two flanking H atoms. These H atoms are generated, sequentially, by translocation of a proton from the protein surface to S3B of FeMo-co and combination with introduced electrons. The first H atom migrates from S3B to exo-Fe6 and the second from S3B to endo-Fe2. Reaction energies and kinetic barriers are reported for all steps. This mechanism accounts for the experimental data: (a) stoichiometry; (b) the N2 -dependence results from promotional N2 bound at exo-Fe2; (c) different N2 binding Km for the HD reaction and the NH3 formation reaction results from involvement of two different sites; (d) inhibition by CO; (e) the non-occurrence of 2HD→H2 +D2 results from the synchronicity of the two transfers of H to D2 ; (f) inhibition of HD production at high pN2 is by competitive binding of N2 at endo-Fe6; (g) the non-leakage of D to solvent follows from the hydrophobic environment and irreversibility of proton introduction.

Understanding the tethered unhooking and rehooking of S2B in the reaction domain of FeMo-co, the active site of nitrogenase.

The active site of the nitrogen fixing enzyme nitrogenase is an Fe7MoS9C cluster, and investigations of the enigmatic chemical mechanism of the enzyme have focussed on a pair of Fe atoms, Fe2 and Fe6, and the S2B atom that bridges them. There are three proposals for the status of the Fe2-S2B-Fe6 bridge during the catalytic cycle: one that it remains intact, another that it is completely labile and absent during catalysis, and a third that S2B is hemilabile, unhooking one of its bonds to Fe2 or Fe6. This report examines the tethered unhooking of S2B and factors that affect it, using DFT calculations of 50 geometric/electronic possibilities with a 485 atom model including all relevant parts of surrounding protein. The outcomes are: (a) unhooking the S2B-Fe2 bond is feasible and favourable, but alternative unhooking of the S2B-Fe6 bond is unlikely for steric reasons, (b) energy differences between hooked and unhooked isomers are generally <10 kcal mol-1, usually with unhooked more stable, (c) ligation at the exo-Fe6 position inhibits unhooking, (d) unhooking of hydrogenated S2B is more favourable than that of bare S2B, (e) hydrogen bonding from the NεH function of His195 to S2B occurs in hooked and unhooked forms, and possibly stabilises unhooking, (f) unhooking is reversible with kinetic barriers ranging 10-13 kcal mol-1. The conclusion is that energetically accessible reversible unhooking of S2B or S2BH, as an intrinsic property of FeMo-co, needs to be considered in the formulation of mechanisms for the reactions of nitrogenase.

13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel".

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe-9S-C-Mo-homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe-C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares 13CFe6 ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E4(4H) Janus intermediate, activated for N2 binding/reduction by accumulation of 4[e-/H+]; (vi) E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S = 1/2 and 3/2 exhibited near-zero 13C isotropic hyperfine coupling constants, Ca = [-1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe-C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6 Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe-C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe-C bonding of the CFe6 core indicate that it does not provide a functionally dynamic (hemilabile) "beating heart"─instead it acts as "a heart of steel", stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.

Calculating the chemical mechanism of nitrogenase: new working hypotheses.

The enzyme nitrogenase converts N2 to NH3 with stoichiometry N2 + 8H+ + 8e- → 2NH3 + H2. The mechanism is chemically complex with multiple steps that must be consistent with much accumulated experimental information, including exchange of H2 and N2 and the N2-dependent hydrogenation of D2 to HD. Previous investigations have developed a collection of working hypotheses that guide ongoing density functional investigations of mechanistic steps and sequences. These include (i) hypotheses about the serial provision of protons and their conversion to H atoms bonded to S and Fe atoms of the FeMo-co catalytic site, (ii) the migration of H atoms over the surface of FeMo-co, (iii) the roles of His195, (iv) identification of three protein channels, one for the ingress of N2, a separate pathway for the passage of exogenous H2 (D2) and product H2 (HD), and a hydrophilic pathway for egress of product NH3. Two additional working hypotheses are described in this paper. N2 passing along the N2 channel approaches and binds end-on to the exo coordination position of Fe2, with favourable energetics when FeMo-co is pre-hydrogenated. This exo-Fe2-N2 is apparently not reduced but has a promotional role by expanding the reaction zone. A second N2 can enter via the N2 ingress channel and bind at the endo-Fe6 position, where it is surrounded by H atom donors suitable for the N2 → NH3 conversion. It is proposed that this endo-Fe6 position is also the binding site for H2 (generated or exogenous), accounting for the competitive inhibition of N2 reduction by H2. The HD reaction occurs at the endo-Fe6 site, promoted by N2 at the exo-Fe2 site. The second hypothesis concerns the most stable electronic states of FeMo-co with ligands bound at Fe2 and Fe6, and provides a protocol for management of electronic states in mechanism calculations.

Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe-9S-C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe-S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe-4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.

Proton Transfer Pathways in Nitrogenase with and without Dissociated S2B.

Nitrogenase is the only enzyme that can convert N2 to NH3 . Crystallographic structures have indicated that one of the sulfide ligands of the active-site FeMo cluster, S2B, can be replaced by an inhibitor, like CO and OH- , and it has been suggested that it may be displaced also during the normal reaction. We have investigated possible proton transfer pathways within the FeMo cluster during the conversion of N2 H2 to two molecules of NH3 , assuming that the protons enter the cluster at the S3B, S4B or S5A sulfide ions and are then transferred to the substrate. We use combined quantum mechanical and molecular mechanical (QM/MM) calculations with the TPSS and B3LYP functionals. The calculations indicate that the barriers for these reactions are reasonable if the S2B ligand remains bound to the cluster, but they become prohibitively high if S2B has dissociated. This suggests that it is unlikely that S2B reversibly dissociates during the normal reaction cycle.

Structures of the nitrogenase complex prepared under catalytic turnover conditions.

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.