On the path to [Fe-S] protein maturation: A personal perspective.

Azotobacter vinelandii is a genetically tractable Gram-negative proteobacterium able to fix nitrogen (N2) under aerobic growth conditions. This narrative describes how biochemical-genetic approaches using A. vinelandii to study nitrogen fixation led to the formulation of the "scaffold hypothesis" for the assembly of both simple and complex [Fe-S] clusters associated with biological nitrogen fixation. These studies also led to the discovery of a parallel, but genetically distinct, pathway for maturation of [Fe-S] proteins that support central metabolic processes.

Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of Saccharomyces cerevisiae.

Biological nitrogen fixation, the conversion of inert N2 to metabolically tractable NH3, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo-R-homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N2 reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as Saccharomyces cerevisiae, represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation. A central player in FeMo-co assembly is the scaffold protein NifEN upon which processing of NifB-co, an [8Fe-9S-C] precursor produced by NifB, occurs. Prior work established that NifB-co can be produced in S. cerevisiae mitochondria. In the present work, a library of nifEN genes from diverse diazotrophs was expressed in S. cerevisiae, targeted to mitochondria, and surveyed for their ability to produce soluble NifEN protein complexes. Many such NifEN variants supported FeMo-co formation when heterologously produced in the diazotroph A. vinelandii. However, only three of them accumulated in soluble forms in mitochondria of aerobically cultured S. cerevisiae. Of these, two variants were active in the in vitro FeMo-co synthesis assay. NifEN, NifB, and NifH proteins from different species, all of them produced in and purified from S. cerevisiae mitochondria, were combined to establish successful FeMo-co biosynthetic pathways. These findings demonstrate that combining diverse interspecies nitrogenase FeMo-co assembly components could be an effective and, perhaps, the only approach to achieve and optimize nitrogen fixation in a eukaryotic organism.IMPORTANCEBiological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo-R-homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae. NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae. FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.

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.

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.

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster.

Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.

The One-Electron Reduced Active-Site FeFe-Cofactor of Fe-Nitrogenase Contains a Hydride Bound to a Formally Oxidized Metal-Ion Core.

The nitrogenase active-site cofactor must accumulate 4e-/4H+ (E4(4H) state) before N2 can bind and be reduced. Earlier studies demonstrated that this E4(4H) state stores the reducing-equivalents as two hydrides, with the cofactor metal-ion core formally at its resting-state redox level. This led to the understanding that N2 binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H2. The state having acquired 2e-/2H+ (E2(2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e-/H+ (E1(H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E1(H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E1(H) state of Fe-nitrogenase shows an S = 1/2 EPR spectrum with g = [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic intracavity, 450 nm photolysis converts to a new and likewise photoactive S = 1/2 state (denoted E1(H)*) with g = [2.009, 1.950, 1.860], which results in a photostationary state, with E1(H)* relaxing to E1(H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E1(H)/E1(H)* photointerconversion. These observations indicate that the addition of the first e-/H+ to the FeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core is formally one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.

AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV-cofactor.

Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2 . These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.

Overview of physiological, biochemical, and regulatory aspects of nitrogen fixation in Azotobacter vinelandii.

Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as well as for the development of improved synthetic catalysts based on the biological mechanism. Over the past 30 years, the free-living nitrogen-fixing bacterium Azotobacter vinelandii emerged as a preferred model organism for mechanistic, structural, genetic, and physiological studies aimed at understanding biological nitrogen fixation. This review provides a contemporary overview of these studies and places them within the context of their historical development.

Specificity of NifEN and VnfEN for the Assembly of Nitrogenase Active Site Cofactors in Azotobacter vinelandii.

The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.

The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase.

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V3+, 3Fe3+, 4Fe2+]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined 51V (I = 7/2) hyperfine splitting, a iso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V3+, 4Fe3+, 3Fe2+]. Our findings suggest that the V3+ does not change valency throughout the catalytic cycle.

Exploring the Role of the Central Carbide of the Nitrogenase Active-Site FeMo-cofactor through Targeted 13C Labeling and ENDOR Spectroscopy.

Mo-dependent nitrogenase is a major contributor to global biological N2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4-) centered within a trigonal prismatic CFe6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S = 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca = -1.30 MHz. This indicates that both the E0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide-Fe bonds.

Comment on "Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase".

Kang et al (Reports, 19 June 2020, p. 1381) report a structure of the nitrogenase MoFe protein that is interpreted to indicate binding of N2 or an N2-derived species to the active-site FeMo cofactor. Independent refinement of the structure and consideration of biochemical evidence do not support this claim.

Electron Redistribution within the Nitrogenase Active Site FeMo-Cofactor During Reductive Elimination of H2 to Achieve N≡N Triple-Bond Activation.

Nitrogen fixation by nitrogenase begins with the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co), generating a state (denoted E4(4H)) with two [Fe-H-Fe] bridging hydrides. Recently, photolytic reductive elimination (re) of the E4(4H) hydrides showed that enzymatic re of E4(4H) hydride yields an H2-bound complex (E4(H2,2H)), in a process corresponding to a formal 2-electron reduction of the metal-ion core of FeMo-co. The resulting electron-density redistribution from Fe-H bonds to the metal ions themselves enables N2 to bind with concomitant H2 release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E4(H2,2H) has not been trapped, cryogenic photolysis of E4(4H) provides a means to address this question. Photolysis of E4(4H) causes hydride-re with release of H2, generating doubly reduced FeMo-co (denoted E4(2H)*), the extreme limit of the electron-density redistribution upon formation of E4(H2,2H). Here we examine the doubly reduced FeMo-co core of the E4(2H)* limiting-state by 1H, 57Fe, and 95Mo ENDOR to illuminate the partial electron-density redistribution upon E4(H2,2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E4(2H)* ENDOR results as extended by DFT computations include (i) the Mo-site participates negligibly, and overall it is unlikely that Mo changes valency throughout the catalytic cycle; and (ii) two distinctive E4(4H) 57Fe signals are suggested as associated with structurally identified "anchors" of one bridging hydride, two others with identified anchors of the second, with NBO-analysis further identifying one anchor of each hydride as a major recipient of electrons released upon breaking Fe-H bonds.

CO as a substrate and inhibitor of H+ reduction for the Mo-, V-, and Fe-nitrogenase isozymes.

Three known nitrogenase isozymes, Mo-, V-, and Fe-, catalyze biological reduction of dinitrogen (N2) to ammonia (NH3). All three utilize the same reductive elimination mechanism: an intermediate with two metal-bound hydrides reductively-eliminates hydrogen gas (H2) in a reaction coupled to binding and activation of N2. Nonetheless, the three isozymes show dramatically different relative rates of H2 formation and N2 reduction, revealing important differences in reactivity with substrates. Carbon monoxide (CO) has been characterized as both an inhibitor and substrate for Mo- and V­nitrogenases, but not for the Fe­nitrogenase. Here, we present a comparative study of the reactivity of the three isozymes with CO, examining CO both as a substrate and as an inhibitor of proton (H+) reduction under steady-state conditions. For Mo­nitrogenase, there is neither detectable reduction of CO nor inhibition of H+ reduction. Fe- and V­nitrogenase show CO reduction and inhibition of H+ reduction that depends on the CO partial pressure. For V­nitrogenase, ethylene (C2H4) is the major reduction product with a maximum specific activity of ~7.5 nmol C2H4/nmol VFe protein/min at 1 atm CO. The major product of CO reduction for Fe­nitrogenase is methane (CH4) with a maximum specific activity of ~4.8 nmol CH4/nmol FeFe protein/min at 0.05 atm CO. The rate of CH4 production by Fe­nitrogenase progressively increases to a maximum at 0.05 atm CO and then declines by ~90% with increasing CO partial pressure up to 1 atm. CO does not inhibit proton reduction in Mo­nitrogenase but shows 16% inhibition for V­nitrogenase and 35% for Fe­nitrogenase.

Reduction of Substrates by Nitrogenases.

Nitrogenase is the enzyme that catalyzes biological N2 reduction to NH3. This enzyme achieves an impressive rate enhancement over the uncatalyzed reaction. Given the high demand for N2 fixation to support food and chemical production and the heavy reliance of the industrial Haber-Bosch nitrogen fixation reaction on fossil fuels, there is a strong need to elucidate how nitrogenase achieves this difficult reaction under benign conditions as a means of informing the design of next generation synthetic catalysts. This Review summarizes recent progress in addressing how nitrogenase catalyzes the reduction of an array of substrates. New insights into the mechanism of N2 and proton reduction are first considered. This is followed by a summary of recent gains in understanding the reduction of a number of other nitrogenous compounds not considered to be physiological substrates. Progress in understanding the reduction of a wide range of C-based substrates, including CO and CO2, is also discussed, and remaining challenges in understanding nitrogenase substrate reduction are considered.

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae.

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.

Time-Resolved EPR Study of H2 Reductive Elimination from the Photoexcited Nitrogenase Janus E4(4H) Intermediate.

Nitrogenase is activated for N2 reduction through the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co: Fe7S9MoC; homocitrate) to form the key Janus intermediate, denoted E4(4H), whose lowest-energy structure contains two [Fe-H-Fe] bridging hydrides and two protons bound to the sulfurs that also bridge the Fe pairs. In the critical step of catalysis, a H2 complex transiently produced by reductive elimination (re) of the hydrides of E4(4H), denoted E4(H2;2H), undergoes H2 displacement by N2, which then undergoes the otherwise energetically unfavorable cleavage of the N≡N triple bond. In pursuing the study of the re activation process, we have employed a photochemical approach to obtaining its atomic-level details. Continuous 450 nm irradiation of the ground state of the dihydride Janus intermediate, denoted E4(4H)a, in an EPR cavity at cryogenic temperatures causes photoinduced re of H2 to generate E4(H2;2H). We here extend this photochemical approach with time-resolved EPR studies of the photolysis process on the ns time scale. These studies reveal an additional intermediate in the catalytic reductive elimination process, an isomer of the E4(4H) FeMo-co metal-ion core that is formed prior to E4(H2;2H) and is thought to be created by breaking an Fe-SH bond, thus further integrating the calculational and structural studies into the experimentally determined mechanism by which nitrogenase is activated to cleave the N≡N triple bond.

Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism To Achieve N2 Reduction.

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) ("Janus") state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2 re with N2 binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (kHP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.