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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Cryo-EM structure of a methanogen nitrogenase-PII protein supercomplex

Abstract Nitrogenases are metalloenzymes that catalyze the reduction of atmospheric dinitrogen to ammonia, sustaining the global nitrogen cycle. While bacterial nitrogenase has been extensively characterized, the architecture and regulation of archaeal nitrogenases remain unknown despite longstanding evidence of nitrogen fixation in methanogens. Here we report a 3.1 Å cryo-electron microscopy structure of a native nitrogenase–PII protein supercomplex from Methanosarcina acetivorans. The structure reveals an unprecedented assembly of three NifDK heterotetramers bridged by six NifI1,2 heterotrimeric PII complexes, which sterically block NifH association and lock the enzyme in an inactive state. The NifI complexes display asymmetric binding of ADP and 2-oxoglutarate, coupling nitrogenase inhibition directly to cellular energy and nitrogen status. Addition of 2-oxoglutarate and ATP releases the NifI complexes, stimulating a threefold increase in NifDK activity in vitro. This higher-order architecture uncovers a previously unrecognized regulatory strategy in methanogens, in which PII proteins drive nitrogenase oligomerization to control activity. The discovery that nitrogenase activity may be modulated through direct assembly into higher-order structures opens new avenues for exploring nitrogenase evolution, regulation, and biotechnological applications. One sentence summary Discovery of a nitrogenase-PII protein supercomplex in methanogens, uncovering a metabolite-gated assembly mechanism for nitrogenase inhibition.

Nitrogenase, Electron Transfer, Methanogen↗
DOE OSTI2026

Predicting compatibility between ferredoxins and the Fe protein of nitrogenase using in silico protein modeling

Biological nitrogen fixation is the process by which certain bacteria and archaea use the enzyme nitrogenase to reduce atmospheric nitrogen into bioavailable ammonium. Engineering non‐nitrogen‐fixing organisms, like plants, to use nitrogenase could reduce dependency on synthetic fertilizer and mitigate the environmental impacts of industrial fertilizer production. However, nitrogenase activity requires delivery of reducing power by small electron carrying proteins known as ferredoxins and flavodoxins, and successfully engineering nitrogenase into new systems will require a mechanistic understanding of electron delivery by these proteins. Most organisms often have multiple ferredoxins, raising the question of which ferredoxin can support nitrogenase activity. The purpose of this study is to gain insight into how we can predict which ferredoxin is compatible with the Fe protein, the component of nitrogenase that interacts with ferredoxin or flavodoxin. Our in silico protein–protein docking simulations reveal that most ferredoxins and flavodoxins involved in nitrogen fixation have the shortest distance (≤10 Å) between their redox cofactor and the [4Fe‐4S] cluster of the Fe protein. We found shorter cofactor distance contributes to faster intermolecular electron tunneling rates. Bacterial ferredoxins that play a role in nitrogen fixation also exhibit more complementary interactions with the Fe protein than bacterial and plant ferredoxins not involved in this process. Heterologous expression of a set of ferredoxins from both nitrogen‐fixing and non‐nitrogen‐fixing bacteria in the diazotroph Rhodopseudomonas palustris supports our model‐derived prediction that shorter distances between the electron‐carrying cofactors favor nitrogenase compatibility. These findings offer a framework to predict and potentially enhance ferredoxin–nitrogenase compatibility, which will help to improve our ability to engineer nitrogen fixation into non‐nitrogen‐fixing organisms like plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Proton transfer during reduction of the catalytic metallo-cofactors of the three nitrogenase isozymes

Nitrogenase catalyzes biological nitrogen fixation, the conversion of atmospheric N 2 into bioavailable ammonia. The three nitrogenase isozymes—Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase—utilize catalytic cofactors distinguished by their metal composition (Fe 7 M, M = Mo, V, or Fe; denoted FeM-co). Their catalytic cycles involve stepwise addition of 8[e−/H+] to FeM-co, generating intermediates designated E n , where n is the number of [e − /H + ] delivered. The electron-transfer has been extensively characterized, but the proton delivery has not. Here, we investigate [e − /H + ] delivery during early-stage conversions, primarily E 0 → E 1 (H), for each of the three nitrogenases, using as reductants γ-ray-generated thermolyzed, mobile electrons at 77 K, and radiation-generated solvent radicals during subsequent annealing to higher temperatures. Our results show E 0 → E 1 (H) conversion differs among the three MFe-proteins. The FeMo-co of MoFe-protein accepts an electron (ET) during 77 K γ-irradiation, but proton transfer (PT) to generate E 1 (H) is only enabled by conformational or thermodynamic activation upon cryoannealing to ∼200 K(ET/PT). For VFe-protein, E 1 (H) forms during annealing at-and-above 210 K by electron-transfer to FeV-co from radicals through proton-coupled electron transfer (PCET), which too is enabled by activated proton transfer. FeFe-protein differs in directly exhibiting delivery of protons at 77 K, which together with the mobile electrons react to form E 1 (H). This could well occur by PCET at 77 K, but does not preclude the possibility of sequential 77 K electron/proton transfer (ET/PT). In addition, 450 nm photolysis reveals the E 1 (H) state of FeV-co, like that of FeFe-co, contains a hydride bound to a formally oxidized cofactor. The mechanistic differences observed here provide a contribution towards understanding the sources of catalytic differences among the three nitrogenase isozymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

ModE Regulates Alternative Nitrogenase Expression in the Methanogen Methanosarcina acetivorans

ABSTRACT All methanogens that can fix nitrogen use molybdenum (Mo) nitrogenase. Some methanogens, including Methanosarcina acetivorans , also contain alternative vanadium‐ and iron‐nitrogenases, encoded by the vnf and anf operons, respectively. These nitrogenases are produced when there is insufficient Mo to support Mo‐nitrogenase activity. The factors that control the expression of the alternative nitrogenases in response to Mo availability are unknown in methanogens. Here we show that ModE is the regulator that represses transcription of the vnf and anf operons in M. acetivorans when cells are grown with Mo. CRISPRi repression of modE results in a significant increase in the transcription of the vnf and anf operons as well as the detection of Fe‐nitrogenase during nitrogen fixation in the presence of Mo. Gel shift assays with recombinant ModE demonstrated that ModE binds a specific sequence motif upstream of the vnf and anf operons, as well as other genes and operons related to nitrogen fixation and Mo transport. However, purified ModE does not contain Mo, and the addition of Mo does not alter the affinity of ModE for DNA, indicating M. acetivorans ModE may not directly bind Mo. This study shows that ModE is the primary Mo‐responsive regulator of alternative nitrogenase expression in M. acetivorans , but other factor(s) are likely involved in directly sensing Mo.

Chanderban, Melissa [Department of Biological Scie↗
DOE OSTI2025

Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen-fixing photoautotrophs

ABSTRACT The nitrogenase gene cluster of unicellular diazotrophic cyanobacteria, such asCyanothece, is frequently selected by nature for nitrogen-fixing partnerships with eukaryotic phototrophs. The essential cluster components that confer an advantage in such partnerships remain underexplored. To use this cluster for the development of synthetic, phototrophic nitrogen-fixing systems, a thorough and systematic analysis of its constituent genes is necessary. An initial effort to assess the possibility of engineering this cluster into non-diazotrophic phototrophs led to the generation of aSynechocystis6803 strain with significant nitrogenase activity. In the current study, a refactoring approach was taken to determine the dispensability of the non-structural genes in the cluster and define a minimal gene set for constructing a functional nitrogenase for phototrophs. Using a bottom-up strategy, thenifgenes fromCyanothece51142 were re-organized to form new operons. The genes were then seamlessly removed to determine their essentiality in the nitrogen fixation process. We demonstrate that besides the structural genesnifHDK,nifBSUENPVZTXW, as well ashesAB, are important for optimal nitrogenase function in a phototroph. We also show that optimal expression of these genes is crucial for efficient nitrogenase activity. Our findings provide a solid foundation for generating synthetic systems that will facilitate solar-powered conversion of atmospheric nitrogen into nitrogen-rich compounds, a stride toward a greener world. IMPORTANCE Integrating nitrogen fixation genes into various photosynthetic organisms is an exciting strategy for converting atmospheric nitrogen into nitrogen-rich products in a green and energy-efficient way. In order to facilitate this process, it is essential that we understand the fundamentals of the functioning of a prokaryotic nitrogen-fixing machinery in a non-diazotrophic, photoautotrophic cell. This study examines a nitrogenase gene cluster that has been naturally selected on multiple occasions for a nitrogen-fixing partnership by eukaryotic photoautotrophs and provides a basic blueprint for designing a photosynthetic organism with nitrogen-fixing ability.

Microbiology↗
DOE OSTI2025

Reductant‐ or Light‐Driven ATP‐Independent Reduction of CO 2 by Nitrogenase MoFe Protein

Nitrogenase is a versatile metalloenzyme that activates and reduces small molecules like N 2 , CO, and CO 2 into value-added chemicals at ambient conditions. Previously, it is shown that the Mo-nitrogenase could reduce CO 2 to CO, but not to hydrocarbons, in an ATP-dependent reaction. Here, it is reported that the ability of the catalytic component of Mo-nitrogenase (MoFe protein) enables ATP-independent reduction of CO 2 to up to C 4 hydrocarbons in room-temperature reactions driven by a chemical reductant (Eu II –DTPA) or visible light (via CdS@ZnS (CZS) quantum dots). Moreover, an opposite deuterium isotope effect is observed on the Eu II –DTPA driven reactions of CO 2 reduction by MoFe protein and its V-counterpart (VFe protein), in that the former displays higher activities in H 2 O, and the latter displays higher activities in D 2 O. Furthermore, these results provide an important foundation for further mechanistic exploration of the nitrogenase-enabled, atypical Fischer–Tropsch type reaction that uses CO 2 instead of CO as a substrate; moreover, they serves as a potential template for the future development of nitrogenase-based applications that effectively recycle the greenhouse gas CO 2 into valuable fuel products.

C-C coupling↗
DOE OSTI2026

Mechanistic Insights into Dinitrogen Reduction to Ammonia in Light-Controlled Nanocrystal:Nitrogenase Complexes

Developing systems that can efficiently capture photon energy and convert this energy into fuels and chemicals requires understanding how to assemble molecular components with diverse functions into complete systems possessing selectivity and efficiency in directing charge carriers to catalytic reactions. There are many challenges to achieving this goal. One promising approach is the development of hybrid systems that combine semiconductor nanocrystals (NCs) for light capture and enzymes as efficient catalysts. Such biohybrid systems capitalize on the tunable electronic and optical properties of NCs while leveraging the unmatched specificity and efficiency of enzymes in catalyzing chemical reactions, thereby offering opportunities to surpass the limitations of each component alone. Here, we focus on recent progress in developing a biohybrid system that combines CdS NCs for photon capture with the enzyme nitrogenase to accomplish light-driven dinitrogen (N 2 ) reduction to ammonia (NH 3 ). Integrating light-harvesting materials with biological catalysts requires a deep understanding of NC properties, protein stability, and electron transfer (ET), making it an inherently multidisciplinary problem. The reduction of N 2 to NH 3 is a challenging reaction, with a high demand in both agriculture and industrial chemical production. This reaction is intrinsically energy intensive, due to the need to activate the N≡N triple bond. The current standard industrial approach to N 2 reduction, the Haber−Bosch reaction, obtains the necessary energy input from fossil fuels, whereas biological systems capable of N 2 reduction utilize the hydrolysis of ATP as their energy source. Replacing these costly, energy-intensive inputs with renewable light energy represents a critical step toward sustainable NH 3 production. Recent progress has demonstrated that semiconductor CdS NCs can be coupled to the catalytic component of nitrogenase, the MoFe protein, to form a biohybrid CdS NC:MoFe protein complex, enabling light-driven N 2 reduction rather than energy input from fossil fuels or ATP. This illustrates how inorganic NCs can functionally replace the natural Fe protein partner, yielding a biohybrid catalyst that enables controlled electron delivery and provides not only light-driven NH 3 production but also new approaches for probing enzyme catalytic function. The CdS NC:MoFe protein biohybrid system enables light-initiated electron delivery at ambient temperature, as well as temperatures below freezing, allowing for stabilization and spectroscopic characterization of key reaction intermediates. These findings highlight how photochemical biohybrids can serve as both functional catalysts and mechanistic probes. Beyond studies of the nitrogenase mechanism, studies of the CdS NC:MoFe system reveal how variables such as NC size, electrostatic binding interactions, and sacrificial electron donors (SEDs) govern complex stability, charge transfer efficiency, and catalytic performance. In addition, studies of nitrogenase and the high activation barrier for N 2 reduction are enabling investigations of new and interesting questions regarding the properties and limitations of NC biocatalysis. In this Account, we describe the key features of CdS NC:MoFe protein biohybrids and the parameters for optimal light-driven N 2 reduction, and how controlling ET with light illuminates the path to new insights into the nitrogenase mechanism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Sulfite Is Not Required for N 2 Reduction Catalyzed by Mo-Nitrogenase

Mo-nitrogenase catalyzes the reduction of dinitrogen (N 2 ) to two ammonia (NH 3 ) at the active-site FeMo-cofactor. Substrate activation requires the accumulation of three or four electrons and protons as two Fe-bound hydrides and is coupled to obligatory H 2 release through reductive hydride elimination. Subsequent delivery of four or five additional electrons and protons to the bound N 2 yields two NH 3 molecules. Increasing evidence suggests that at least one belt sulfide within FeMo-cofactor is dynamically involved in the catalytic cycle. A recent report further proposed that sulfite (SO 3 2− ) is required for N 2 reduction, with sulfite binding required for NH 3 release and a subsequent six-electron reduction of the bound sulfite to regenerate the resting cofactor. To test this proposal, we conducted turnover studies of Mo-nitrogenase under sulfite-free conditions using a reduced viologen as reductant and protein preparations devoid of dithionite or sulfite. Under these conditions, nitrogenase effectively catalyzed both N 2 reduction and proton reduction, exhibiting steady-state turnover under N 2 for 6 min, with a turnover number exceeding 150, approaching that observed with dithionite as reductant. The same H 2 -formed/N 2 -reduced ratio was observed whether dithionite or the viologen species was used as reductant. Further, EPR spectroscopic analyses showed that the FeMo-cofactor returned to its resting state after multiple catalytic cycles in the absence of sulfite. Finally, physiological bypass of sulfite formation does not affect the capacity for diazotrophic growth of the model nitrogen-fixing organism Azotobacter vinelandii . Finally, these results demonstrate that sulfite is not required for Mo-nitrogenase-catalyzed N 2 reduction either in vitro or in vivo .

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN

The maturation and installation of the active site metal cluster (FeMo-co, Fe 7 S 9 CMo- R -homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe 8 S 9 C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. However, some putative diazotrophic bacteria, like Roseiflexus sp. RS-1, lack the nifEN genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the Roseiflexus NifH, NifB, and apo-NifDK proteins produced in Escherichia coli are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The E. coli expressed NifDK RS contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDK RS ). Apo-NifDK RS could be activated for N 2 reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDK RS plus E. coli produced NifB RS and NifH RS were sufficient to yield active NifDK RS when incubated with the necessary substrates (homocitrate, molybdate, and S -adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The E. coli produced NifH RS and NifB RS proteins were independently shown to be functional. The reconstituted NifDK RS demonstrated reduction of N 2 , protons, and acetylene in ratios observed for Azotobacter vinelandii NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifH RS , NifB RS , and apo-NifDK RS .

Payá-Tormo, Lucía↗
DOE OSTI2025

Heterologous synthesis of a simplified nitrogenase analog in Escherichia coli

The heterologous synthesis of a nitrogen-fixing system in a non-diazotrophic organism is a long-sought-after goal because of the crucial importance of nitrogenase for agronomy, energy, and the environment. Here, we report the heterologous synthesis of a two-component nitrogenase analog fromAzotobacter vinelandii, which consists of the reductase component (NifH) and the cofactor maturase (NifEN), inEscherichia coli. Metal, electron paramagnetic resonance, and activity analyses verify the cluster composition and functional competence of the heterologously expressed NifH and NifEN. Nuclear magnetic resonance, nanoscale secondary ion mass spectrometry, and growth experiments further illustrate the ability of the NifH/NifEN system to reduce N 2 and incorporate the reduced N into the cellular mass. These results establish NifEN/NifH as a simplified nitrogenase analog that could be optimized and engineered to facilitate transgenic expression and biotechnological adaptations of this important metalloenzyme.

Science & Technology - Other Topics↗
DOE OSTI2025

Toward a Unified Kinetic Model of Nitrogenase Catalysis

The microbial enzyme nitrogenase catalyzes the MgATP-dependent reduction of N 2 to 2NH 3 , a transformation central to the global nitrogen cycle. While the canonical Thorneley−Lowe (TL) kinetic model has long served as a mechanistic framework, it does not incorporate several recent insights. Here, we present an updated kinetic model for Monitrogenase that incorporates these new findings. A significant insight is that electron transfer (ET) from the reduced Fe protein to the FeMo-cofactor is gated by MgATP-dependent conformational transitions and can be described as a probabilistic event that is dependent on the ligand bound to the active-site metallocofactor. The updated kinetic model quantitatively reproduces steady-state product formation rates across a broad range of experimental conditions, yielding revised estimates for key rate constants. It is demonstrated that under N 2 turnover, the probability of productive ET to the active site decreases by ∼60%, resulting in a significant fraction of Fe protein cycles that are unproductive for electron delivery. This mechanistic feature explains the observed rate limitation in N 2 reduction and implies a revised minimum energetic cost of approximately 25 MgATP per N 2 reduced. Integrating these new features into the revised kinetic model provides a more complete and usable foundation for understanding nitrogenase catalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Bacterial synergies amplify nitrogenase activity in diverse systems

Endophytes are microbes living within plant tissue, with some having the capacity to fix atmospheric nitrogen in both a free-living state and within their plant host. They are part of a diverse microbial community whose interactions sometimes result in a more productive symbiosis with the host plant. Here, we report the co-isolation of diazotrophic endophytes with synergistic partners sourced from two separate nutrient-limited sites. In the presence of these synergistic strains, the nitrogen-fixing activity of the diazotroph is amplified. One such partnership was co-isolated from extracts of plants from a nutrient-limited Hawaiian lava field and another from the roots of Populus trees on a nutrient-limited gravel bar in the Pacific Northwest. The synergistic strains were capable of increasing the nitrogenase activity of different diazotrophic species from other environments, perhaps indicating that these endophytic microbial interactions are common to environments where nutrients are particularly limited. Multiple overlapping mechanisms seem to be involved in this interaction. Though synergistic strains are likely capable of protecting nitrogenase from oxygen, another mechanism seems evident in both environments. The synergies do not depend exclusively on physical contact, indicating a secreted compound may be involved. This work offers insights into beneficial microbial interactions, providing potential avenues for optimizing inocula for use in agriculture.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Carbon monoxide chemistry of α-V70I Mo-nitrogenase: Evidence from EPR- and IR-monitored photolysis – or, what a difference a methyl makes

A critical step in the global nitrogen cycle is the conversion of dinitrogen into biologically accessible ammonia. In Nature this is accomplished by the nitrogenase (N 2 ase) family of enzymes. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction by N 2 ase, but it can also be a substrate of the enzyme, when it is catalytically reduced to hydrocarbons. Understanding the CO interactions with N 2 ases are thus relevant to both dinitrogen fixation and Fischer-Tropsch-like chemistry. Here, in this work, the interaction of CO with the α-V70I variant of Azotobacter vinelandii MoFe N 2 ase was investigated using electron paramagnetic resonance (EPR) and infrared (IR) monitored photolysis of bound CO under cryogenic conditions. This was supplemented by further analysis of stopped-flow Fourier transform IR (SF-FT-IR) data under turnover conditions. The α-V70I variant adds a single methyl group close to the FeMo-cofactor active site, and the results show that this inhibits and slows, but does not substantially chemically change, the binding of CO to the FeMocofactor. The EPR spectra of both the hi-CO and lo-CO states closely resemble those from the wild-type enzyme. Similarly, the SF-FT-IR spectra of CO inhibited α-V70I and wild-type enzyme are strikingly similar, showing only small shifts in band energies which allow better interpretation of the published wild-type spectra. The extra carbon does, however, impact and inhibit the photochemical release and migration of CO at cryogenic temperatures, resulting in novel CO-bound species. These include a product species, termed Lo-1*, which may involve CO photochemically migrating on the FeMo-cofactor.

Carbon monoxide↗
DOE OSTI2025

Pre-steady-state kinetics of nanocrystal:molybdenum nitrogenase biohybrids reveals hole-scavenging efficiency is critical to N 2 reduction

Molybdenum (Mo) nitrogenase is a two-component enzyme complex that catalyzes the reduction of dinitrogen to ammonia and protons to hydrogen gas. We have shown that electrons for dinitrogen reduction can be delivered photochemically to the catalytic MoFe protein component by cadmium sulfide (CdS) nanocrystals. In this study, we used electron paramagnetic resonance spectroscopy to measure the transient populations of catalytic intermediates. We fit the populations with a pre-steady-state kinetic model, which allowed us to distinguish between productive and non-productive reaction pathways and extract the rate constants for the reaction. Our results demonstrated that the rate of catalytic electron delivery into MoFe protein increased with the concentration of the sacrificial electron donor. This enabled electron delivery to exceed the rate of hydride protonation, a relaxation pathway that competes with N 2 binding. Thus, managing the balance between electron transfer and hole transfer reactions is required to achieve a kinetic regime that favors N 2 reduction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Electrochemical Characterization of Photo-Driven Hole-Scavenging by Cadmium Sulfide Quantum Dot-Nitrogenase Biohybrid Complexes

Light-driven biohybrid systems that couple semiconductor nanocrystals with enzymes offer a promising strategy for solar-to-chemical energy-conserving reduction reactions, yet are often limited by inefficient hole scavenging. Hole scavenging is critical for maintaining charge separation in the light-absorbing electron donor molecule and, thus, for sustaining catalytic turnover, making quantification essential to improving system efficiency. Here, we introduce a photoelectrochemical approach for real-time monitoring of hole scavenging in cadmium sulfide (CdS) quantum dot (QD)-nitrogenase MoFe protein biohybrids that catalyze the reduction of H+ and N2. Using hydroquinone (HQ) as the sacrificial electron donor (SED), oxidation of HQ to benzoquinone (BQ) by photogenerated valence-band holes is coupled to the electrochemical reduction of BQ at an electrode, enabling quantification of the hole-scavenging reaction via chronoamperometry. This approach provides a continuous, real-time readout of charge-transfer dynamics under illumination. Systematic variation of light intensity and donor concentration reveals that hole scavenging scales with photon flux and exhibits a non-linear dependence on SED concentration, with an optimum at intermediate HQ concentrations. These results establish chronoamperometry as a powerful tool for probing photoinduced charge transfer, advancing the quantitative understanding of hole-scavenging in biohybrid systems, and offering a generalizable framework for optimizing solar-driven biocatalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

EPR and 31 P ENDOR Characterization of Pseudo-Jahn–Teller Dynamics and N 2 Activation in Functional Nitrogenase Models, P 3 E M(N 2 ) (M = Fe, Co; E = Si, B, C)

Here, the nominally trigonal, pseudo-Jahn-Teller (PJT)-active, S = ½ N 2 -bound transition-metal complexes, P 3 E M(N 2 ), M = Fe, Co, with three in-plane phosphine-ligands and axial donors, E = Si, B, C, include functional nitrogenase models that catalyze reduction of N 2 to NH 3 . We applied EPR, 31 P ENDOR spectroscopy and DFT computations to characterize the PJT-induced distortions of four selected P 3 E M(N 2 ), revealing how the metal-ion and axial ligand E together tune both PJT dynamics and N 2 activation for reduction. Comparisons reveal an unrecognized correlation between PJT distortion, M-E bond elasticity, and N 2 activation, providing guidelines for designing bioinspired N 2 -reduction catalysts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Trafficking of a nitrogenase FeMo-cofactor assembly intermediate

The maturation of the unique FeMo-cofactor of molybdenum nitrogenase is a multistep process requiring the sequential action of a series of maturase complexes. As a final step, the NifEN complex forms FeMo-cofactor from the precursor NifB-co, also called L-cluster, through replacement of an apical iron ion by molybdenum and the attachment of an organic homocitrate ligand. NifB-co is delivered by a small cofactor chaperone, NifX, and initially bound near the surface of the maturase NifEN. Here, we report high-resolution cryo-electron microscopy structures of NifEN in complex with NifX, showing NifB-co binding to NifEN in full detail, capturing both interacting partners in the act of cluster transfer. In a dynamic transfer complex, the large metal cluster is coordinated by single residues from both NifEN and NifX. In silico studies concur with these structures but suggest a third, internal conversion site where cluster maturation likely takes place.

metalloproteins↗