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Nitrogenases are the only enzymes that activate the strong triple bond in N 2 . The mechanism for the activation has been very difficult to determine in spite of decades of work. In previous modeling studies it has been suggested that the mechanism for nitrogen activation starts out by four pre-activation steps (A 0 -A 4 ) before catalysis. That suggestion led to excellent agreement with experimental Elecrtron Paramagnetic Resonance (EPR) observations in the step where N 2 becomes protonated (E 4 ). An important part of the pre-activation is that a sulfide is released. In the present paper, the details of the pre-activation are modeled, including the release of the sulfide. Several possible transition states for the release have been obtained. An A 4 (E 0 ) state is reached which is very similar to the E 4 state. For completeness, the steps going back from A 4 (E 0 ) to A 0 after catalysis are also modeled, including the insertion of a sulfide., (© 2024 The Author(s). Journal of Computational Chemistry published by Wiley Periodicals LLC.)

Biological nitrogen fixation, performed by the enzyme nitrogenase, supplies nearly 50% of the bioavailable nitrogen pool on Earth, yet the structural nature of the enzyme intermediates involved in this cycle remains ambiguous. Here we present four high resolution cryoEM structures of the nitrogenase MoFe-protein, sampled along a time course of alkaline reaction mixtures under an acetylene atmosphere. This series of structures reveals a sequence of salient changes including perturbations to the inorganic framework of the FeMo-cofactor; depletion of the homocitrate moiety; diminished density around the S2B belt sulfur of the FeMo-cofactor; rearrangements of cluster-adjacent side chains; and the asymmetric displacement of the FeMo-cofactor. We further demonstrate that the nitrogenase associated factor T protein can recognize and bind an alkaline inactivated MoFe-protein in vitro. These time-resolved structures provide experimental support for the displacement of S2B and distortions of the FeMo-cofactor at the E 0 -E 3 intermediates of the substrate reduction mechanism, prior to nitrogen binding, highlighting cluster rearrangements potentially relevant to nitrogen fixation by biological and synthetic clusters., Competing Interests: Competing interests: All authors declare no competing interests., (© 2024. The Author(s).)

Nitrogenases catalyze dinitrogen (N 2 ) fixation to ammonia (NH 3 ). While these enzymes are highly sensitive to deactivation by molecular oxygen (O 2 ) they can be produced by obligate aerobes for diazotrophy, necessitating a mechanism by which nitrogenase can be protected from deactivation. In the bacterium Azotobacter vinelandii, one mode of such protection involves an O 2 -responsive ferredoxin-type protein ("Shethna protein II", or "FeSII") which is thought to bind with Mo-dependent nitrogenase's two component proteins (NifH and NifDK) to form a catalytically stalled yet O 2 -tolerant tripartite protein complex. This protection mechanism has been reported for Mo-nitrogenase, however, in vitro assays with V-nitrogenase suggest that this mechanism is not universal to the three known nitrogenase isoforms. Here we report that the reductase of the V-nitrogenase (VnfH) can engage in this FeSII-mediated protection mechanism when cross-coupled with Mo-nitrogenase NifDK. Interestingly, the cross-coupling of the Mo-nitrogenase reductase NifH with the V-nitrogenase VnfDGK protein does not yield such protection., (© 2024 Wiley-VCH GmbH.)

The production of ammonia (NH 3 ) from nitrogen sources involves competitive adsorption of different intermediates and multiple electron and proton transfers, presenting grand challenges in catalyst design. In nature nitrogenases reduce dinitrogen to NH 3 using two component proteins, in which electrons and protons are delivered from Fe protein to the active site in MoFe protein for transfer to the bound N 2 . We draw inspiration from this structural enzymology, and design a two-component metal-sulfur-carbon (M-S-C) catalyst composed of sulfur-doped carbon-supported ruthenium (Ru) single atoms (SAs) and nanoparticles (NPs) for the electrochemical reduction of nitrate (NO 3 - ) to NH 3 . The catalyst demonstrates a remarkable NH 3 yield rate of ~37 mg L -1  h -1 and a Faradaic efficiency of ~97 % for over 200 hours, outperforming those consisting solely of SAs or NPs, and even surpassing most reported electrocatalysts. Our experimental and theoretical investigations reveal the critical role of Ru SAs with the coordination of S in promoting the formation of the HONO intermediate and the subsequent reduction reaction over the NP-surface nearby. Such process results in a more energetically accessible pathway for NO 3 - reduction on Ru NPs co-existing with SAs. This study proves a better understanding of how M-S-Cs act as a synthetic nitrogenase mimic during ammonia synthesis, and contributes to the future mechanism-based catalyst design., (© 2024 Wiley-VCH GmbH.)

Nitrogenase converts nitrogen in the air to ammonia. It is often regarded as the second most important enzyme in nature after photosystem II. The mechanism for how nitrogenase is able to perform the difficult task of cleaving the strong bond in N 2 is debated. It is known that for every electron that is donated to N 2 , two ATP are hydrolyzed. In the experimentally suggested mechanism, the activation occurs after four reductions of the ground state, but there is no suggestion for how the enzyme uses the hydrolysis energy to perform catalysis. In the theoretical mechanism, it is suggested that hydrolysis is used to reduce the electron donor. In previous papers, the steps leading to the activation of N 2 in the so-called E 4 state has been investigated, using both the experimental and theoretical mechanism, showing that only the theoretical one leads to agreement with EPR observations for E 4 . In the present paper, the four steps following E 4 , leading to the release of two ammonia molecules, are described using the same methodology as used in the previous studies.

From the reduction of dinitrogen to the oxidation of water, the chemical transformations catalysed by metalloenzymes underlie global geochemical and biochemical cycles. These reactions represent some of the most kinetically and thermodynamically challenging processes known and require the complex choreography of the fundamental building blocks of nature, electrons and protons, to be carried out with utmost precision and accuracy. The rate-determining step of catalysis in many metalloenzymes consists of a protein structural rearrangement, suggesting that nature has evolved to leverage macroscopic changes in protein molecular structure to control subatomic changes in metallocofactor electronic structure. The proton-coupled electron transfer mechanisms operative in nitrogenase, photosystem II and ribonucleotide reductase exemplify this interplay between molecular and electronic structural control. We present the culmination of decades of study on each of these systems and clarify what is known regarding the interplay between structural changes and functional outcomes in these metalloenzyme linchpins., (© 2024. Springer Nature Limited.)

The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N 2 ) fixation. While NifHs have been extensively studied in bacteria, structural information about their archaeal counterparts is limited. Archaeal NifHs are considered more ancient, particularly those from Methanococcales, a group of marine hydrogenotrophic methanogens, which includes diazotrophs growing at temperatures near 92 °C. Here, we structurally and biochemically analyse NifHs from three Methanococcales, offering the X-ray crystal structures from meso-, thermo-, and hyperthermophilic methanogens. While NifH from Methanococcus maripaludis (37 °C) was obtained through heterologous recombinant expression, the proteins from Methanothermococcus thermolithotrophicus (65 °C) and Methanocaldococcus infernus (85 °C) were natively purified from the diazotrophic archaea. The structures from M. thermolithotrophicus crystallised as isolated exhibit high flexibility. In contrast, the complexes of NifH with MgADP obtained from the three methanogens are superposable, more rigid, and present remarkable structural conservation with their homologues. They retain key structural features of P-loop NTPases and share similar electrostatic profiles with the counterpart from the bacterial model organism Azotobacter vinelandii. In comparison to the NifH from the phylogenetically distant Methanosarcina acetivorans, these reductases do not cross-react significantly with Mo-nitrogenase from A. vinelandii. However, they associate with bacterial nitrogenase when ADP· AlF 4 - is added to mimic a transient reactive state. Accordingly, detailed surface analyses suggest that subtle substitutions would affect optimal binding during the catalytic cycle between the NifH from Methanococcales and the bacterial nitrogenase, implying differences in the N 2 -machinery from these ancient archaea., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg 2+ , can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the N 2 -reducing enzyme nitrogenase. The Proterozoic ancestor is predicted to be ~540-2,300 million years old, post-dating the Great Oxidation Event. Growth rates under nitrogen-fixing conditions are ~80% of those of wild type in Azotobacter vinelandii . In the extant enzyme, the hydrolysis of two MgATP is coupled to electron transfer to support substrate reduction. The ancestor has a strict requirement for ATP with no other nucleotide triphosphate analogs (GTP, ITP, and UTP) supporting activity. Alternative divalent metal ions (Fe 2+ , Co 2+ , and Mn 2+ ) support activity with ATP but with diminished activities compared to Mg 2+ , similar to the extant enzyme. Additionally, it is shown that the ancestor has an identical efficiency in ATP hydrolyzed per electron transferred to the extant of two. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme.IMPORTANCELife depends on energy-carrying molecules to power many sustaining processes. There is evidence that these molecules may predate the rise of life on Earth, but how and when these dependencies formed is unknown. The resurrection of ancient enzymes provides a unique tool to probe the enzyme's function and usage of energy-carrying molecules, shedding light on their biochemical origins. Through experimental reconstruction, this research investigates the ancestral dependence of a nitrogen-fixing enzyme on the energy carrier ATP, a requirement for function in the modern enzyme. We show that the resurrected ancestor does not have generalist nucleotide specificity. Rather, the ancestor has a strict requirement for ATP, like the modern enzyme, with similar function and efficiency. The findings elucidate the early-evolved necessity of energy-yielding molecules, delineating their role in ancient biochemical processes. Ultimately, these insights contribute to unraveling the intricate tapestry of evolutionary biology and the origins of life-sustaining dependencies., Competing Interests: The authors declare no conflict of interest.

We have studied whether dissociation of the S2B sulfide ligand from one of its two coordinating Fe ions may affect the later parts of the reaction mechanism of nitrogenase. Such dissociation has been shown to be favourable for the E 2 -E 4 states in the reaction mechanism, but previous studies have assumed that S2B either remains bridging or has fully dissociated from the active-site FeMo cluster. We employ combined quantum mechanical and molecular mechanical (QM/MM) calculations with two density-functional theory methods, r 2 SCAN and TPSSh. To make dissociation of S2B possible, we have added a proton to this group throughout the reaction. We study the reaction starting from the E 4 state with N 2 H 2 bound to the cluster. Our results indicate that half-dissociation of S2B is unfavourable in most steps of the reaction mechanism. We observe favourable half-dissociation of S2B only when NH or NH 2 is bound to the cluster, bridging Fe2 and Fe6. However, the former state is most likely not involved in the reaction mechanism and the latter state is only an intermittent intermediate of the E 7 state. Therefore, half-dissociation of S2B seems to play only a minor role in the later parts of the reaction mechanism of nitrogenase. Our suggested mechanism with a protonated S2B is alternating (the two N atoms of the substrate is protonated in an alternating manner) and the substrate prefers to bind to Fe2, in contrast to the preferred binding to Fe6 observed when S2B is unprotonated and bridging Fe2 and Fe6.

Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments., (© 2024. Springer Nature Limited.)

Nitrogen fixation is a key process that sustains life on Earth. Nitrogenase is the sole enzyme capable of fixing nitrogen under ambient conditions. Extensive research efforts have been dedicated to elucidating the enzyme mechanism and its artificial activation through high applied voltage, photochemistry, or strong reducing agents. Harnessing light irradiation to minimize the required external bias can lower the process's high energy investment. Herein, we present the development of photo-bioelectrochemical cells (PBECs) utilizing BiVO 4 /CoP or CdS/NiO photoanodes for nitrogenase activation toward N 2 fixation. The constructed PBEC based on BiVO 4 /CoP photoanode requires minimal external bias (200 mV) and suppresses O 2 generation that allows efficient activation of the nitrogenase enzyme, using glucose as an electron donor. In a second developed PBEC configuration, CdS/NiO photoanode was used, enabling bias-free activation of the nitrogenase-based cathode to produce 100 μM of ammonia at a faradaic efficiency (FE) of 12%. The ammonia production was determined by a commonly used fluorescence probe and further validated using 1 H-NMR spectroscopy. The presented PBECs lay the foundation for biotic-abiotic systems to directly activate enzymes toward value-added chemicals by light-driven reactions., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Coenzyme F 430 is a nickel-containing tetrapyrrole, serving as the prosthetic group of methyl-coenzyme M reductase in methanogenic and methanotrophic archaea. During coenzyme F 430 biosynthesis, the tetrapyrrole macrocycle is reduced by the nitrogenase-like CfbC/D system consisting of the reductase component CfbC and the catalytic component CfbD. Both components are homodimeric proteins, each carrying a [4Fe-4S] cluster. Here, the ligands of the [4Fe-4S] clusters of CfbC 2 and CfbD 2 were identified revealing an all cysteine ligation of both clusters. Moreover, the midpoint potentials of the [4Fe-4S] clusters were determined to be -256 mV for CfbC 2 and -407 mV for CfbD 2 . These midpoint potentials indicate that the consecutive thermodynamically unfavorable 6 individual "up-hill" electron transfers to the organic moiety of the Ni 2+ -sirohydrochlorin a,c-diamide substrate require an intricate interplay of ATP-binding, hydrolysis, protein complex formation and release to drive product formation, which is a common theme in nitrogenase-like systems., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Nitrogenase plays a key role in the global nitrogen cycle; yet, the evolutionary history of nitrogenase and, particularly, the sequence of appearance between the homologous, yet distinct NifDK (the catalytic component) and NifEN (the cofactor maturase) of the extant molybdenum nitrogenase, remains elusive. Here, we report the ability of NifEN to reduce N 2 at its surface-exposed L-cluster ([Fe 8 S 9 C]), a structural/functional homolog of the M-cluster (or cofactor; [( R -homocitrate)MoFe 7 S 9 C]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N 2 reduction. These observations, coupled with phylogenetic, ecological, and mechanistic considerations, lead to the proposal of a NifEN-like, L-cluster-carrying protein as an ancient nitrogenase, the exploration of which could shed crucial light on the evolutionary origin of nitrogenase and related enzymes.

Biological nitrogen fixation is crucial for agriculture and improving fertilizer efficiency, but organic fertilizers in enhancing this process remain debated. Here, we investigate the impact of organic fertilizers on biological nitrogen fixation through experiments and propose a new model where bacterial interactions with complex carbon sources enhance nitrogen fixation. Field experiments showed that adding organic fertilizers increased the nitrogenase activity by 57.85%. Subculture experiments revealed that organic fertilizer addition enriched genes corresponding to complex carbon and energy metabolism, as well as nifJ involved in electron transfer for nitrogenase. It also enhanced bacterial interactions and enhanced connectors associated with complex carbon degradation. Validation experiments demonstrated that combinations increased nitrogenase activity by 2.98 times compared to the single. Our findings suggest that organic fertilizers promoted nitrogen fixation by enhancing microbial cooperation, improved the degradation of complex carbon sources, and thereby provided utilizable carbon sources, energy, and electrons to N-fixers, thus increasing nitrogenase activity and nitrogen fixation.

Nitrogenase reduces N 2 to NH 3 at its active-site cofactor. Previous studies of an N 2 -bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N 2 species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N 2 reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N 2 species on Av1* remains elusive. Here we report a study of ATP-independent, Eu II /SO 3 2- -driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D 2 -derived D by Av1* into the products of C 2 H 2 - and H + -reduction, and decreased formation of NH 3 by Av1* concomitant with the release of N 2 under H 2 ; moreover, they reveal a strict dependence of these activities on SO 3 2- . These observations point to the presence of distinct N 2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N 2 via belt-sulfur mobilization., (© 2024 Wiley-VCH GmbH.)

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 E 0 . Two of these species were preliminary assigned to hydrogenated E 2 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 33 S and 77 Se 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., (© 2024. The Author(s).)

In converting N 2 to NH 3 the enzyme nitrogenase utilises 8 electrons and 8 protons in the complete catalytic cycle. The source of the electrons is an Fe 4 S 4 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 [CFe 7 S 9 Mo(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 H 3 O + 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.

Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in environmental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The molybdenum-iron protein (MoFeP), a component of the enzyme nitrogenase, catalyzes the reduction of an array of small molecules, including N 2 to NH 3 . In microorganisms, during the catalytic cycle, MoFeP receives electrons from the obligate biological redox partner iron protein (FeP) in a process coupled to the hydrolysis of two MgATP per one electron transferred. Despite the favorable redox properties of the cofactors, the requirement of the MgATP hydrolysis significantly decreases the energy efficiency of MoFeP. Therefore, remarkable efforts have been devoted to electrochemically activating MoFeP without FeP and MgATP. Previously, MoFeP was adsorbed on an electrode surface and revealed a slow catalysis with and without electron transfer mediators. However, enzyme adsorption can cause conformational and structural changes in a fragile protein molecule and alter its catalytic activity. In this work, MoFeP was electrochemically studied in solution. Various electron transfer mediators with potentials ranging from -0.3 V to -1 V (vs. NHE) were examined with MoFeP using cyclic voltammetry. No significant catalytic activity of the MoFeP was observed with any of the tested mediators. This indicates that efficient electrochemical activation of MoFeP cannot be achieved exclusively by increasing the driving force between the MoFeP redox cofactors and an electron donor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The mechanism for N 2 activation in the E 4 state of nitrogenase was investigated by model calculations. In the experimentally suggested mechanism, the E 4 state is obtained after four reductions to the ground state. In a recent theoretical study, results for a different mechanism have been found in excellent agreement with available Electron Paramagnetic Resonance (EPR) experiments for E 4 . The two hydrides in E 4 leave as H 2 concertedly with the binding of N 2 . The mechanism suggested differs from the experimentally suggested one by a requirement for four activation steps prior to catalysis. In the present study, the experimentally suggested mechanism is studied using the same methods as those used in the previous study on the theoretical mechanism. The computed results make it very unlikely that a structure obtained after four reductions from the ground state has two hydrides, and the experimentally suggested mechanism does therefore not agree with the EPR experiments for E 4 . Another structure with only one hydride is here suggested to be the one that has been observed to bind N 2 after only four reductions of the ground state.

Nitrogenase is the only enzyme that can cleave the strong triple bond in N 2 , making nitrogen available for biological lifeforms. The active site is a MoFe 7 S 9 C cluster (the FeMo cluster) that binds eight electrons and protons during one catalytic cycle, giving rise to eight intermediate states E 0 -E 7 . It is experimentally known that N 2 binds to the E 4 state and that H 2 is a compulsory byproduct of the reaction. However, formation of H 2 is also an unproductive side reaction that should be avoided, especially in the early steps of the reaction mechanism (E 2 and E 3 ). Here, we study the formation of H 2 for various structural interpretations of the E 2 -E 4 states using combined quantum mechanical and molecular mechanical (QM/MM) calculations and four different density-functional theory methods. We find large differences in the predictions of the different methods. B3LYP strongly favours protonation of the central carbide ion and H 2 cannot form from such structures. On the other hand, with TPSS, r 2 SCAN and TPSSh, H 2 formation is strongly exothermic for all structures and E n and therefore need strict kinetic control to be avoided. For the E 2 state, the kinetic barriers for the low-energy structures are high enough to avoid H 2 formation. However, for both the E 3 and E 4 states, all three methods predict that the best structure has two hydride ions bridging the same pair of Fe ions (Fe2 and Fe6) and these two ions can combine to form H 2 with an activation barrier of only 29-57 kJ mol -1 , corresponding to rates of 7 × 10 2 to 5 × 10 7 s -1 , i.e. much faster than the turnover rate of the enzyme (1-5 s -1 ). We have also studied H-atom movements within the FeMo cluster, showing that the various protonation states can quite freely be interconverted (activation barriers of 12-69 kJ mol -1 ).

Nitrogenase is the only enzyme that catalyzes the reduction of nitrogen gas into ammonia. Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Many nitrogen fixing bacteria protect nitrogenase from CO inhibition using the protective protein CowN. This work demonstrates that a conserved glutamic acid residue near the C-terminus of Gluconacetobacter diazotrophicus CowN is necessary for its function. Mutation of the glutamic acid residue abolishes both CowN's protection against CO inhibition and the ability of CowN to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection by CowN. Overall, this work uncovers structural features in CowN that are required for its function and provides new insights into its nitrogenase binding and CO protection mechanism.

Nitrogenases are best known for catalyzing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO 2 ) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the three nitrogenase isozymes, the iron nitrogenase has the highest wild-type activity for the reduction of CO 2 , but the molecular architecture facilitating these activities has remained unknown. Here, we report a 2.35-Å cryogenic electron microscopy structure of the ADP·AlF 3 -stabilized iron nitrogenase complex from Rhodobacter capsulatus, revealing an [Fe 8 S 9 C-(R)-homocitrate] cluster in the active site. The enzyme complex suggests that the iron nitrogenase G subunit is involved in cluster stabilization and substrate channeling and confers specificity between nitrogenase reductase and catalytic component proteins. Moreover, the structure highlights a different interface between the two catalytic halves of the iron and the molybdenum nitrogenase, potentially influencing the intrasubunit 'communication' and thus the nitrogenase mechanism., (© 2023. The Author(s).)

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 (N 2 ) to NH 3 . 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 [Fe 4 S 4 ] 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.

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., (© 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).)

Nitrogenase is the only enzyme that can cleave the strong triple bond in N 2 , making nitrogen available for biological life. There are three isozymes of nitrogenase, differing in the composition of the active site, viz., Mo, V, and Fe-nitrogenase. Recently, the first crystal structure of Fe-nitrogenase was presented. We have performed the first combined quantum mechanical and molecular mechanical (QM/MM) study of Fe-nitrogenase. We show with QM/MM and quantum-refinement calculations that the homocitrate ligand is most likely protonated on the alcohol oxygen in the resting E 0 state. The most stable broken-symmetry (BS) states are the same as for Mo-nitrogenase, i.e., the three Noodleman BS7-type states (with a surplus of β spin on the eighth Fe ion), which maximize the number of nearby antiferromagnetically coupled Fe-Fe pairs. For the E 1 state, we find that protonation of the S2B μ 2 belt sulfide ion is most favorable, 14-117 kJ/mol more stable than structures with a Fe-bound hydride ion (the best has a hydride ion on the Fe2 ion) calculated with four different density-functional theory methods. This is similar to what was found for Mo-nitrogenase, but it does not explain the recent EPR observation that the E 1 state of Fe-nitrogenase should contain a photolyzable hydride ion. For the E 1 state, many BS states are close in energy, and the preferred BS state differs depending on the position of the extra proton and which density functional is used.

In providing bioavailable nitrogen as building blocks for all classes of biomacromolecules, biological nitrogen fixation is an essential process for all organismic life. Only a single enzyme, nitrogenase, performs this task at ambient conditions and with ATP as an energy source. The assembly of the complex iron-sulfur enzyme nitrogenase and its catalytic mechanism remains a matter of intense study. Recent progress in the structural analysis of the three known isoforms of nitrogenase-differentiated primarily by the heterometal in their active site cofactor-has revealed a degree of structural plasticity of these clusters that suggest two distinct binding sites for substrates and reaction intermediates. A mechanistic proposal based on this finding integrates most of the available experimental data. Furthermore, the first applications of high-resolution cryo-electron microscopy have highlighted further dynamic conformational changes. Structures obtained under turnover conditions support the proposed alternating half-site reactivity in the C 2 -symmetric nitrogenase complex., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Nitrogenase MoFe protein can be coupled with CdS nanocrystals (NCs) to enable photocatalytic N 2 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.

Enzymatic Fisher-Tropsch (FT) process catalyzed by vanadium (V)-nitrogenase can convert carbon monoxide (CO) to longer-chain hydrocarbons (>C2) under ambient conditions, although this process requires high-cost reducing agent(s) and/or the ATP-dependent reductase as electron and energy sources. Using visible light-activated CdS@ZnS (CZS) core-shell quantum dots (QDs) as alternative reducing equivalent for the catalytic component (VFe protein) of V-nitrogenase, we first report a CZS : VFe biohybrid system that enables effective photo-enzymatic C-C coupling reactions, hydrogenating CO into hydrocarbon fuels (up to C4) that can be hardly achieved with conventional inorganic photocatalysts. Surface ligand engineering optimizes molecular and opto-electronic coupling between QDs and the VFe protein, realizing high efficiency (internal quantum yield >56 %), ATP-independent, photon-to-fuel production, achieving an electron turnover number of >900, that is 72 % compared to the natural ATP-coupled transformation of CO into hydrocarbons by V-nitrogenase. The selectivity of products can be controlled by irradiation conditions, with higher photon flux favoring (longer-chain) hydrocarbon generation. The CZS : VFe biohybrids not only can find applications in industrial CO removal for high-value-added chemical production by using the cheap, renewable solar energy, but also will inspire related research interests in understanding the molecular and electronic processes in photo-biocatalytic systems., (© 2023 Wiley-VCH GmbH.)

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.

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe 7 S 9 MoC-homocitrate) as a critical N 2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E 4 (4H), which has accumulated 4[e - /H + ] as two bridging hydrides, Fe2-H-Fe6 and Fe3-H-Fe7, and protons bound to two sulfurs. E 4 (4H) is poised to bind/reduce N 2 as driven by mechanistically-coupled H 2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H 2 as the enzyme relaxes to state E 2 (2H), containing 2[e - /H + ] as a hydride and sulfur-bound proton; accumulation of E 4 (4H) in α-V70I is enhanced by HP suppression. EPR and 95 Mo 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 E 0 state of the WT MoFe protein and to both α-V70I conformations generating E 2 (2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e - /H + ] generates E 4 (4H) with Fe2-H-Fe6 as the second hydride. E 4 (4H) in WT enzyme and a minority α-V70I E 4 (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 E 2 (2H) containing Fe3-H-Fe7. In the dominant α-V70I E 4 (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 E 2 (2H) contains Fe2-H-Fe6. It is this HP suppression in E 4 (4H) that enables α-V70I MoFe to accumulate E 4 (4H) in high occupancy. In addition, HP suppression in α-V70I E 4 (4H) kinetically unmasks hydride reductive-elimination without N 2 -binding, a process that is precluded in WT enzyme.

The biological conversion of N 2 to NH 3 is accomplished by the nitrogenase family, which is collectively comprised of three closely related but unique metalloenzymes. In the present study, we have employed a combination of the synchrotron-based technique of 57 Fe nuclear resonance vibrational spectroscopy together with DFT-based quantum mechanics/molecular mechanics (QM/MM) calculations to probe the electronic structure and dynamics of the catalytic components of each of the three unique M N 2 ase enzymes (M = Mo, V, Fe) in both the presence (holo-) and absence (apo-) of the catalytic FeMco clusters (FeMoco, FeVco and FeFeco). The results described herein provide vibrational mode assignments for important fingerprint regions of the FeMco clusters, and demonstrate the sensitivity of the calculated partial vibrational density of states (PVDOS) to the geometric and electronic structures of these clusters. Furthermore, we discuss the challenges that are faced when employing NRVS to investigate large, multi-component metalloenzymatic systems, and outline the scope and limitations of current state-of-the-art theory in reproducing complex spectra.

Nitrogenase is the only enzyme that can convert N 2 into NH 3 . The reaction requires the addition of eight electrons and protons to the enzyme and the mechanism is normally described by nine states, E 0 -E 8 , 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 N 2 . We have used combined quantum mechanical and molecular mechanics methods to study the binding of N 2 to the E 0 -E 4 states of nitrogenase, using four different density functional theory (DFT) methods. We test many different structures for the E 2 -E 4 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 N 2 binding to Fe6. It is the only method that reproduces the experimental observation of unfavourable binding to the E 0 -E 2 states and favourable binding to E 3 and E 4 . 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 E 2 -E 4 states. Moreover, such structures with two hydride ions both bridging Fe2 and Fe6 are the best models for E 4 and also for the N 2 -bound E 3 and E 4 states. However, for E 4 , 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 H 2 from the two bridging hydride ions in the E 4 state would enhance the binding of N 2 .

The Fischer-Tropsch (FT) process converts a mixture of CO and H 2 into liquid hydrocarbons as a major component of the gas-to-liquid technology for the production of synthetic fuels. Contrary to the energy-demanding chemical FT process, the enzymatic FT-type reactions catalyzed by nitrogenase enzymes, their metalloclusters, and synthetic mimics utilize H + and e - as the reducing equivalents to reduce CO, CO 2 , and CN - into hydrocarbons under ambient conditions. The C 1 chemistry exemplified by these FT-type reactions is underscored by the structural and electronic properties of the nitrogenase-associated metallocenters, and recent studies have pointed to the potential relevance of this reactivity to nitrogenase mechanism, prebiotic chemistry, and biotechnological applications. This review will provide an overview of the features of nitrogenase enzymes and associated metalloclusters, followed by a detailed discussion of the activities of various nitrogenase-derived FT systems and plausible mechanisms of the enzymatic FT reactions, highlighting the versatility of this unique reactivity while providing perspectives onto its mechanistic, evolutionary, and biotechnological implications.

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 57 Fe-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 57 Fe 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

26 well selected oxidized P-clusters (P 2+ ) 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 P 2+ clusters correspond to Fe 2 3+ Fe 6 2+ with high electron delocalization, showing the same oxidation states as the resting states of P-clusters (P N ) in nitrogenases. The previously uncertain reduction of P 2+ to P N clusters by two electrons was assigned as a double protonation of P 2+ , 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 P 2+ clusters and longer α-hydroxy C-O bond (average of 1.422 Å) in P N clusters, while no change is observed in the electronic structures of Fe 8 S 7 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 Å from the homocitrate in the FeMo cofactor and 14.947 Å from the [Fe 4 S 4 ] cluster, respectively, and may well function as important electron-transport sites.

When moving protons in enzymes, water molecules are often used as intermediates. The water molecules used are not necessarily seen in the crystal structures if they move around at high rates. In a different situation, for metal containing cofactors in enzymes, it is sometimes necessary to move protons on the cofactor from the position they enter the cofactor to another position where the energy is lower. That is, for example, the situation in nitrogenase. In recent studies on that enzyme, prohibitively high barriers were sometimes found for transferring protons, and that was used as a strong argument against mechanisms where a sulfide is lost in the mechanism. A high barrier could be due to nonoptimal distances and angles at the transition state. In the present study, possibilities are investigated to use water molecules to reduce these barriers. The study is very general and could have been done for many other enzymes. The effect of water was found to be very large in the case of nitrogenase with a lowering of one barrier from 15.6 kcal/mol down to essentially zero. It is concluded that the effect of water molecules must be taken into account for meaningful results.

A central feature of the current understanding of dinitrogen (N 2 ) 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.

The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, which is also exhibited by extant A. vinelandii nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism., Competing Interests: AG, DH, AR, BC, AP, LS, BK No competing interests declared, (© 2023, Garcia et al.)

The binding of N 2 to FeMo-co, the catalytic site of the enzyme nitrogenase, is central to the conversion to NH 3 , but also has a separate role in promoting the N 2 -dependent HD reaction (D 2 + 2H + + 2e - → 2HD). The protein surrounding FeMo-co contains a clear channel for ingress of N 2 , 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' N 2 bound at exo -Fe2, and a second 'reducible' N 2 bound in the reaction domain, specifically the endo -coordination position of Fe2 or Fe6. The range of possibilities for the binding of reducible N 2 in the presence of bound promotional N 2 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 N 2 through protein, past the first N 2 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 (η 1 ) binding of N 2 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 N 2 binding from a diffusible position within the protein is estimated to be 4 kcal mol -1 , and so the binding free energies for reducible N 2 are suitably negative. N 2 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 N 2 is expanded. These results inform computational simulation of the subsequent steps in which surrounding H atoms transfer to reducible N 2 .

Nitrogenase is the enzyme that converts N 2 to NH 3 under ambient conditions. The chemical mechanism of this catalysis at the active site FeMo-co [Fe 7 S 9 CMo(homocitrate)] is unknown. An obligatory co-product is H 2 , while exogenous H 2 is a competitive inhibitor. Isotopic substitution using exogenous D 2 revealed the N 2 -dependent reaction D 2 +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 D 2 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 D 2 , 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 N 2 -dependence results from promotional N 2 bound at exo-Fe2; (c) different N 2 binding K m for the HD reaction and the NH 3 formation reaction results from involvement of two different sites; (d) inhibition by CO; (e) the non-occurrence of 2HD→H 2 +D 2 results from the synchronicity of the two transfers of H to D 2 ; (f) inhibition of HD production at high pN 2 is by competitive binding of N 2 at endo-Fe6; (g) the non-leakage of D to solvent follows from the hydrophobic environment and irreversibility of proton introduction., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The factors/structural features which are responsible for the binding, activation and reduction of N 2 to NH 3 by FeMoco of nitrogenase have not been completely understood well. Several relevant model complexes by Holland et al. and Peters et al. have been synthesized, characterized and studied by theoretical calculations. For a matter of fact, those complexes are much different than real active N 2 -binding Fe-sites of FeMoco, which possesses a central C(4-) ion having an eight valence electrons as an μ 6 -bridge. Here, a series of [(S 3 C(0))Fe(II/I/0)-N 2 ] n- complexes in different charged/spin states containing a coordinated σ- and π-donor C(0)-atom which possesses eight outer shell electrons [carbone, (Ph 3 P) 2 C(0); Ph 3 P→C(0)←PPh 3 ] and three S-donor sites (i.e. - S-Ar), have been studied by DFT, QTAIM, and EDA-NOCV calculations. The effect of the weak field ligand on Fe-centres and the subsequent N 2 -binding has been studied by EDA-NOCV analysis. The role of the oxidation state of Fe and N 2 -binding in different charged and spin states of the complex have been investigated by EDA-NOCV analyses. The intrinsic interaction energies of the Fe-N 2 bond are in the range from -42/-35 to -67 kcal/mol in their corresponding ground states. The S 3 C(0) donor set is argued here to be closer to the actual coordination environment of one of the six Fe-centres of nitrogenase. In comparison, the captivating model complexes reported by Holland et al. and Peter et al. possess a stronger π-acceptor C-ring (S 2 C ring donor, π-C donor) and stronger donor set like CP 3 (σ-C donor) ligands, respectively., (© 2022 Wiley Periodicals LLC.)

Nitrogenase is the sole enzyme responsible for the ATP-dependent conversion of atmospheric dinitrogen into the bioavailable form of ammonia (NH 3 ), making this protein essential for the maintenance of the nitrogen cycle and thus life itself. Despite the widespread use of the Haber-Bosch process to industrially produce NH 3 , biological nitrogen fixation still accounts for half of the bioavailable nitrogen on Earth. An important feature of nitrogenase is that it operates under physiological conditions, where the equilibrium strongly favours ammonia production. This biological, multielectron reduction is a complex catalytic reaction that has perplexed scientists for decades. In this review, we explore the current understanding of the molybdenum nitrogenase system based on experimental and computational research, as well as the limitations of the crystallographic, spectroscopic, and computational techniques employed. Finally, essential outstanding questions regarding the nitrogenase system will be highlighted alongside suggestions for future experimental and computational work to elucidate this essential yet elusive process., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Nitrogenase is the only enzyme that can cleave the triple bond in N 2 , making nitrogen available for all lifeforms. Previous computational studies have given widely diverging results regarding the reaction mechanism of the enzyme. For example, some recent studies have suggested that one of the μ 2 -bridging sulfide ligands (S2B) may dissociate from one of the Fe ions when protonated in the doubly reduced and protonated E 2 state, whereas other studies indicated that such half-dissociated states are unfavorable. We have examined how the relative energies of 26 structures of the E 2 state depend on details of combined quantum mechanical and molecular mechanical (QM/MM) calculations. We show that the selection of the broken-symmetry state, the basis set, relativistic effects, the size of the QM system, relaxation of the surroundings, and the conformations of the bound protons may affect the relative energies of the various structures by up to 12, 22, 9, 20, 37, and 33 kJ/mol, respectively. However, they do not change the preferred type of structures. On the other hand, the choice of the DFT functional strongly affects the preferences. The hybrid B3LYP functional strongly prefers doubly protonation of the central carbide ion, but such a structure is not consistent with experimental EPR data. Other functionals suggest structures with a hydride ion, in agreement with the experiments, and show that the ion bridges between Fe2 and Fe6. Moreover, there are two structures of the same type that are degenerate within 1-5 kJ/mol, in agreement with the observation of two EPR signals. However, the pure generalized gradient approximation (GGA) functional TPSS favors structures with a protonated S2B also bridging Fe2 and Fe6, whereas r 2 SCAN (meta-GGA) and TPSSh (hybrid) prefer structures with S2B dissociated from Fe2 (but remaining bound to Fe6). The energy difference between the two types of structure is so small (7-18 kJ/mol) that both types need to be considered in future investigations of the mechanism of nitrogenase.

Ancestral sequence reconstruction (ASR) infers predicted ancestral states for sites within sequences and can constrain the functions and properties of ancestors of extant protein families. Here, we compare the likely sequences of inferred nitrogenase ancestors to extant nitrogenase sequence diversity. We show that the most-likely combinations of ancestral states for key substrate channel residues are not represented in extant sequence space, and rarely found within a more broadly defined physiochemical space-supporting that the earliest ancestors of extant nitrogenases likely had alternative substrate channel composition. These differences may indicate differing environmental selection pressures acting on nitrogenase substrate specificity in ancient environments. These results highlight ASR's potential as an in silico tool for developing hypotheses about ancestral enzyme functions, as well as improving hypothesis testing through more targeted in vitro and in vivo experiments., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The active site of the nitrogen fixing enzyme nitrogenase is an Fe 7 MoS 9 C 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.

Nitrogen fixation at iron centres is a fundamental catalytic step for N 2 utilisation, relevant to biological (nitrogenase) and industrial (Haber-Bosch) processes. This step is coupled with important electronic structure changes which are currently poorly understood. We show here for the first time that terminal dinitrogen dissociation from iron complexes that coordinate N 2 in a terminal and bridging fashion leaves the Fe-N 2 -Fe unit intact but significantly enhances the degree of N 2 activation (Δν≈180 cm -1 , Raman spectroscopy) through charge redistribution. The transformation proceeds with local spin state change at the iron centre (S= 1 / 2 ${{ 1/2 }}$ →S= 3 / 2 ). Further dissociation of the bridging N 2 can be induced under thermolytic conditions, triggering a disproportionation reaction, from which the tetrahedral (PNN) 2 Fe could be isolated. This work shows that dinitrogen activation can be induced in the absence of external chemical stimuli such as reducing agents or Lewis acids., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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 CFe 6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13 C 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 13 CFe 6 ENDOR measurements for (i) the wild-type protein resting state ( E 0 ; α-Val 70 ) to those of (ii) α-Ile 70 , (iii) α-Ala 70 -substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E 4 (4H) Janus intermediate, activated for N 2 binding/reduction by accumulation of 4[e - /H + ]; (vi) E 4 (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 13 C isotropic hyperfine coupling constants, C a = [-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 CFe 6 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 CFe 6 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.

The biological process of dinitrogen reduction to ammonium occurs at the cofactors of nitrogenases, the only enzymes that catalyze this challenging chemical reaction. Three types of nitrogenases have been described, named according to the heterometal in their cofactor: molybdenum, vanadium or iron nitrogenases. Spectroscopic and structural characterization allowed the unambiguous identification of the cofactors of molybdenum and vanadium nitrogenases and revealed a central μ 6 -carbide in both of them. Although genetic studies suggested that the cofactor of the iron nitrogenase contains a similar Fe 6 C core, this has not been experimentally demonstrated. Here we report Valence-to-Core X-ray Emission Spectroscopy providing experimental evidence that this cofactor contains a carbide, thereby making the Fe 6 C core a feature of all nitrogenase cofactors., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Mo-nitrogenase catalyzes the challenging N 2 -to-NH 3 reduction. This complex reaction proceeds through a series of intermediate states (E n ) of its active site FeMo-cofactor. An understanding of the kinetics of the conversion between E n states is central to defining the mechanism of nitrogenase. Here, rate constants of key steps have been determined through a steady-state kinetic model with fits to experimental data. The model reveals that the rate for H 2 formation from the early electron populated state E 2 (2H) is much slower than that from the more reduced E 4 (4H) state. Further, it is found that the competing reactions of H 2 formation and N 2 binding at the E 4 (4H) state occur with equal rate constants. The H 2 -dependent reverse reaction of the N 2 binding step is found to have a rate constant of 5.5 ± 0.2 (atm H 2 ) -1 s -1 (7.2 ± 0.3 (mM H 2 ) -1 s -1 ). Importantly, the reduction of N 2 bound to FeMo-cofactor proceeds with a rate constant of 1 ± 0.1 s -1 , revealing a previously unrecognized slow step in the Mo-nitrogenase catalytic cycle associated with the chemical transformation of N 2 to 2 NH 3 . Finally, the populations of E n states under different reaction conditions are predicted, providing a powerful tool to guide the spectroscopic and mechanistic studies of Mo-nitrogenase.