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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. [ABSTRACT FROM AUTHOR]

We have designed a [Fe(SH)4H]− model with the fifth proton binding either to Fe or S. We show that the energy difference between these two isomers (∆E) is hard to estimate with quantum-mechanical (QM) methods. For example, different density functional theory (DFT) methods give ∆E estimates that vary by almost 140 kJ/mol, mainly depending on the amount of exact Hartree–Fock included (0%–54%). The model is so small that it can be treated by many high-level QM methods, including coupled-cluster (CC) and multiconfigurational perturbation theory approaches. With extrapolated CC series (up to fully connected coupled-cluster calculations with singles, doubles, and triples) and semistochastic heat-bath configuration interaction methods, we obtain results that seem to be converged to full configuration interaction results within 5 kJ/mol. Our best result for ∆E is 101 kJ/mol. With this reference, we show that M06 and B3LYP-D3 give the best results among 35 DFT methods tested for this system. Brueckner doubles coupled cluster with perturbaitve triples seems to be the most accurate coupled-cluster approach with approximate triples. CCSD(T) with Kohn–Sham orbitals gives results within 4–11 kJ/mol of the extrapolated CC results, depending on the DFT method. Single-reference CC calculations seem to be reasonably accurate (giving an error of ∼5 kJ/mol compared to multireference methods), even if the D1 diagnostic is quite high (0.25) for one of the two isomers. [ABSTRACT FROM AUTHOR]

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 N2 at its surface-exposed L-cluster ([Fe8S9C]), a structural/functional homolog of the M-cluster (or cofactor; [(R-homocitrate) MoFe7S9C]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N2 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. [ABSTRACT FROM AUTHOR]

The biohydrogen production in Dark fermentation (DF) could be enhanced by ZnFe 2 O 4 nanomaterials (NPs). The optimum conditions were obtained by a response surface method. Eventually, a biohydrogen production of 2645 mL/L was obtained after 48 h of batch culture, which is 116% higher than control without ZnFe 2 O 4 NPs. The mechanism was investigated by a multi-scale method. In bioreactor scale, ZnFe 2 O 4 NPs could enhance the glucose metabolic ability of C. pasteurianum to promote cell growth and biohydrogen production. The metabolic pathways of biohydrogen production were shifted from the butyric acid-type to the ethanol-type. In cellular scale, ZnFe 2 O 4 NPs could reduce bacterial flagella assembly. In macromolecule scale, the activities of hydrogenase and ethanol dehydrogenase were increased. In genetic scale, the differentially expressed genes mainly involved in biohydrogen production, like those encoded Fe-only hydrogenase, ethanol dehydrogenase, and nitrogenase were up-regulated. The results could provide theoretical and technical supports for the development of commercial biohydrogen process. • ZnFe2O4 NPs can effectively promote biohydrogen production. • Optimal enhancement conditions determined through response surface optimization. • ZnFe2O4 NPs enhance glucose utilization by C. pasteurianum. • ZnFe2O4 NPs boost hydrogenase & ethanol dehydrogenase in C. pasteurianum. • Mechanism of NPs promoting biohydrogen production revealed by multi-scale analysis. [ABSTRACT FROM AUTHOR]

We describe the genome of an Eremiobacterota population from tundra soil that contains the minimal set of nif genes needed to fix atmospheric N2. This putative diazotroph population, which we name Candidatus Lamibacter sapmiensis, links for the first time Eremiobacterota and N2 fixation. The integrity of the genome and its nif genes are well supported by both environmental and taxonomic signals. Ca. Lamibacter sapmiensis contains three nifH homologues and the complementary set of nifDKENB genes that are needed to assemble a functional nitrogenase. The putative diazotrophic role of Ca. Lamibacter sapmiensis is supported by the presence of genes that regulate N2 fixation and other genes involved in downstream processes such as ammonia assimilation. Similar to other Eremiobacterota, Ca. Lamibacter sapmiensis encodes the potential for atmospheric chemosynthesis via CO2 fixation coupled with H2 and CO oxidation. Interestingly, the presence of a N2O reductase indicates that this population could play a role as a N2O sink in tundra soils. Due to the lack of activity data, it remains uncertain if Ca. Lamibacter sapmiensis is able to assemble a functional nitrogenase and participate in N2 fixation. Confirmation of this ability would be a testament to the great metabolic versatility of Eremiobacterota, which appears to underlie their ecological success in cold and oligotrophic environments. [ABSTRACT FROM AUTHOR]

Previous research has mainly focused on breeding, timber supply, and physiological–biochemical characteristics of Paulownia fortunei. However, there has been limited attention given to its endophytic rhizobia and its potential benefits for plant growth due to a lack of knowledge about the existence of root nodules. In this study, we isolated nine bacterial strains from the root nodules after occasionally discovering the nodular‐like structure in uncultivated three year P. fortunei roots and then sequenced the 16S rDNA and nifA sequencing to conduct the bacterial strain identifications. We then determined the carbon use capability and nitrogenase activity of the bacterial strains. The result of 16S rDNA sequencing indicated that the endophytic rhizobia belongs to the genus Agrobacterium, Rhizobium, Herbaspirillum and Burkholderia. The results of nifA sequencing indicated that the nifA gene was sequenced in strain PG‐3, PG‐5, PG‐6, PG‐7, PG‐8 and PG‐9 rather than in strain PG‐1, PG‐2 and PG‐4. Besides, the nitrogenase activity of strain PG‐9 was significantly higher than other bacterial strains. Redundancy analysis (RDA) results indicated that fructose, glucose, and sucrose were significantly related to the plant height, diameter, and biomass rather than other carbon sources and nitrogenase activity. Our research revealed that the majority of bacterial strains isolated from P. fortunei exhibited a broad carbon utilization pathway and have the potential to promote plant growth after re‐Inoculation. This study aims to investigate the phylogeny of endophytic rhizobia in P. fortunei, and expand the range of non‐legume hosts studied for future research on biological N‐fixation. [ABSTRACT FROM AUTHOR]

Although commonly cited to be out of balance, the N cycle is balanced over long time scales and is likely not currently significantly out of balance within the errors of our geochemical estimates or biological extrapolations. Habitats for N 2 fixation have recently been discovered (polar regions and deep sea) or proposed (oxygen-depleted zones, ODZs), although their significance remains unresolved. There are currently significant issues for measuring the basic characteristics of marine N 2 fixation ranging from determination of in situ rates to identification and quantification of species. There continue to be discoveries of new diazotrophs, although their significance and relative contributions remain to be resolved, from haptophyte and diatom symbionts to non-cyanobacterial diazotrophs. N 2 fixation in Trichodesmium continues to be incompletely understood, with implications for cell biology and N 2 fixation in plants. Biological nitrogen (N 2) fixation is critical in global biogeochemical cycles and in sustaining the productivity of the oceans. There remain many unanswered questions, unresolved hypotheses, and unchallenged paradigms. The fundamental balance of N input and losses has not been fully resolved. One of the major N 2 -fixers, Trichodesmium , remains an enigma with intriguing biological and ecological secrets. Cyanobacterial N 2 fixation, once thought to be primarily due to free-living cyanobacteria, now also appears to be dependent on microbial interactions, from microbiomes to unicellular symbioses, which remain poorly characterized. Nitrogenase genes associated with diverse non-cyanobacterial diazotrophs (NCDs) are prevalent, but their significance remains a huge knowledge gap. Answering questions, new and old, such as those discussed here, is needed to understand the ocean's N and C cycles and their responses to environmental change. [ABSTRACT FROM AUTHOR]

Nitrogenase, the key enzyme for biological nitrogen fixation, is an evolutionary singularity, as only one mechanism capable of catalyzing the reduction of atmospheric N 2 into fixed nitrogen is known to have evolved. Nitrogenase has three metal isoforms (iron, molybdenum, and vanadium) and is thus connected and dependent upon biogeochemical cycles for metal availability. Molybdenum is abundant in modern oceans, was very scarce in ancient oceans and remains scarce in modern terrestrial environments. Despite its scarcity, it is proposed that early nitrogenases first utilized Mo-cofactors given its catalytic advantages over abundant Fe. To understand the evolution and diversification of the nitrogen cycle, we must first understand the paleoecological and planetary conditions that give rise to biological nitrogen fixation. The evolution of nitrogen fixation undoubtedly altered nearly all corners of the biosphere, given the essential role of nitrogen in the synthesis of biomass. To date, there is no unified view on what planetary conditions gave rise to nitrogen fixation or how these conditions have sustained it evolutionarily. Intriguingly, the concentrations of metals that nitrogenases require to function have changed throughout Earth's history. In this review, we describe the interconnection of the metal and nitrogen cycles with nitrogenase evolution and the importance of ancient ecology in the formation of the modern nitrogen cycle. We argue that exploration of the nitrogen cycle's deep past will provide insights into humanity's immediate environmental challenges centered on nitrogen availability. [ABSTRACT FROM AUTHOR]

Nitrogenase reduces N2 to NH3 at its active‐site cofactor. Previous studies of an N2‐bound Mo‐nitrogenase from Azotobacter vinelandii suggest binding of three N2 species via asymmetric belt‐sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N2 reduction involving all cofactor belt‐sulfur sites; yet, the evidence for the existence of multiple N2 species on Av1* remains elusive. Here we report a study of ATP‐independent, EuII/SO32−‐driven turnover of Av1* using GC‐MS and frequency‐selective pulse NMR techniques. Our data demonstrate incorporation of D2‐derived D by Av1* into the products of C2H2‐ and H+‐reduction, and decreased formation of NH3 by Av1* concomitant with the release of N2 under H2; moreover, they reveal a strict dependence of these activities on SO32−. These observations point to the presence of distinct N2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N2 via belt‐sulfur mobilization. [ABSTRACT FROM AUTHOR]

Nitrogenase reduces N2 to NH3 at its active‐site cofactor. Previous studies of an N2‐bound Mo‐nitrogenase from Azotobacter vinelandii suggest binding of three N2 species via asymmetric belt‐sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N2 reduction involving all cofactor belt‐sulfur sites; yet, the evidence for the existence of multiple N2 species on Av1* remains elusive. Here we report a study of ATP‐independent, EuII/SO32−‐driven turnover of Av1* using GC‐MS and frequency‐selective pulse NMR techniques. Our data demonstrate incorporation of D2‐derived D by Av1* into the products of C2H2‐ and H+‐reduction, and decreased formation of NH3 by Av1* concomitant with the release of N2 under H2; moreover, they reveal a strict dependence of these activities on SO32−. These observations point to the presence of distinct N2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N2 via belt‐sulfur mobilization. [ABSTRACT FROM AUTHOR]

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

In many frankia, the ability to nodulate host plants (Nod+) and fix nitrogen (Fix+) is a common strategy. However, some frankia within the Pseudofrankia genus lack one or two of these traits. This phenomenon has been consistently observed across various actinorhizal nodule isolates, displaying Nod- and/or Fix-phenotypes. Yet, the mechanisms supporting the colonization and persistence of these inefficient frankia within nodules, both with and without symbiotic strains (Nod+/Fix+), remain unclear. It is also uncertain whether these associations burden or benefit host plants. This study delves into the ecological interactions between Parafrankia EUN1f and Pseudofrankia inefficax EuI1c, isolated from Elaeagnus umbellata nodules. EUN1f (Nod+/Fix+) and EuI1c (Nod+/Fix-) display contrasting symbiotic traits. While the prediction suggests a competitive scenario, the absence of direct interaction evidence implies that the competitive advantage of EUN1f and EuI1c is likely contingent on contextual factors such as substrate availability and the specific nature of stressors in their respective habitats. In co-culture, EUN1f outperforms EuI1c, especially under specific conditions, driven by its nitrogenase activity. Iron-depleted conditions favor EUN1f, emphasizing iron's role in microbial competition. Both strains benefit from host root exudates in pure culture, but EUN1f dominates in co-culture, enhancing its competitive traits. Nodulation experiments show that host plant preferences align with inoculum strain abundance under nitrogendepleted conditions, while consistently favoring EUN1f in nitrogen-supplied media. This study unveils competitive dynamics and niche exclusion between EUN1f and EuI1c, suggesting that host plant may penalize less effective strains and even all strains. These findings highlight the complex interplay between strain competition and host selective pressure, warranting further research into the underlying mechanisms shaping plant-microbe-microbe interactions in diverse ecosystems. [ABSTRACT FROM AUTHOR]

The electronic structures of FeFe-cofactors (FeFe-cos) in resting and turnover states, together with their PN clusters from iron-only nitrogenases, have been calculated using the bond valence method, and their crystallographic data were reported recently and deposited in the Protein Data Bank (PDB codes: 8BOQ and 8OIE). The calculated results have also been compared with those of their homologous Mo- and V-nitrogenases. For FeFe-cos in the resting state, Fe1/2/4/5/6/7/8 atoms are prone to Fe3+, while the Fe3 atom shows different degrees of mixed valences. The results support that the Fe8 atom at the terminal positions of FeFe-cos possesses the same oxidation states as the Mo3+/V3+ atoms of FeMo-/FeV-cos. In the turnover state, the overall oxidation state of FeFe-co is slightly reduced than those in the resting species, and its electronic configuration is rearranged after the substitution of S2B with OH, compatible with those found in CO-bound FeV-co. Moreover, the calculations give the formal oxidation states of 6Fe2+–2Fe3+ for the electronic structures of PN clusters in Fe-nitrogenases. By the comparison of Mo-, V- and Fe-nitrogenases, the overall oxidation levels of 7Fe atoms (Fe1–Fe7) for both FeFe- and FeMo-cos in resting states are found to be higher than that of FeV-co. For the PN clusters in MoFe-, VFe- and FeFe-proteins, they all exhibit a strong reductive character. [ABSTRACT FROM AUTHOR]

The novel genus Aquibium that lacks nitrogenase was recently reclassified from the Mesorhizobium genus. The genomes of Aquibium species isolated from water were smaller and had higher GC contents than those of Mesorhizobium species. Six Mesorhizobium species lacking nitrogenase were found to exhibit low similarity in the average nucleotide identity values to the other 24 Mesorhizobium species. Therefore, they were classified as the non-N2-fixing Mesorhizobium lineage (N-ML), an evolutionary intermediate species. The results of our phylogenomic analyses and the loss of Rhizobiales-specific fur/mur indicated that Mesorhizobium species may have evolved from Aquibium species through an ecological transition. Halotolerant and alkali-resistant Aquibium and Mesorhizobium microcysteis belonging to N-ML possessed many tripartite ATP-independent periplasmic transporter and sodium/proton antiporter subunits composed of seven genes (mrpABCDEFG). These genes were not present in the N2-fixing Mesorhizobium lineage (ML), suggesting that genes acquired for adaptation to highly saline and alkaline environments were lost during the evolution of ML as the habitat changed to soil. Land-to-water habitat changes in Aquibium species, close relatives of Mesorhizobium species, could have influenced their genomic evolution by the gain and loss of genes. Our study indicated that lineage-specific evolution could have played a significant role in shaping their genome architecture and conferring their ability to thrive in different habitats.IMPORTANCEPhylogenetic analyses revealed that the Aquibium lineage (AL) and non-N2-fixing Mesorhizobium lineage (N-ML) were monophyletically grouped into distinct clusters separate from the N2-fixing Mesorhizobium lineage (ML). The N-ML, an evolutionary intermediate species having characteristics of both ancestral and descendant species, could provide a genomic snapshot of the genetic changes that occur during adaptation. Genomic analyses of AL, N-ML, and ML revealed that changes in the levels of genes related to transporters, chemotaxis, and nitrogen fixation likely reflect adaptations to different environmental conditions. Our study sheds light on the complex and dynamic nature of the evolution of rhizobia in response to changes in their environment and highlights the crucial role of genomic analysis in understanding these processes. [ABSTRACT FROM AUTHOR]

Developing efficient nanostructured electrocatalysts for N2 reduction to NH3 under mild conditions remains a major challenge. The Fe−Mo cofactor serves as the archetypal active site in nitrogenase. Inspired by nitrogenase, we designed a series of heteronuclear dual‐atom catalysts (DACs) labeled as FeMoN6−aXa (a=1, 2, 3; X=B, C, O, S) anchored on the pore of g‐C3N4 to probe the impact of coordination on FeMo‐catalyzed nitrogen fixation. The stability, reaction paths, activity, and selectivity of 12 different FeMoN6−aXa DACs have been systematically studied using density functional theory. Of these, four DACs (FeMoN5B1, FeMoN5O1, FeMoN4O2, and FeMoN3C3) displayed promising nitrogen reduction reaction (NRR) performance. Notably, FeMoN5O1 stands out with an ultralow limiting potential of −0.11 V and high selectivity. Analysis of the density of states and charge/spin changes shows FeMoN5O1′s high activity arises from optimal N2 binding on Fe initially and synergy of the FeMo dimer enabling protonation in NRR. This work contributes to the advancement of rational design for efficient NRR catalysts by regulating atomic coordination environments. [ABSTRACT FROM AUTHOR]

Nitrogenase is a complicated enzyme that actives the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3). The aim of this manuscript is to review the nitrogenase evolution with considering nitrogenase, structure of nitrogenase, action mechanism of nitrogenase and oxygen sensitive mechanism of nitrogenase. The searches focused on publications from 1980 to February 2023, using PubMed, Google Scholar, Science Direct, and Scopus databases. In the term of evolution, the nitrogen cycle has experienced highly changes; at the beginning of life and suggested the exact anoxic scenario, the comparatively sufficient ammonium was possibly used in an assimilation/mineralization cycle by protocellular organisms. The main nif gene products which are active in nitrogen fixation are nifH, nifD, nifK, nifT, nifY/nafY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifL, nifA, nifB, fdxN, nifQ, and nifJ. The main vnf gene products which are active in nitrogen fixation are vnfA, vnfE, vnfN, vnfX, vnfH, vnfFd, vnfD, vnfG, vnfK, and vnfY. Oxygen can be either detrimental or beneficial for diazotrophs in organisms suitable for an aerobic catabolism, and it supports the production of a substrate for nitrogenase (ATP), but it can also impede the activity and suppress the synthesis of this enzyme. [ABSTRACT FROM AUTHOR]

In Rhodobacter capsulatus , hydrogen production is predominantly limited by nitrogenase activity and light conversion efficiency. Modifying related genes can potentially mitigate these limitations. In this study, partial deletions of the mopAB and mdtB genes were performed in the R. capsulatus JL1 (WT) genome. Resultant mutants, JL11 (mopAB - ) and JL15 (mopAB - , mdtB - ), demonstrated favorable growth on MedA in photosynthetic anaerobic conditions. However, the growth of JL12 (mdtB - ) was inhibited. Batch fermentation experiments revealed an augmented hydrogen production in JL15, achieving a remarkable 177.89 ± 9.98 mmol L−1. Furthermore, the light conversion efficiencies of JL15 reached 4.6% and 3.3% under 3 ± 0.5 klux and 7 ± 0.5 klux, respectively. In comparison, JL1 (WT) achieved efficiencies of 3.3% and 1.8% with nitrogenase activities of 6.16 ± 0.11 and 6.90 ± 0.30 nmol min−1·mg−1. These findings highlight the promising potential of co-deletion as a strategy for enhancing hydrogen production. • Double-gene inactivation of mopAB and mdtB was carried out to construct mutant JL15 with improved H 2 production. • The LCE of JL15 was increased up to 83.3% compared with that of wild-type at high light conditon. • The nitrogenease activity 6.90 ± 0.30 nmol min−1·mg−1 of JL15 was increased by 55.4% to JL1 (WT) at high light conditon. [ABSTRACT FROM AUTHOR]

Only a single enzyme system—nitrogenase—carries out the conversion of atmospheric N2 into bioavailable ammonium, an essential prerequisite for all organismic life. The reduction of this inert substrate at ambient conditions poses unique catalytic challenges that strain our mechanistic understanding even after decades of intense research. Structural biology has added its part to this greater tapestry, and in this review, I provide a personal (and highly biased) summary of the parts of the story to which I had the privilege to contribute. It focuses on the crystallographic analysis of the three isoforms of nitrogenases at high resolution and the binding of ligands and inhibitors to the active-site cofactors of the enzyme. In conjunction with the wealth of available biochemical, biophysical, and spectroscopic data on the protein, this has led us to a mechanistic hypothesis based on an elementary mechanism of repetitive hydride formation and insertion. [ABSTRACT FROM AUTHOR]

Nitrogenases have the remarkable ability to catalyze the reduction of dinitrogen to ammonia under physiological conditions. How does this happen? The current view of the nitrogenase mechanism focuses on the role of hydrides, the binding of dinitrogen in a reductive elimination process coupled to loss of dihydrogen, and the binding of substrates to a binuclear site on the active site cofactor. This review focuses on recent experimental characterizations of turnover relevant forms of the enzyme determined by cryo-electron microscopy and other approaches, and comparison of these forms to the resting state enzyme and the broader family of iron sulfur clusters. Emerging themes include the following: (i) The obligatory coupling of protein and electron transfers does not occur in synthetic and small-molecule iron–sulfur clusters. The coupling of these processes in nitrogenase suggests that they may involve unique features of the cofactor, such as hydride formation on the trigonal prismatic arrangement of irons, protonation of belt sulfurs, and/or protonation of the interstitial carbon. (ii) Both the active site cofactor and protein are dynamic under turnover conditions; the changes are such that more highly reduced forms may differ in key ways from the resting-state structure. Homocitrate appears to play a key role in coupling cofactor and protein dynamics. (iii) Structural asymmetries are observed in nitrogenase under turnover-relevant conditions by cryo-electron microscopy, although the mechanistic relevance of these states (such as half-of-sites reactivity) remains to be established. [ABSTRACT FROM AUTHOR]

Nitrogen fixation can be a substantial flux of new biologically available nitrogen (N) into some aquatic ecosystems. The most commonly used method for measuring N2-fixation is the acetylene reduction method, which measures nitrogenase activity. This method requires the assumption of a theoretical ratio of ethylene produced to N2-fixed unless the estimates are calibrated using 15N2 as a tracer. We measured N2-fixation using both the acetylene reduction method and 15N2 uptake methods to examine the ratio of ethylene produced to N2-fixed (C2H4:N2), estimate controls on variation in this ratio, and quantify the immediate release of N2-fixed as dissolved N. We estimated N2-fixation for benthic biofilms and separated Nostoc in four streams in Wyoming, Little Laramie River, Ditch Creek, Spread Creek, and Polecat Creek. Estimates of the C2H4:N2 ratio for any one stream and date ranged from 1.6 to 7.1 with a group-level mean of 3.8. Immediate release of N2-fixed as dissolved organic and inorganic N was < 1% of N2-fixation. Ethylene production increased 1.8 times faster than N2-fixation as temperature increased showing strong control of temperature in the C2H4:N2 ratio. Temperature contributed substantially to the variability in ratios of ethylene produced to N2-fixed. Slight differences in the C2H4:N2 ratio can considerably alter N2-fixation estimates particularly in N2-fixation dominated stream biofilms, thus when calibrating using a range of temperatures may be essential. [ABSTRACT FROM AUTHOR]

NifA is a σ54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein's central AAA+ domain. In this work, we examine if an additional Cys pair that is part of a C(X)5 C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues' redox state may directly influence the DNA binding domain's DNA binding affinity and/or alter the protein's oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner. [ABSTRACT FROM AUTHOR]

The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal clusters for N2 reduction. Early evidence shows that an essential part of the mechanism involves transducing information between the nitrogenase component proteins through conformational dynamics. Here, millisecond time-resolved hydrogen-deuterium exchange mass spectrometry was used to unravel peptide-level protein motion on the time scale of catalysis of Mo-dependent nitrogenase from Azotobacter vinelandii. Normal mode analysis calculations complemented this data, providing insights into the specific signal transduction pathways that relay information across protein interfaces at distances spanning 100 Å. Together, these results show that conformational changes induced by protein docking are rapidly transduced to the active site, suggesting a specific mechanism for activating the metal cofactor in the enzyme active site. The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves long-range conformational changes within the enzyme complex, however, direct biophysical evidence of communication between the Fe protein and the MoFe protein is lacking. Here, the authors combine millisecond time-resolved hydrogen-deuterium exchange mass spectrometry and normal mode analysis, revealing molecular-level insights into how the Fe protein alters the stability and dynamics of the MoFe protein near the active site in a nucleotide-dependent manner. [ABSTRACT FROM AUTHOR]

Nitrogen fixation is often cited as an important nitrogen source for termites. However, the rate of nitrogen fixation between experiments and termite species is highly variable due to termite diversity and sampling limitations. New evidence suggests that subterranean termites (Coptotermes formosanus Shiraki) (Blattodea, Rhinotermitidae) seek alternative dietary nitrogen sources for colony growth and development. This study compares N2 fixation rates and nitrogenase gene expression rates to a diverse assemblage of feeding groups. It was observed that nitrogenase expression was much higher in one-piece nesting termites from the family Kalotermitidae which lack access to soil. On the other hand, subterranean termites from the family Rhinotermitidae fix nitrogen at much lower rates, not differing from ambient nitrogenase activity in soil. [ABSTRACT FROM AUTHOR]

Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe8S7]) and M-cluster core (or L-cluster; [Fe8S9C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD, nifK, nifH, nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB, nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli. Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme. [ABSTRACT FROM AUTHOR]

Numerous synthetic models of the FeMo‐co cluster of nitrogenases have been proposed to find the simplest structure with relevant reactivity. Indeed, such structures are able to perform multi‐electrons reduction processes, such as the conversion of N2 to ammonia, and of CO2 into methane and alkenes. The most challenging parameter to imitate is indeed the central carbide ligand, which is believed to maintain the integrity of iron sulfide assembly during the course of catalytic cycles. The study proposes the use of bis(diphenylthiophosphinoyl)methanediide (SCS)2− as an ideal platform for the synthesis of bi‐ and tetra‐metallic iron complexes, in which the iron‐carbon interaction is maintained upon structural diversification and redox state changes. [ABSTRACT FROM AUTHOR]

Nitrogen (N) is an essential macronutrient for plant growth and productivity. Leguminous plants establish symbiotic relationships with nitrogen-fixing rhizobial bacteria to use atmospheric dinitrogen gas to meet high N demand under low-N conditions. Nodule formation and N fixation are energy-consuming processes and are inhibited by nitrate present in the environment. Previous studies in model leguminous plants characterized NIN-LIKE PROTEIN (NLP) proteins that mediate nitrate control of root nodule symbiosis, but the mechanism by which nitrate regulates soybean root nodules via NLP remains unclear. In the soybean genome we found four homologs of AtNLP7, named GmNLP7a-GmNLP7d. We showed that the expression of GmNLP7s is responsive to nitrate but not to rhizobial infection and localized GmNLP7a to the nucleus. Downregulation of GmNLP7s increased nodule number, and overexpression of GmNLP7a (GmNLP7aOE) reduced nodule number regardless of nitrate availability, suggesting a negative role for GmNLP7s in nodulation. Nitrogenase activity in the GmNLP7aOE line was comparable to that of the wild type, indicating that GmNLP7a does not affect mature nodule activity. Overexpression of GmNLP7a downregulated the expression of GmNIN1a and GmENOD40-1. GmNLP7a interacted with GmNIN1a via the PB1 domain. Our results reveal a new regulator of GmNLP7 in nodulation and a molecular mechanism by which nitrate affects nodule number in soybean. [ABSTRACT FROM AUTHOR]

SUMMARY: Although vacuolar phosphate transporters (VPTs) are essential for plant phosphorus adaptation, their role in Rhizobium–legume symbiosis is unclear. In this study, homologous genes of VPT1 (MtVPTs) were identified in Medicago truncatula to assess their roles in Rhizobium–legume symbiosis and phosphorus adaptation. MtVPT2 and MtVPT3 mainly positively responded to low and high phosphate, respectively. However, both mtvpt2 and mtvpt3 mutants displayed shoot phenotypes with high phosphate sensitivity and low phosphate tolerance. The root‐to‐shoot phosphate transfer efficiency was significantly enhanced in mtvpt3 but weakened in mtvpt2, accompanied by lower and higher root cytosolic inorganic phosphate (Pi) concentration, respectively. Low phosphate induced MtVPT2 and MtVPT3 expressions in nodules. MtVPT2 and MtVPT3 mutations markedly reduced the nodule number and nitrogenase activity under different phosphate conditions. Cytosolic Pi concentration in nodules was significantly lower in mtvpt2 and mtvpt3 than in the wildtype, especially in tissues near the base of nodules, probably due to inhibition of long‐distance Pi transport and cytosolic Pi supply. Also, mtvpt2 and mtvpt3 could not maintain a stable cytosolic Pi level in the nodule fixation zone as the wildtype under low phosphate stress. These findings show that MtVPT2 and MtVPT3 modulate phosphorus adaptation and rhizobia–legume symbiosis, possibly by regulating long‐distance Pi transport. Significance Statement: These data highlight that MtVPT2 and MtVPT3 play a crucial role in phosphorus adaption and nodule symbiosis in Medicago truncatula. Furthermore, genetic manipulation of MtVPT2 and MtVPT3 can improve plant traits. [ABSTRACT FROM AUTHOR]

Während Ansätze zur selektiven Erzeugung des I E i - oder I Z i -Isomers eines Alkens in hohem Maße von der Transformation abhängen, die zur Bildung der C=C-Bindung verwendet wird, zeigte sein Team, dass die I E i -zu- I Z i -Isomerisierung unter Verwendung günstiger Photosensitizer und operativ einfacher Verfahren für eine Reihe von Olefinen mit unterschiedlichen Substitutionsmustern verallgemeinert werden kann.[9] Geeignete Substrate tragen Substituenten, die das -System für das I E i -Isomer effektiver erweitern als für das I Z i -Isomer. Sie betonte auch, dass dies zur Entwicklung robusterer, skalierbarer, nachhaltiger und schnellerer Synthesewege für die Herstellung von Wirkstoffen führen kann.[9] Ryan Gilmour (Universität Münster) erläuterte, wie sowohl die Schweiz als auch die Stereochemie, traditionell das zentrale Thema der Bürgenstock-Konferenz,[8] sein aktuelles Forschungsprogramm zur kontra-thermodynamischen Alken-Isomerisierung geprägt haben. So führte der Reaktionspfad der inneren Sphäre zur Entwicklung allgemeiner und milder C-H-Amidierungsreaktionen, wodurch die Verwendung risikoreicher Acylazid-Reagenzien umgangen werden konnte,[55] während die Verwendung des Reaktionspfads der äußeren Sphäre beispielsweise die Synthese von - oder -Lactamen ermöglichte.[29] Didier Bourissou (Universität Toulouse & CNRS) zeigte, wie ein rationales Ligandendesign anspruchsvolle Redoxprozesse, wie z. B. die oxidative Addition von Au SP I sp -Spezies an Csp SP 2 sp -Halogenbindungen, fördern kann. [Extracted from the article]

NifA is the primary activator of nitrogenase, and the N-terminal domain of nifA is sensitive to ammonium concentration. In this work, a mutant Rhodobacter capsulatus ZX01 with a genetically engineered deletion in the N-terminal region of nifA1 was constructed by employing overlap extension PCR to mitigate the inhibition of ammonium on nitrogenase expression in photosynthetic bacteria. The effects of different ammonium ion concentrations on the growth and photo-fermentative hydrogen production performance of wild-type strain R. capsulatus SB1003 and mutant ZX01 with glucose and volatile fatty acids as the carbon sources were studied, respectively. When the ratio of NH 4 +-N was 20% and 30%, the hydrogen yield of the mutant ZX01 was enhanced by 14.8% and 20.9% compared with that of R. capsulatus SB1003 using 25 mM acetic acid and 34 mM butyric acid as the carbon source, respectively. In comparison, using 30 mM glucose as the carbon source, the hydrogen yield of ZX01 was increased by 17.7% and 22.2% compared with that of R. capsulatus SB1003 when the ratio of NH 4 +-N was 20% and 30%, and the nitrogenase activity of ZX01 was also enhanced by 38.0% and 47.6%, respectively. When using 10 mM NH 4 + as a single nitrogen source, ZX01 showed a 2.6-fold increase in H 2 production. These results indicated that ZX01 demonstrated higher ammonium tolerance and better hydrogen production performance than the wild-type. The deletion in the N-terminal region of nifA1 could partially de-repress the nitrogenase activity inhibited by ammonium. [Display omitted] • The mutant with a deletion in the N-terminal region of nifA1 was constructed by overlap extension PCR. • The deletion in the N-terminal of nifA1 appeared to partially derepress the NH 4 + inhibition on nitrogenase activity. • The H 2 yield of ZX01 was increased by up to 22.3% compared with that of wild-type in the presence of ammonium. [ABSTRACT FROM AUTHOR]

Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants. [ABSTRACT FROM AUTHOR]

A conductive polymer‐based photosynthetic biohybrid is constructed to enhance biological nitrogen fixation by increasing nitrogenase activity in the non‐photosynthetic bacterium Azotobacter Chroococcum (A. Chroococcum). The light‐harvesting cationic poly(fluorene‐alt‐phenylene) (PFP) electrostatically binds to the surface of the bacteria and possesses satisfactory conductivity to facilitate electron transfer to the bacterium, promoting the nitrogen fixation pathway through redox proteins on the surface of the bacteria when under illumination. Therefore, the nitrogenase activity, hydrogen, NH4+‐N and L‐amino acids production are increased by 260 %, 37 %, 44 %, and 47 %, respectively. The expression levels of nifD and nifK encoding molybdenum‐iron (MoFe) protein and relevant nitrogen‐fixing proteins are up‐regulated. These photoactive conductive polymer‐bacteria biohybrids provide a new method for improving the biological nitrogen fixation capability of non‐photosynthetic nitrogen‐fixing bacteria. [ABSTRACT FROM AUTHOR]

A conductive polymer‐based photosynthetic biohybrid is constructed to enhance biological nitrogen fixation by increasing nitrogenase activity in the non‐photosynthetic bacterium Azotobacter Chroococcum (A. Chroococcum). The light‐harvesting cationic poly(fluorene‐alt‐phenylene) (PFP) electrostatically binds to the surface of the bacteria and possesses satisfactory conductivity to facilitate electron transfer to the bacterium, promoting the nitrogen fixation pathway through redox proteins on the surface of the bacteria when under illumination. Therefore, the nitrogenase activity, hydrogen, NH4+‐N and L‐amino acids production are increased by 260 %, 37 %, 44 %, and 47 %, respectively. The expression levels of nifD and nifK encoding molybdenum‐iron (MoFe) protein and relevant nitrogen‐fixing proteins are up‐regulated. These photoactive conductive polymer‐bacteria biohybrids provide a new method for improving the biological nitrogen fixation capability of non‐photosynthetic nitrogen‐fixing bacteria. [ABSTRACT FROM AUTHOR]

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

Unicellular diazotrophs are recognized as important contributors to the aquatic nitrogen cycle. Yet, quantifying the number of aquatic diazotrophs, especially the unicellular fraction, is highly challenging and relies mostly on microscopical and molecular approaches. In this study, a new flow cytometry-based method was developed to quantify unicellular diazotrophs by immunolabeling the nitrogenase enzyme. The quantification method was initially developed using a diazotrophic monoculture (Vibrio natriegens) and verified by different controls as well as various auxiliary approaches such as N2 fixation and confocal laser scanning microscopy. It was evident that only 15–20% of the total number of V. natriegens cells synthesized the nitrogenase enzyme, even though the media was N-limited and anaerobic. This approach was further tested on field samples collected from marine and freshwater environments. Overall, the number of unicellular diazotrophs measured in various aquatic environments ranged from 1 to 6 × 107 cells L−1. However, the fraction of these diazotrophs compared to total bacteria was significantly lower in the Eastern Mediterranean Sea (0.1%) than that collected along the Jordan River (4.7%). Nevertheless, N2 fixation rates per cell were highest in the Mediterranean Sea (~ 90 attomole N cell−1d−1), while lower in the lake and the river measured herein (1.4 to 0.3 attomole N cell−1 d−1, respectively). Following the above, this direct quantification approach can provide new insights on the number and specific contribution of unicellular diazotrophs to total N2 fixation in marine and freshwater environments. [ABSTRACT FROM AUTHOR]

Soybean is a rich source of protein and oil for humans. It forms a symbiotic association with rhizobia to fix atmospheric nitrogen for its growth and development. Rhizobial type‐III effectors act as important signaling molecules in symbiosis. However, they are poorly studied. In this study, mutation of TtsI in Sinorhizobium fredii HH103 reduced nodule numbers and dry weights in SN14 (cultivated soybean) and ZYD00006 (wild soybean). Moreover, it reduced nitrogenase activity in SN14 but not in ZYD00006. Based on the differences in nitrogenase activity between SN14 and ZYD00006 inoculated with HH103 and the TtsI mutant, two conditional quantitative trait loci associated with nitrogenase activity were identified in chromosome segment substitution lines derived from SN14 and ZYD00006. Using transcriptome analysis, single‐nucleotide polymorphism (SNP) analysis, and qRT‐PCR, Glyma.13G150000 (GmTNRP1) was identified as a candidate gene in soybean that was associated with nitrogenase activity, along with NopT (a type‐III effector). In addition, GmTNRP1 (an LRR‐RK family protein located on the cell membrane) was observed to be induced by NopT to negatively regulate nitrogenase activity. In transgenic hairy roots with GmTNRP1 silencing, no significant difference was observed in terms of nitrogenase activity between nodules inoculated with the NopT mutant and those inoculated with HH103 (wild type). However, the nitrogenase activity in the nodules decreased after overexpression of GmTNRP1. SNP located in the GmTNRP1 promoter affected its expression in response to NopT. Haplotype analysis revealed that SN14 belonged to Hap1 and ZYD00006 belonged to Hap2, which explained the different results in the two varieties. To the best of our knowledge, this is the first study to identify a soybean gene associated with a rhizobial type‐III effector in the nodules. GmTNRP1 can negatively regulate nitrogenase activity in the nodules. This study enhanced the understanding of the mechanism underlying symbiotic association between soybean and rhizobia. [ABSTRACT FROM AUTHOR]

Life on Earth depends on N2‐fixing microbes to make ammonia from atmospheric N2 gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N2 fixation was once believed to have evolved during the Archean‐Proterozoic times using Fe as a cofactor. However, δ15N values of paleo‐ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo‐oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral‐associated trace metals to a model N2‐fixing bacterium Azotobacter vinelandii. N2 fixation was observed when Mo in molybdenite, V in cavansite, and Fe in ferrihydrite were used as the sole sources of cofactors, but the rate of N2 fixation was greatly reduced. A physical separation between minerals and cells further reduced the rate of N2 fixation. Biochemical assays detected five siderophores, including aminochelin, azotochelin, azotobactin, protochelin, and vibrioferrin, as possible chelators to extract metals from minerals. The results of this study demonstrate that mineral‐associated trace metals are bioavailable as cofactors of nitrogenases to support N2 fixation in those environments that lack soluble trace metals and may offer a partial answer to the paradox. [ABSTRACT FROM AUTHOR]

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles' pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages. [ABSTRACT FROM AUTHOR]

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

Under nitrogen‐limiting conditions, the filamentous cyanobacterium Nostoc PCC7120 differentiates nitrogen‐fixing heterocysts at semi‐regular intervals along filaments generating a periodic pattern of two distinct cell types. Heterocysts are micro‐oxic cells that host the oxygen‐sensitive nitrogenase allowing two antagonistic activities to take place simultaneously. Although several factors required to control the differentiation process are known, the molecular mechanisms engaged have only been elucidated for a few of them. The patB (cnfR) gene has been shown to be essential for heterocyst formation and nitrogen fixation in this cyanobacterium, but its function remains to be clarified. Here, we show that PatB acts as a direct transcriptional regulator of genes required for nitrogenase production and activity. The DNA‐binding activity of PatB does not depend on micro‐oxia as it interacts with its target promoters under aerobic conditions both in vitro and in vivo. The absence of the DNA‐binding domain of PatB can be rescued in the heterocyst but not in the vegetative cell. Furthermore, the putative ferredoxin domain of PatB is not essential to its interaction with DNA. The patB gene is widely conserved in cyanobacterial genomes and its function can be pleiotropic since it is not limited to nitrogen fixation control. [ABSTRACT FROM AUTHOR]

Molybdenum dinitrogen complexes have played a major role as catalytic model systems of nitrogenase. In comparison, analogous tungsten complexes have in most cases found to be catalytically inactive. Herein, a tungsten complex was shown to be supported by a pentadentate tetrapodal (pentaPod) phosphine ligand, under conditions of N2 fixation, primarily catalyzes the hydrogen evolution reaction (HER), in contrast to its Mo analogue, which catalytically mediates the nitrogen‐reduction reaction (N2RR). DFT calculations were employed to evaluate possible mechanisms and identify the most likely pathways of N2RR and HER activities exhibited by Mo‐ and W‐pentaPod complexes. Two mechanisms for N2RR by PCET are considered, starting from neutral (M(0) cycle) and cationic (M(I) cycle) dinitrogen complexes (M=Mo, W). The latter was found to be energetically more favorable. For HER three scenarios are treated; that is, through bimolecular reactions of early M‐NxHy intermediates, pure hydride intermediates or mixed M(H)(NxHy) species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

Hydrogen with high energy content is considered to be a promising alternative clean energy source. Biohydrogen production through microbes provides a renewable and immense hydrogen supply by utilizing raw materials such as inexhaustible natural sunlight, water, and even organic waste, which is supposed to solve the two problems of "energy supply and environment protection" at the same time. Hydrogenases and nitrogenases are two classes of key enzymes involved in biohydrogen production and can be applied under different biological conditions. Both the research on enzymatic catalytic mechanisms and the innovations of enzymatic techniques are important and necessary for the application of biohydrogen production. In this review, we introduce the enzymatic structures related to biohydrogen production, summarize recent enzymatic and genetic engineering works to enhance hydrogen production, and describe the chemical efforts of novel synthetic artificial enzymes inspired by the two biocatalysts. Continual studies on the two types of enzymes in the future will further improve the efficiency of biohydrogen production and contribute to the economic feasibility of biohydrogen as an energy source. [ABSTRACT FROM AUTHOR]

The circumboreal/circumpolar N2‐fixing lichen Stereocaulon vesuvianum is among the most widespread and abundant fruticose species in montane Britain but has lost the capacity to fix N2 over large areas of the country.To investigate whether loss of N2‐fixation in S. vesuvianum is linked to increased N deposition, we examined thallus morphology, physiology and chemistry at twelve locations representing an N deposition gradient of 3–40 kg ha−1 year−1. Measurements were made in parallel on a non‐N2‐fixing reference species (Parmelia saxatilis). The presence or absence of cephalodia (N2‐fixing nodules containing the cyanobacterium Stigonema sp) was recorded in over 500 herbarium specimens of S. vesuvianum dating back to 1820.Cephalodium abundance in S. vesuvianum, and 15N concentration in S. vesuvianum and P. saxatilis, were strongly negatively correlated with N deposition and particularly with dry deposited N; cephalodia do not form at total N deposition rates ≥8–9 kg ha−1 year−1. Other morphological oddities in S. vesuvianum at N‐polluted sites include increased apothecium (fungal reproductive structure) production and green algal biofilm development. Biofilm covered thalli without cephalodia lacked nitrogenase activity and cephalodia at sites where they rarely develop had nitrogenase activities typical for this species. The presence or absence of cephalodia in herbarium specimens of S. vesuvianum suggest that the present‐day N‐deposition linked gradient in N2‐fixing capacity did not exist in the 19th century and largely developed between 1900–1940.Synthesis. We provide clear evidence that N2‐fixing capacity in S. vesuvianum has been lost in regions subjected to many decades of enhanced atmospheric N deposition. This loss is consistent with established models of diazotrophy, which identify supply of combined N as an inhibitor of N2‐fixation. Progressive depletion of thallus 15N with increasing N deposition is in line with available data indicating that much atmospheric N pollution is 15N‐depleted. Rates of nitrogenase activity in S. vesuvianum are low compared to other symbiotic systems and perhaps more likely supplanted by elevated N deposition. We suggest that other ecosystem compartments with low rates of fixation (e.g. soils) might also be susceptible to N pollution and merit investigation. [ABSTRACT FROM AUTHOR]

Spin-state regulation of the Mo center from high-spin to medium-spin assisted by the neighboring Fe center relieves the strong overlapping between Mo 4 d orbital and the key intermediates, realizing enhanced NRR activity. [Display omitted] • Spin-state regulation of Mo from high-spin to medium-spin by the neighboring Fe. • The medium-spin of Mo promotes the protonation of *NH2. • The decreasing of spin state leads to moderate binding strength of·NH3. • Fe/Mo N C is a novel NRR catalyst with low limiting potentials and high selectivity. Electrocatalytic nitrogen reduction reaction (eNRR) is a promising method for the sustainable production of ammonia as an alternative to the traditional energy-intensive Haber-Bosch process. In this work, an efficient strategy by atomic spin regulation to promote NRR through Fe-transition metal (TM) hybrid heteronuclear dual-atom catalysts has been studied. By means of DFT computations, the stability, activity, and selectivity of 30 kinds of Fe-based dual-atoms anchored on N -doped porous graphene are systematically investigated to evaluate their catalytic performance. Fe/Mo N C is screened as an excellent NRR catalyst with the limiting potentials of 0.63 V, and also suppresses HER. In the Fe/Mo N C, the neighboring Fe atom regulates the spin state of the Mo center in MoN 4 from high-spin state (2.63 μ B) to medium-spin state (0.74 μ B), which can effectively relieve the strong overlapping between Mo 4 d orbital with the N x H y intermediates, promote the desorption of reaction product, and eventually achieve a lower limiting potential. Interestingly, the archetype of the active center of nitrogenase is also a FeMo-cofactor, which is consistent with our screening results. The work may provide new insight into the mechanism of nitrogenase, and promote the rational design of efficient NRR catalysts by atomic spin regulation. [ABSTRACT FROM AUTHOR]

Nitrogenase is the enzyme that converts N2 to NH3 under ambient conditions. The chemical mechanism of this catalysis at the active site FeMo‐co [Fe7S9CMo(homocitrate)] is unknown. An obligatory co‐product is H2, while exogenous H2 is a competitive inhibitor. Isotopic substitution using exogenous D2 revealed the N2‐dependent reaction D2+2H++2e−→2HD (the 'HD reaction'), together with a collection of additional experimental characteristics and requirements. This paper describes a detailed mechanism for the HD reaction, developed and elaborated using density functional simulations with a 486‐atom model of the active site and surrounding protein. First D2 binds at one Fe atom (endo‐Fe6 coordination position), where it is flanked by H−Fe6 (exo position) and H−Fe2 (endo position). Then there is synchronous transfer of these two H atoms to bound D2, forming one HD bound to Fe2 and a second HD bound to Fe6. These two HD dissociate sequentially. The final phase is recovery of the two flanking H atoms. These H atoms are generated, sequentially, by translocation of a proton from the protein surface to S3B of FeMo‐co and combination with introduced electrons. The first H atom migrates from S3B to exo‐Fe6 and the second from S3B to endo‐Fe2. Reaction energies and kinetic barriers are reported for all steps. This mechanism accounts for the experimental data: (a) stoichiometry; (b) the N2‐dependence results from promotional N2 bound at exo‐Fe2; (c) different N2 binding Km for the HD reaction and the NH3 formation reaction results from involvement of two different sites; (d) inhibition by CO; (e) the non‐occurrence of 2HD→H2+D2 results from the synchronicity of the two transfers of H to D2; (f) inhibition of HD production at high pN2 is by competitive binding of N2 at endo‐Fe6; (g) the non‐leakage of D to solvent follows from the hydrophobic environment and irreversibility of proton introduction. [ABSTRACT FROM AUTHOR]

Nitrogen–fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N2) in the atmosphere into bioavailable nitrogen. Elaborating the molecular mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural production can alleviate the "nitrogen problem". Azotobacter vinelandii is a well–established model bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the nif gene cluster to fix nitrogen. In Azotobacter vinelandii, the NifA–NifL system fine–tunes the nif gene cluster transcription by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript, we investigate the transcriptional regulation mechanism of the nif gene in autogenous nitrogen–fixing bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture, providing preliminary comprehensive data for the study of autogenous nitrogen–fixing regulation. [ABSTRACT FROM AUTHOR]

The factors/structural features which are responsible for the binding, activation and reduction of N2 to NH3 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 N2‐binding Fe‐sites of FeMoco, which possesses a central C(4‐) ion having an eight valence electrons as an μ6‐bridge. Here, a series of [(S3C(0))Fe(II/I/0)‐N2]n‐ complexes in different charged/spin states containing a coordinated σ‐ and π‐donor C(0)‐atom which possesses eight outer shell electrons [carbone, (Ph3P)2C(0); Ph3P→C(0)←PPh3] 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 N2‐binding has been studied by EDA‐NOCV analysis. The role of the oxidation state of Fe and N2‐binding in different charged and spin states of the complex have been investigated by EDA‐NOCV analyses. The intrinsic interaction energies of the Fe−N2 bond are in the range from −42/−35 to −67 kcal/mol in their corresponding ground states. The S3C(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 (S2Cring donor, π‐C donor) and stronger donor set like CP3 (σ‐C donor) ligands, respectively. [ABSTRACT FROM AUTHOR]

We have calculated redox potentials of the two metal clusters in Mo-nitrogenase with quantum mechanical (QM) calculations. We employ an approach calibrated for iron–sulfur clusters with 1–4 Fe ions, involving QM-cluster calculations in continuum solvent and large QM systems (400–500 atoms), based on structures from combined QM and molecular mechanics (QM/MM) geometry optimisations. Calculations on the P-cluster show that we can reproduce the experimental redox potentials within 0.33 V. This is similar to the accuracy obtained for the smaller clusters, although two of the redox reactions involve also proton transfer. The calculated P1+/PN redox potential is nearly the same independently of whether P1+ is protonated or deprotonated, explaining why redox titrations do not show any pH dependence. For the FeMo cluster, the calculations clearly show that the formal oxidation state of the cluster in the resting E0 state is Mo III Fe 3 II Fe 4 III , in agreement with previous experimental studies and QM calculations. Moreover, the redox potentials of the first five E0–E4 states are nearly constant, as is expected if the electrons are delivered by the same site (the P-cluster). However, the redox potentials are insensitive to the formal oxidation states of the Fe ion (i.e., whether the added protons bind to sulfide or Fe ions). Finally, we show that the later (E4–E8) states of the reaction mechanism have redox potential that are more positive (i.e., more exothermic) than that of the E0/E1 couple. [ABSTRACT FROM AUTHOR]

The Trichodesmium genus comprises some of the most abundant N2-fixing organisms in oligotrophic marine ecosystems. Since nitrogenase, the key enzyme for N2 fixation, is irreversibly inhibited upon O2 exposure, these organisms have to coordinate their N2-fixing ability with simultaneous photosynthetic O2 production. Although being the principal object of many laboratory and field studies, the overall process of how Trichodesmium reconciles these two mutually exclusive processes remains unresolved. This is in part due to contradictory results that fuel the Trichodesmium enigma. In this review, we sift through methodological details that could potentially explain the discrepancy between findings related to Trichodesmium's physiology. In doing so, we exhaustively contrast studies concerning both spatial and temporal nitrogenase protective strategies, with particular attention to more recent insights. Finally, we suggest new experimental approaches for solving the complex orchestration of N2 fixation and photosynthesis in Trichodesmium. [ABSTRACT FROM AUTHOR]