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The transfer of nitrogen fixation (nif) genes from diazotrophs to non-diazotrophic hosts is of increasing interest for engineering biological nitrogen fixation. A recombinant Escherichia coli strain expressing Azotobacter vinelandii 18 nif genes (nifHDKBUSVQENXYWZMF, nif iscA, and nafU) were previously constructed and showed nitrogenase activity. In the present study, we constructed several E. coli strain derivatives in which all or some of the 18 nif genes were additionally integrated into the fliK locus of the chromosome in various combinations. E. coli derivatives with the chromosomal integration of nif iscA, nifU, and nifS, which are involved in the biosynthesis of the [4Fe-4S] cluster of dinitrogenase reductase, exhibited enhanced nitrogenase activity. We also revealed that overexpression of E. coli fldA and ydbK, which encode flavodoxin and flavodoxin-reducing enzyme, respectively, enhanced nitrogenase activity, likely by facilitating electron transfer to dinitrogenase reductase. The additional expression of nifM, putatively involved in maturation of dinitrogenase reductase, further enhanced nitrogenase activity and the amount of soluble NifH. By combining these factors, we successfully improved nitrogenase activity 10-fold., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB., (© 2024. The Author(s).)

Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living soil diazotroph Azotobacter vinelandii transfers electrons from the key metabolite NADH to the low-potential ferredoxin FdxA that serves as a direct electron donor to the dinitrogenase reductases. This process is mediated by the RNF complex that exploits the proton motive force over the cytoplasmic membrane to lower the midpoint potential of the transferred electron. Here we report the cryogenic electron microscopy structure of the nitrogenase-associated RNF complex of A. vinelandii, a seven-subunit membrane protein assembly that contains four flavin cofactors and six iron-sulfur centers. Its function requires the strict coupling of electron and proton transfer but also involves major conformational changes within the assembly that can be traced with a combination of electron microscopy and modeling., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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.

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).)

Importance: The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase., Competing Interests: The authors declare no conflict of interest.

Biological nitrogen fixation, the conversion of atmospheric dinitrogen into bioavailable ammonium, is exclusively catalyzed by the enzyme nitrogenase that is present in nitrogen-fixing organisms, the diazotrophs. So far, three different nitrogenase variants, encoded in their corresponding, distinct gene clusters, have been found in nature. Each one of these consists of a catalytic dinitrogenase component and a unique, ATP-dependent reductase, the Fe protein. The three variant nitrogenases differ in the composition of the active site and contain either molybdenum, vanadium or only iron in the dinitrogenase component. Here we present the 2.0 Å resolution crystal structure of the ADP-bound reductase component AnfH of the iron-only nitrogenase from the model diazotroph Azotobacter vinelandii. A comparison of this structure with the ones reported for the two other Fe protein homologs NifH and VnfH in the ADP-bound state shows that all are adopting the same conformation. However, cross-reactivity assays with the three nitrogenase homologs revealed AnfH to be compatible with iron-only nitrogenase and to a lesser degree with the vanadium-containing enzyme, but not with molybdenum nitrogenase., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin), acting as an electron donor for nitrogenase. However, the relative contribution of Rnf to nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defects under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of a fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between flavin mononucleotide and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The MoFe protein component of the nitrogenase enzyme complex is the substrate reducing site and contains two sets of symmetrically arrayed metallo centers called the P (Fe 8 S 7 ) and the FeMoco (MoFe 7 S 9 -C-homocitrate) centers. The ATP-binding Fe protein is the specific reductant for the MoFe protein. Both symmetrical halves of the MoFe protein are thought to function independently during nitrogenase catalysis. Forming [AlF 4 ] - transition-state complexes between the MoFe protein and the Fe protein of Azotobacter vinelandii ranging from 0 to 2 Fe protein/MoFe protein produced a series of complexes whose specific activity decreases with increase in bound Fe protein/MoFe protein ratio. Reduction of 2H + to H 2 was inhibited in a linear manner with an x-intercept at 2.0 with increasing Fe protein binding, whereas acetylene reduction to ethylene decreased more rapidly with an x-intercept near 1.5. H + reduction is a distinct process occurring independently at each half of the MoFe protein but acetylene reduction decreases more rapidly than H + reduction with increasing Fe protein/MoFe protein ratio, suggesting that a response is transmitted between the two αβ halves of the MoFe protein for acetylene reduction as Fe protein is bound. A mechanistic model is derived to investigate this behavior. The model predicts that each site functions independently for 2H + reduction to H 2 . For acetylene reduction, the model predicts positive (synchronous) not negative cooperativity arising from acetylene binding to both sites before substrate reduction occurs. When this model is applied to inhibition by Cp2 and modified Av2 protein (L127∆) that form strong, non-dissociable complexes, positive cooperativity is absent and each site acts independently. The results suggest a new paradigm for the catalytic function of the MoFe protein during nitrogenase catalysis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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

Mo-dependent nitrogenase is a major contributor to global biological N 2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe 7 MoS 9 C-homocitrate) contains a carbide (C 4- ) centered within a trigonal prismatic CFe 6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with 13 C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13 C-carbide ENDOR of the S = 3/2 resting state (E 0 ) is remarkable, with an extremely small isotropic hyperfine coupling constant, C a = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13 C carbide isotropic hyperfine-coupling constant essentially unchanged, C a = -1.30 MHz. This indicates that both the E 0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to C a from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E 0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe 6 core structures of E 0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide-Fe bonds.

The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation (nif) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii. The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A. vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family., Competing Interests: The authors have declared that no competing interests exist

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue β-188Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing β-188Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing β-188Ser) are important for communication between the two halves., Competing Interests: The authors have declared that no competing interests exist.

Several microorganisms can utilize l-rhamnose as a carbon and energy source through the nonphosphorylative metabolic pathway, in which l-rhamnose 1-dehydrogenase (RhaDH) catalyzes the NAD(P) + -dependent oxidization of l-rhamnose to l-rhamnono-1,4-lactone. We herein investigated the crystal structures of RhaDH from Azotobacter vinelandii in ligand-free, NAD + -bound, NADP + -bound, and l-rhamnose- and NAD + -bound forms at 1.9, 2.1, 2.4, and 1.6 Å resolution, respectively. The significant interactions with the 2'-phosphate group of NADP + , but not the 2'-hydroxyl group of NAD + , were consistent with a preference for NADP + over NAD + . The C5-OH and C6-methyl groups of l-rhamnose were recognized by specific residues of RhaDH through hydrogen bonds and hydrophobic contact, respectively, which contribute to the different substrate specificities from other aldose 1-dehydrogenases in the short-chain dehydrogenase/reductase superfamily., (© 2021 Federation of European Biochemical Societies.)

Nitrogenase converts N 2 to NH 3 , and CO to hydrocarbons, at its cofactor site. Herein, we report a biochemical and spectroscopic characterization of a Mo-nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDK Cit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDK Cit reveals a shift of CO reduction from H 2 evolution toward hydrocarbon formation and an opposite shift of N 2 reduction from NH 3 formation toward H 2 evolution. Consistent with a shift in the Mo K-edge energy of NifDK Cit relative to that of its wild-type counterpart, EPR analysis demonstrates a broadening of the line-shape and a decrease in the intensity of the cofactor-originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution., (© 2020 Wiley-VCH GmbH.)

Azotobacter vinelandii contains three forms of nitrogenase known as the Mo-, V-, and Fe-nitrogenases. They are all two-component enzyme systems, where the catalytic component, referred to as NifDK, VnfDGK, and AnfDGK, associates with the reductase component, the Fe protein or NifH, VnfH, and AnfH respectively. AnfDGK and VnfDGK have an additional subunit compared to NifDK, termed gamma or AnfG and VnfG, whose role is unknown. The expression of each nitrogenase is tightly regulated by metal availability, however it is known that there is crosstalk between the Mo- and V‑nitrogenases but the Fe‑nitrogenase components cannot support substrate reduction with its Mo‑nitrogenase counterparts. Here, docking models for the nitrogenase complexes were generated in ClusPro 2.0 based on the crystal structure of the Mo‑nitrogenase and refined using the HADDOCK 2.2 refinement interface to identify structural determinants that enable crosstalk between the Mo- and V‑nitrogenase but not the Fe‑nitrogenase. Differing salt bridge interactions were identified at the binding interface of each complex. Specifically, positively charged residues of VnfG enable complementary interactions with NifH and VnfH but not AnfH. Similarly, negatively charged residues of AnfG enable interactions with AnfH but not NifH or VnfH. A role for the G subunit is revealed where VnfG could be mediating crosstalk between the Mo- and V‑nitrogenases while the AnfG subunit on AnfDGK makes interactions with NifH and VnfH unfavorable, reducing competition with NifDK and funneling electrons to the most efficient nitrogenase., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O 2 -binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa , MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR. IMPORTANCE A. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell., (Copyright © 2020 American Society for Microbiology.)

Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii , Av GReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMP-synthesizing contributor to alginate production. Overproduction of Av GReg in the parental strain phenocopied a Δ mucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC Av GReg. IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications., (Copyright © 2020 American Society for Microbiology.)

We report an organic redox-polymer-based electroenzymatic nitrogen fixation system using a metal-free redox polymer, namely neutral-red-modified poly(glycidyl methacrylate-co-methylmethacrylate-co-poly(ethyleneglycol)methacrylate) with a low redox potential of -0.58 V vs. SCE. The stable and efficient electric wiring of nitrogenase within the redox polymer matrix enables mediated bioelectrocatalysis of N 3 - , NO 2 - and N 2 to NH 3 catalyzed by the MoFe protein via the polymer-bound redox moieties distributed in the polymer matrix in the absence of the Fe protein. Bulk bioelectrosynthetic experiments produced 209±30 nmol NH 3  nmol MoFe -1  h -1 from N 2 reduction. 15 N 2 labeling experiments and NMR analysis were performed to confirm biosynthetic N 2 reduction to NH 3 ., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Coupling the nitrogenase MoFe protein to light-harvesting semiconductor nanomaterials replaces the natural electron transfer complex of Fe protein and ATP and provides low-potential photoexcited electrons for photocatalytic N 2 reduction. A central question is how direct photochemical electron delivery from nanocrystals to MoFe protein is able to support the multielectron ammonia production reaction. In this study, low photon flux conditions were used to identify the initial reaction intermediates of CdS quantum dot (QD):MoFe protein nitrogenase complexes under photochemical activation using EPR. Illumination of CdS QD:MoFe protein complexes led to redox changes in the MoFe protein active site FeMo-co observed as the gradual decline in the E 0 resting state intensity that was accompanied by an increase in the intensity of a new " g eff = 4.5" EPR signal. The magnetic properties of the g eff = 4.5 signal support assignment as a reduced S = 3/2 state, and reaction modeling was used to define it as a two-electron-reduced "E 2 " intermediate. Use of a MoFe protein variant, β-188 Cys , which poises the P cluster in the oxidized P + state, demonstrated that the P cluster can function as a site of photoexcited electron delivery from CdS to MoFe protein. Overall, the results establish the initial steps for how photoexcited CdS delivers electrons into the MoFe protein during reduction of N 2 to ammonia and the role of electron flux in the photochemical reaction cycle.

Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N 2 ) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N 2 turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αβ dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo-O5 (carboxyl) or Mo-O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Biological nitrogen fixation is an inherent trait exclusive to a select number of prokaryotes. Although molybdenum nitrogenase is the dominant catalyst for dinitrogen reduction, some diazotrophs also contain one or two additional types of nitrogenase that use alternative metal content as the active-site cofactor. The occurrence of alternative nitrogenases has not been well studied due to the discriminatory expression of the molybdenum nitrogenase and lack of comprehensive genomic data. This study reports on the genomic analysis of 87 unique species containing alternative nitrogenase sequences. The distribution of nitrogen-fixing genes within these species from distinct taxonomic groups shows the presence of the minimum gene set required for nitrogen fixation, including catalytic and biosynthetic enzymes of the Mo-dependent system (NifHDKENB) and the varying occurrence of additional Nif-dedicated components. These include NifS and NifU, found primarily in aerobic species, thus suggesting that these genes are necessary to accommodate the high demand for Fe-S clusters during aerobic nitrogen fixation., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nitrogenases catalyze the ambient reduction of N 2 and CO at its cofactor site. Herein we present a biochemical and spectroscopic characterization of an Azotobacter vinelandii V nitrogenase variant expressing a citrate-substituted cofactor. Designated VnfDGK Cit , the catalytic component of this V nitrogenase variant has an αβ 2 (δ) subunit composition and carries an 8Fe P* cluster and a citrate-substituted V cluster analogue in the αβ dimer, as well as a 4Fe cluster in the "orphaned" β-subunit. Interestingly, when normalized based on the amount of cofactor, VnfDGK Cit shows a shift of N 2 reduction from H 2 evolution toward NH 3 formation and an opposite shift of CO reduction from hydrocarbon formation toward H 2 evolution. These observations point to a role of the organic ligand in proton delivery during catalysis and imply the use of different reaction sites/mechanisms by nitrogenase for different substrate reductions. Moreover, the increased NH 3 /H 2 ratio upon citrate substitution suggests the possibility to modify the organic ligand for improved ammonia synthesis in the future., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The obligate aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated., Results: In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions., Conclusions: These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.

We have made a systematic combined quantum mechanical and molecular mechanical (QM/MM) investigation of possible structures of the N 2 bound state of nitrogenase. We assume that N 2 is immediately protonated to a N 2 H 2 state, thereby avoiding the problem of determining the position of the protons in the cluster. We have systematically studied both end-on and side-on structures, as well as both HNNH and NNH 2 states. Our results indicate that the binding of N 2 H 2 is determined more by interactions and steric clashes with the surrounding protein than by the intrinsic preferences of the ligand and the cluster. The best binding mode with both the TPSS and B3LYP density-functional theory methods has trans-HNNH terminally bound to Fe2. It is stabilised by stacking of the substrate with His-195 and Ser-278. However, several other structures come rather close in energy (within 3-35 kJ/mol) at least in some calculations: The corresponding cis-HNNH structure terminally bound to Fe2 is second best with B3LYP. A structure with HNNH 2 terminally bound to Fe6 is second most stable with TPSS (where the third proton is transferred to the substrate from the homocitrate ligand). Structures with trans-HNNH, bound to Fe4 or Fe6, or cis-HNNH bound to Fe6 are also rather stable. Finally, with the TPSS functional, a structure with cis-HNNH side-on binding to the Fe3-Fe4-Fe5-Fe7 face of the cluster is also rather low in energy, but all side-on structures are strongly disfavoured by the B3LYP method.

Biological nitrogen fixation (BNF) is accomplished through the action of the oxygen-sensitive enzyme nitrogenase. One unique caveat of this reaction is the inclusion of hydrogen gas (H 2 ) evolution as a requirement of the reaction mechanism. In the absence of nitrogen gas as a substrate, nitrogenase will reduce available protons to become a directional ATP-dependent hydrogenase. Aerobic nitrogen-fixing microbes are of particular interest, because these organisms have evolved to perform these reactions with oxygen-sensitive enzymes in an environment surrounded by oxygen. The ability to maintain a functioning nitrogenase in aerobic conditions facilitates the application of these organisms under conditions where most anaerobic nitrogen fixers are excluded. In recent years, questions related to the potential yields of the nitrogenase-derived products ammonium and H 2 have grown more approachable to experimentation based on efforts to construct increasingly more complicated strains of aerobic nitrogen fixers such as the obligate aerobe Azotobacter vinelandii. This mini-review provides perspectives of recent and historical efforts to understand and quantify the yields of ammonium and H 2 that can be obtained through the model aerobe A. vinelandii, and outstanding questions that remain to be answered to fully realize the potential of nitrogenase in these applications with model aerobic bacteria.

The radical S -adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes ( nifUSXB and fdxN ) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN , and a library consisting of 28 different nifB genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Nitrogenase catalyzes the reduction of N 2 to NH 3 , supporting all biological nitrogen fixation. Electron donors to this enzyme are ferredoxin or flavodoxin ( in vivo ) and sodium dithionite ( in vitro ). Features of these electron donors put a limit on spectrophotometric studies and electrocatalytic applications of nitrogenase. Although it is common to use methyl viologen as an electron donor for many low-potential oxidoreductases, decreased nitrogenase activity is observed with an increasing concentration of methyl viologen, limiting its utility under many circumstances. In this work, we suggest that this concentration-dependent decrease in activity can be explained by the formation of a dimer of the radical cation of methyl viologen (Me 2 V •+ ) 2 at higher methyl viologen concentrations. In addition, viologens functionalized with positively and negatively charged groups were synthesized and studied using spectroscopy and cyclic voltammetry. A sulfonated viologen derivative, 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium radical {[(SPr) 2 V • ] - }, was found to support full nitrogenase activity up to a mediator concentration of 3 mM, while the positively charged viologen derivative was not an efficient reductant of nitrogenase due to the high standard redox potential. The utility of [(SPr) 2 V • ] - as an electron donor for nitrogenase was demonstrated by a simple, sensitive spectrophotometric assay for nitrogenase activity that can provide accurate values for the specific activity and turnover rate constant under argon. Under N 2 , the formation of ammonia was confirmed. Because of the observed full activity of nitrogenase and low overpotential, [(SPr) 2 V • ] - should also prove to be valuable for nitrogenase electrocatalysis, including bioelectrosynthetic N 2 reduction.

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

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N 2 reduction to 2NH 3 : Mo-, V-, and Fe-nitrogenase, named respectively for the metal ( M ) in their active site metallocofactors (metal-ion composition, M Fe 7 ). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N 2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E 4 (4H) ("Janus") state. N 2 binding/reduction in this state is coupled to reductive elimination ( re ) of the two hydrides as H 2 , the forward direction of a reductive-elimination/oxidative-addition ( re/oa ) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N 2 /D 2 . Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H 2 formation by hydride protonolysis ( k HP ) but significant differences in the rate constant for H 2 re with N 2 binding/reduction ( k re ). We now report that V-nitrogenase also exhibits HD formation during N 2 /D 2 turnover (and H 2 inhibition of N 2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N 2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant ( k HP ) for H 2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant ( k re ) for the re of H 2 coupled to N 2 binding/reduction, which decreases in the order Mo > V > Fe.

Molybdenum nitrogenase catalyzes the reduction of dinitrogen into ammonia, which requires the coordinated transfer of eight electrons to the active site cofactor (FeMoco) through the intermediacy of an [8Fe-7S] cluster (P-cluster), both housed in the molybdenum-iron protein (MoFeP). Previous studies on MoFeP from two different organisms, Azotobacter vinelandii ( Av) and Gluconacetobacter diazotrophicus ( Gd), have established that the P-cluster is conformationally flexible and can undergo substantial structural changes upon two-electron oxidation to the P OX state, whereby a backbone amidate and an oxygenic residue (Ser or Tyr) ligate to two of the cluster's Fe centers. This redox-dependent change in coordination has been implicated in the conformationally gated electron transfer in nitrogenase. Here, we have investigated the role of the oxygenic ligand in Av MoFeP, which natively contains a Ser ligand (βSer188) to the P-cluster. Three variants were generated in which (1) the oxygenic ligand was eliminated (βSer188Ala), (2) the P-cluster environment was converted to the one in Gd MoFeP (βPhe99Tyr/βSer188Ala), and (3) two oxygenic ligands were simultaneously included (βPhe99Tyr). Our studies have revealed that the P-cluster can become compositionally labile upon oxidation and reversibly lose one or two Fe centers in the absence of the oxygenic ligand, while still retaining wild-type-like dinitrogen reduction activity. Our findings also suggest that Av and Gd MoFePs evolved with specific preferences for Ser and Tyr ligands, respectively, and that the structural control of these ligands must extend beyond the primary and secondary coordination spheres of the P-cluster. The P-cluster adds to the increasing number of examples of inherently labile Fe-S clusters whose compositional instability may be an obligatory feature to enable redox-linked conformational changes to facilitate multielectron redox reactions.

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least-studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic ( i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the FAD cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors., (© 2019 Varghese et al.)

FeS proteins are metalloproteins prevalent in the metabolic pathways of most organisms, playing key roles in a wide range of essential cellular processes. A member of this protein family, the Fe protein of nitrogenase, is a homodimer that contains a redox-active [Fe 4 S 4 ] cluster at the subunit interface and an ATP-binding site within each subunit. During catalysis, the Fe protein serves as the obligate electron donor for its catalytic partner, transferring electrons concomitant with ATP hydrolysis to the cofactor site of the catalytic component to enable substrate reduction. The effectiveness of Fe protein in electron transfer is reflected by the unique reactivity of nitrogenase toward small-molecule substrates. Most notably, nitrogenase is capable of catalyzing the ambient reduction of N 2 and CO into NH 4 + and hydrocarbons, respectively, in reactions that parallel the important industrial Haber-Bosch and Fischer-Tropsch processes. Other than participating in nitrogenase catalysis, the Fe protein also functions as an essential factor in nitrogenase assembly, which again highlights its capacity as an effective, ATP-dependent electron donor. Recently, the Fe protein of a soil bacterium, Azotobacter vinelandii, was shown to act as a reductase on its own and catalyze the ambient conversion of CO 2 to CO at its [Fe 4 S 4 ] cluster either under in vitro conditions when a strong reductant is supplied or under in vivo conditions through the action of an unknown electron donor(s) in the cell. Subsequently, the Fe protein of a mesophilic methanogenic organism, Methanosarcina acetivorans, was shown to catalyze the in vitro reduction of CO 2 and CO into hydrocarbons under ambient conditions, illustrating an impact of protein scaffold on the redox properties of the [Fe 4 S 4 ] cluster and the reactivity of the cluster toward C1 substrates. This reactivity was further traced to the [Fe 4 S 4 ] cluster itself, as a synthetic [Fe 4 S 4 ] compound was shown to catalyze the reduction of CO 2 and CO to hydrocarbons in solutions in the presence of a strong reductant. Together, these observations pointed to an inherent ability of the [Fe 4 S 4 ] clusters and, possibly, the FeS clusters in general to catalyze C1-substrate reduction. Theoretical calculations have led to the proposal of a plausible reaction pathway that involves the formation of hydrocarbons via aldehyde-like intermediates, providing an important framework for further mechanistic investigations of FeS-based activation and reduction of C1 substrates. In this Account, we summarize the recent work leading to the discovery of C1-substrate reduction by protein-bound and free [Fe 4 S 4 ] clusters as well as the current mechanistic understanding of this FeS-based reactivity. In addition, we briefly discuss the evolutionary implications of this discovery and potential applications that could be developed to enable FeS-based strategies for the ambient recycling of unwanted C1 waste into useful chemical commodities.

A major hurdle in the studies of nitrogenase, one of the most complicated metalloenzymes known to date, is to obtain large amounts of intact, active proteins. Nitrogenase and related proteins are often multimeric and consist of metal centers that are critical for their activities. Most notably, the well-studied MoFe protein of Mo-nitrogenase is a heterotetramer that houses two of the most complicated metal clusters found in nature, the P-cluster and the FeMoco (or M-cluster). The structural complexity of these proteins and the oxygen sensitivity of their associated metal clusters, along with the demand for large amounts of high-quality proteins in most downstream analyses, make large-scale, high-yield purification of fully competent nitrogenase proteins a formidable task and yet, at the same time, a prerequisite for the success of nitrogenase research. This chapter highlights several methods that have been developed over the past few decades chiefly for the purification of naturally expressed nitrogenase in the diazotroph Azotobacter vinelandii. In addition, purification and Fe-S reconstitution strategies are also outlined for the heterologously expressed nitrogenase proteins in Escherichia coli.

Nitrogenase is the only known enzymatic system capable of reducing atmospheric dinitrogen to ammonia. This unique reaction requires tightly choreographed interactions between the nitrogenase component proteins, the molybdenum-iron (MoFe)- and iron (Fe)-proteins, as well as regulation of electron transfer between multiple metal centers that are only found in these components. Several decades of research beginning in the 1950s yielded substantial information of how nitrogenase manages the task of N 2 fixation. However, key mechanistic steps in this highly oxygen-sensitive and ATP-intensive reaction have only recently been identified at an atomic level. A critical part in any mechanistic elucidation is the necessity to connect spectroscopic and functional properties of the component proteins to the detailed three-dimensional structures. Structural information derived from X-ray diffraction (XRD) methods has provided detailed atomic insights into the enzyme system and, in particular, its active site FeMo-cofactor. The following chapter outlines the general protocols for the crystallization of Azotobacter vinelandii (Av) nitrogenase component proteins, with a special emphasis on different applications, such as high-resolution XRD, single-crystal spectroscopy, and the structural characterization of bound inhibitors.

Biological nitrogen fixation (BNF) through the enzyme nitrogenase is performed by a unique class of organisms known as diazotrophs. One interesting facet of BNF is that it produces molecular hydrogen (H 2 ) as a requisite by-product. In the absence of N 2 substrate, or under conditions that limit access of N 2 to the enzyme through modifications of amino acids near the active site, nitrogenase activity can be redirected toward a role as a dedicated hydrogenase. In free-living diazotrophs, nitrogenases are tightly regulated to minimize BNF to meet only the growth requirements of the cell, and are often accompanied by uptake hydrogenases that oxidize the H 2 by-product to recover the electrons from this product. The wild-type strain of Azotobacter vinelandii performs all of the tasks described above to minimize losses of H 2 while also growing as an obligate aerobe. Individual alterations to A. vinelandii have been demonstrated that disrupt key aspects of the N 2 reduction cycle, thereby diverting resources and energy toward the production of H 2 . In this work, we have combined three approaches to override the primary regulation of BNF and redirect metabolism to drive biological H 2 production by nitrogenase in A. vinelandii. The resulting H 2 -producing strain was further utilized as a surrogate to study secondary, post-transcriptional regulation of BNF by several key nitrogen-containing metabolites. The improvement in yields of H 2 that were achieved through various combinations of these three approaches was compared and is presented along with the insights into inhibition of BNF by several nitrogen compounds that are common in various waste streams. The findings indicate that both ammonium and nitrite hinder BNF through this secondary inhibition, but urea and nitrate do not. These results provide essential details to inform future biosynthetic approaches to yield nitrogen products that do not inadvertently inhibit BNF.

The enzyme nitrogenase catalyzes the reduction of N 2 to ammonia but also that of protons to H 2 . These reactions compete at the mechanistically central 'Janus' intermediate, denoted E 4 (4H), which has accumulated 4e - /4H + as two bridging Fe-H-Fe hydrides on the active-site cofactor. This state can lose e - /H + by hydride protonolysis (HP) or become activated by reductive elimination ( re) of the two hydrides and bind N 2 with H 2 loss, yielding an E 4 (2N2H) state that goes on to generate two NH 3 molecules. Thus, E 4 (4H) represents the key branch point for these competing reactions. Here, we present a steady-state kinetic analysis that precisely describes this competition. The analysis demonstrates that steady-state, high-electron flux turnover overwhelmingly populates the E 4 states at the expense of less reduced states, quenching HP at those states. The ratio of rate constants for E 4 (4H) hydride protonolysis ( k HP ) versus reductive elimination ( k re ) provides a sensitive measure of competition between these two processes and thus is a central parameter of nitrogenase catalysis. Analysis of measurements with the three nitrogenase variants (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase) reveals that at a fixed N 2 pressure their tendency to productively react with N 2 to produce two NH 3 molecules and an accompanying H 2 , rather than diverting electrons to the side reaction, HP production of H 2 , decreases with their ratio of rate constants, k re / k HP : Mo-nitrogenase, 5.1 atm -1 ; V-nitrogenase, 2 atm -1 ; and Fe-nitrogenase, 0.77 atm -1 (namely, in a 1:0.39:0.15 ratio). Moreover, the lower catalytic effectiveness of the alternative nitrogenases, with more H 2 production side reaction, is not caused by a higher k HP but by a significantly lower k re .

Azotobacter vinelandii produces differentiated cells, called cysts, surrounded by two alginate layers, which are necessary for their desiccation resistance. This alginate contains variable proportions of guluronate residues, resulting from the activity of seven extracytoplasmic epimerases, AlgE1-7. These enzymes are exported by a system secretion encoded by the eexDEF operon; mutants lacking the AlgE1-7 epimerases, the EexDEF or the RpoS sigma factor produce alginate, but are unable to form desiccation resistant cysts. Herein, we found that RpoS was required for full transcription of the algE1-7 and eexDEF genes. We found that the AlgE1-7 protein levels were diminished in the rpoS mutant strain. In addition, the alginate produced in the absence of RpoS was more viscous in the presence of proteases, a phenotype similar to that of the eexD mutant. Primer extension analysis located two promoters for the eexDEF operon, one of them was RpoS-dependent. Thus, during encysting conditions, RpoS coordinates the expression of both the AlgE1-7 epimerases and the EexDEF protein complex responsible for their transport.

Nitrogenases catalyze the biological fixation of inert N 2 into bioavailable ammonium. They are bipartite systems consisting of the catalytic dinitrogenase and a complementary reductase, the Fe protein that is also the site where ATP is hydrolyzed to drive the reaction forward. Three different subclasses of dinitrogenases are known, employing either molybdenum, vanadium or only iron at their active site cofactor. Although in all these classes the mode and mechanism of interaction with Fe protein is conserved, each one encodes its own orthologue of the reductase in the corresponding gene cluster. Here we present the 2.2 Å resolution structure of VnfH from Azotobacter vinelandii, the Fe protein of the alternative, vanadium-dependent nitrogenase system, in its ADP-bound state. VnfH adopts the same conformation that was observed for NifH, the Fe protein of molybdenum nitrogenase, in complex with ADP, representing a state of the functional cycle that is ready for reduction and subsequent nucleotide exchange. The overall similarity of NifH and VnfH confirms the experimentally determined cross-reactivity of both ATP-hydrolyzing reductases.

The enzyme nitrogenase converts N 2 to NH 3 , and hydrogenates many other small unsaturated molecules, using multiple electrons and multiple protons. The protein contains a number of water structures in the vicinity of the active site, FeMo-co, and functional roles have been assigned to two of these with detailed mechanisms proposed for the serial ingress of protons and the egress of product NH 3 . A separate small water pool (SWP), in a different part of the protein surrounding FeMo-co, has unknown function. A recent investigation of protein crystals soaked in low-pH buffer revealed changes in residues near this SWP, and suggested that it could be involved in proton transfer steps. This paper examines the SWP in three protein crystal structures, Azotobacter vinelandii (Av1) and Clostridium pasterianum (Cp1) in their neutral resting states, and Cp1 at low pH. The H atoms, not observed crystallographically, were patched in through density functional calculations using large protein models. Optimisation of the various possibilities, with assessment against crystal dimensions, yielded the most probable distributions of hydrogen atoms in the hydrogen bonds, and the location of H 3 O + in the low-pH state. These detailed structures vary in water content and water involvement with surrounding residues, and vary also in their hydrogen bonding to S atoms of FeMo-co. A conserved mechanism for proton transfer to FeMo-co is not evident, and it is concluded that the SWP has no role in the mechanism of nitrogenase., (© 2018 Federation of European Biochemical Societies.)

Azotobacter vinelandii selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N 2 , with their expression regulated by the presence or absence of different metal cofactors in its environment. Each alternative nitrogenase isoenzyme is predicted to have different electron flux requirements based on in vitro measurements, with the molybdenum nitrogenase requiring the lowest flux and the iron-only nitrogenase requiring the highest. Here, prior characterized strains, derepressed in nitrogenase synthesis and also deficient in uptake hydrogenase, were further modified to generate new mutants lacking the ability to produce poly-β-hydroxybutyrate (PHB). PHB is a storage polymer generated under oxygen-limiting conditions and can represent up to 70% of the cells' dry weight. The absence of such granules facilitated the study of relationships between catalytic biomass and product molar yields across different adaptive respiration conditions. The released hydrogen gas observed during growth, due to the inability of the mutants to recapture hydrogen, allowed for direct monitoring of in vivo nitrogenase activity for each isoenzyme. The data presented here show that increasing oxygen exposure limits equally the in vivo activities of all nitrogenase isoenzymes, while under comparative conditions, the Mo nitrogenase enzyme evolves more hydrogen per unit of biomass than the alternative isoenzymes. IMPORTANCE A. vinelandii has been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered around A. vinelandii 's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targeted in vitro measurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts the in vivo activity of each nitrogenase isoenzyme are lacking. This article aims to provide in vivo nitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation., (Copyright © 2018 American Society for Microbiology.)

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N 2 ) to ammonia (NH 3 ) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N 2 is reduced to NH 3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (P N ) state to the two-electron oxidized P 2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P 2+ , P 1+ , and P N ) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P 1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P 2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as "assembly factors." Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein-assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein-NafY, MoFe protein-NifY, and MoFe protein-NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Jimenez-Vicente et al.)

The Mo and V nitrogenases are structurally homologous yet catalytically distinct in their abilities to reduce CO to hydrocarbons. Here we report a comparative analysis of the CO-reducing activities of the Mo- and V-nitrogenase cofactors (i.e., the M and V clusters) upon insertion of the respective cofactor into the same, cofactor-deficient MoFe protein scaffold. Our data reveal a combined contribution from the protein environment and cofactor properties to the reactivity of nitrogenase toward CO, thus laying a foundation for further mechanistic investigation of the enzymatic CO reduction, while suggesting the potential of targeting both the protein scaffold and the cofactor species for nitrogenase-based applications in the future., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nitrogenases catalyze biological dinitrogen (N 2 ) reduction to ammonia (NH 3 ), and also reduce a number of non-physiological substrates, including carbon dioxide (CO 2 ) to formate (HCOO - ) and methane (CH 4 ). Three versions of nitrogenase are known (Mo-, V-, and Fe-nitrogenase), each showing different reactivities towards various substrates. Normally, electrons for substrate reduction are delivered by the Fe protein component of nitrogenase, with energy coming from the hydrolysis of 2 ATP to 2 ADP+2 Pi for each electron transferred. Recently, it has been demonstrated that energy and electrons can be delivered from an electrode to the catalytic nitrogenase MoFe-protein without the need for Fe protein or ATP hydrolysis. Here, it is demonstrated that both the MoFe- and FeFe-protein can be immobilized as a polymer layer on an electrode and that electron transfer mediated by cobaltocene can drive CO 2 reduction to formate in this system. It was also found that the FeFe-protein diverts a greater percentage of electrons to CO 2 reduction versus proton reduction compared to the MoFe-protein. Quantification of electron flow to products exhibited Faradaic efficiencies of CO 2 conversion to formate of 9% for MoFe protein and 32% for FeFe-protein, with the remaining electrons going to proton reduction to make H 2 ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of many different biological systems, including enzyme kinetics and the genetic code. It has been especially useful in working out the structures and mechanisms of different nitrogenase enzymes, how they can function in oxic environments and the interactions of nitrogen fixation with other aspects of metabolism. Interest in studying A. vinelandii has waned in recent decades, but this bacterium still possesses great potential for new discoveries in many fields and commercial applications. The species is of interest for research because of its genetic pliability and natural competence. Its features of particular interest to industry are its ability to produce multiple valuable polymers - bioplastic and alginate in particular; its nitrogen-fixing prowess, which could reduce the need for synthetic fertilizer in agriculture and industrial fermentations, via coculture; its production of potentially useful enzymes and metabolic pathways; and even its biofuel production abilities. This review summarizes the history and potential for future research using this versatile microbe.