Explore the vast range of titles available.

The structure of vanadium nitrogenase reveals key differences from its counterpart molybdenum nitrogenase, particularly in the way it ligands its FeV cofactor, that help to explain the basis for the unique properties of these two nitrogenases. Nitrogenases catalyze the reduction of dinitrogen (N 2 ) gas to ammonium at a complex heterometallic cofactor. This most commonly occurs at the FeMo cofactor (FeMoco), a [Mo–7Fe–9S–C] cluster whose exact reactivity and substrate-binding mode remain unknown. Alternative nitrogenases replace molybdenum with either vanadium or iron and differ in reactivity, most prominently in the ability of vanadium nitrogenase to reduce CO to hydrocarbons. Here we report the 1.35-Å structure of vanadium nitrogenase from Azotobacter vinelandii . The 240-kDa protein contains an additional α-helical subunit that is not present in molybdenum nitrogenase. The FeV cofactor (FeVco) is a [V–7Fe–8S–C] cluster with a homocitrate ligand to vanadium. Unexpectedly, it lacks one sulfide ion compared to FeMoco, which is replaced by a bridging ligand, likely a μ-1,3-carbonate. The anion fits into a pocket within the protein that is obstructed in molybdenum nitrogenase, and its different chemical character helps to rationalize the altered chemical properties of this unique N 2 - and CO-fixing enzyme.

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.

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. Biological nitrogen fixation requires low-potential electrons from ferredoxin or flavodoxin. Here the authors show how the soil diazotroph Azotobacter vinelandii employs the NADH:ferredoxin oxidoreductase RNF1 complex to lower the midpoint potential of the electron from NADH to reduce ferredoxin.

ABSTRACT 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. Flavin transferase ApbE is responsible for the flavinylation of ferredoxin:NAD+-oxidoreductase (Rnf).

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. The element nitrogen is required for all forms of life, and is an essential component of important biological molecules such as DNA and proteins. The most abundant form of nitrogen is dinitrogen, which comprises 78% of the Earth’s atmosphere. However, dinitrogen is highly unreactive, and so the nitrogen must be converted into a more reactive form before it can be used biologically. The only known enzyme capable of carrying out this reaction is called nitrogenase, but how this enzyme performs this difficult task is still not understood. Enzymes contain a region known as the active site, to which substrates – the molecules that the enzyme acts upon – bind. The active site of nitrogenase contains a region called the FeMo-cofactor, which must transform from an inactive to an active state to catalyze the conversion of dinitrogen to ammonia. Another substrate of the nitrogenase enzyme is a molecule called selenocyanate, which is made up of atoms of selenium, carbon and nitrogen. Spatzal, Perez et al. examined the structure of the active site of nitrogenase taken from the bacteria species Azotobacter vinelandii while the enzyme transformed selenocyanate. This revealed unexpected structural changes of the FeMo-cofactor that significantly challenge previous assumptions about how the active site works. For example, a single selenium atom from selenocyanate can be incorporated into a specific position of the FeMo-cofactor, which highlights the importance of this position for the enzyme’s initial interaction with substrates. Spatzal, Perez et al. then used the inserted selenium atom as a probe to investigate the changes in the active site structure that occur when either reacting with a substrate called acetylene or being inhibited by carbon monoxide. This revealed that selenium can migrate into the positions taken up by three of the FeMo-cofactor’s nine sulfur atoms (the three “belt-sulfurs”) during these interactions. The active site was not previously thought to be active in this way: this will need to be taken into account in all future models that describe how dinitrogen is converted into a biologically useful form. In the future, Spatzal, Perez et al. will investigate in detail how these “belt-sulfur” atoms exchange with atoms from the substrate, where the removed sulfur is stored, and the pathway by which it returns. Further experiments will also characterize the active site during the transformation of dinitrogen.

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.

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. Here, the authors characterize selenium and sulphur incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii under turnover conditions using EPR.

All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii , expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium- or iron-dependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii , it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM , vnfEN , and anfOR , are not required for the artificial Anf system in E. coli . This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

Life 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.

The oxygen-sensitive molybdenum-dependent nitrogenase of Azotobacter vinelandii is protected from oxidative damage by a reversible ‘switch-off’ mechanism 1 . It forms a complex with a small ferredoxin, FeSII (ref.  2 ) or the ‘Shethna protein II’ 3 , which acts as an O 2 sensor and associates with the two component proteins of nitrogenase when its [2Fe:2S] cluster becomes oxidized 4 , 5 . Here we report the three-dimensional structure of the protective ternary complex of the catalytic subunit of Mo-nitrogenase, its cognate reductase and the FeSII protein, determined by single-particle cryo-electron microscopy. The dimeric FeSII protein associates with two copies of each component to assemble a 620 kDa core complex that then polymerizes into large, filamentous structures. This complex is catalytically inactive, but the enzyme components are quickly released and reactivated upon oxygen depletion. The first step in complex formation is the association of FeSII with the more O 2 -sensitive Fe protein component of nitrogenase during sudden oxidative stress. The action of this small ferredoxin represents a straightforward means of protection from O 2 that may be crucial for the maintenance of recombinant nitrogenase in food crops. A small ferredoxin (Shethna protein II) of Azotobacter vinelandii can provide protection from O 2 stress that may be crucial for the maintenance of recombinant nitrogenase in food crops.