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Markus W Ribbe - One of the best experts on this subject based on the ideXlab platform.
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uncoupling binding of substrate co from turnover by vanadium Nitrogenase
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Chi Chung Lee, Aaron W Fay, Tsuchien Weng, Courtney M Krest, Britt Hedman, Keith O Hodgson, Markus W RibbeAbstract:Biocatalysis by Nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of Nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-Nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-Nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-Nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.
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Nitrogenase and homologs
Journal of Biological Inorganic Chemistry, 2015Co-Authors: Yilin Hu, Markus W RibbeAbstract:Nitrogenase catalyzes biological nitrogen fixation, a key step in the global nitrogen cycle. Three homologous Nitrogenases have been identified to date, along with several structural and/or functional homologs of this enzyme that are involved in Nitrogenase assembly, bacteriochlorophyll biosynthesis and methanogenic process, respectively. In this article, we provide an overview of the structures and functions of Nitrogenase and its homologs, which highlights the similarity and disparity of this uniquely versatile group of enzymes.
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biosynthesis of Nitrogenase metalloclusters
Chemical Reviews, 2014Co-Authors: Markus W Ribbe, Keith O Hodgson, Britt HedmanAbstract:Nitrogenase is a complex metalloenzyme that is best known for its function in biological nitrogen fixation.1,2 Harbored in a group of microbes called diazotrophs, Nitrogenase catalyzes the reduction of nitrogen (N2) to ammonia (NH3) in a reaction that is usually depicted as N2 + 8H+ + 16MgATP + 8e− → 2NH3 + H2 + 16MgADP + 16Pi. This reaction not only represents a key step in the global nitrogen cycle, but also embodies the formidable chemistry of breaking the exceptionally stable N≡N triple bond. Recently, Nitrogenase was shown to reduce carbon monoxide (CO) to hydrocarbons under the same reaction conditions of biological nitrogen fixation,3–6 defining it as a versatile metalloenzyme that is capable of activating N2 and CO and converting them into products of agronomic and economic values. Interestingly, the reactions of N2- and CO-reduction by Nitrogenase parallel two important processes in industry: the Haber-Bosch process, which is used for ammonia production from N2 and hydrogen (H2);7 and the Fischer-Tropsch process, which is used for carbon fuel production from CO and H2.8 However, contrary to the industrial processes, the Nitrogenase-catalyzed reactions occur under ambient conditions, making this enzyme a fascinating subject from the perspective of chemical energy. Three homologous Nitrogenases, namely, the molybdenum (Mo), vanadium (V) and iron (Fe)-only Nitrogenases, have been identified to date.9,10 The best studied among them is the Mo Nitrogenase from Azotobacter vinelandii, which consists of two component proteins. One, designated the Fe protein (NifH), is a γ2-dimer that contains a subunit-bridging [Fe4S4] cluster per dimer and an ATP binding site within each subunit. The other, designated the MoFe protein (NifDK), is an α2β2-tetramer that contains two complex metalloclusters per αβ-dimer: a P-cluster ([Fe8S7]) at the α/β-subunit interface and an M-cluster ([MoFe7S9C-homocitrate]) within the α-subunit.11–14 Catalysis by the Mo Nitrogenase involves the formation of a complex between NifH and NifDK15,16 and the inter-protein transfer of electrons from the [Fe4S4] cluster of NifH, via the P-cluster, to the M-cluster of NifDK, where substrate reduction eventually occurs (Figure 1). Such an electron pathway highlights the functions of the P- and M-clusters in substrate reduction. Both are high-nuclearity metalloclusters with unusual structures not recognized in other biological systems, and both have evaded successful chemical synthesis so far. The unique properties of the P- and M-clusters of Nitrogenase will be discussed below (section 1.1), followed by an overview of proteins involved in the biosynthesis of these clusters (section 1.2). Figure 1 Crystal structure of the ADP•AlF4−-stabilized NifH/NifDK complex (A) and the relative positions of components involved in the transfer of electrons (B). The two subunits of NifH are colored gray and light brown, and the α- and ... 1.1. Properties of the metal clusters in Nitrogenase The P-cluster is bridged between the α- and β-subunits of NifDK at a position that is 10 A below the surface of the protein.11–13 Structurally, it can be viewed as two [Fe4S3] partial cubanes bridged by a μ6-sulfide (Figure 2A and B); whereas chemically, it can exist in three oxidation states (designated the PN, P1+ and POX state, respectively). In the presence of excess dithionite, the P-cluster exists in an all-ferrous, diamagnetic state (designated the PN-cluster). Following the treatment of a dye oxidant [e.g., indigodisulfonate (IDS)], the PN-cluster can be two-electron oxidized to a stable S = integer (3 or 4) state (designated the POX-cluster), which displays a characteristic, parallel-mode electron paramagnetic resonance (EPR) signal at g =11.817–19. Both the PN- and the POX-clusters (Figure 2A and B) are covalently coordinated by six cysteinyl ligands in NifDK, three from the α-subunit (Cysα62, Cysα88 and Cysα154) and three from the β-subunit (Cysβ70, Cysβ95 and Cysβ153). Each of the Cysα62, Cysα154, Cysβ70 and Cysβ153 ligands coordinates one Fe atom, and each of the Cysα88 and Cysβ95 ligands coordinates two Fe atoms of the P-cluster.20,21 However, the core structures of the PN- and POX-clusters are different, with one half of the POX-cluster present in a more open conformation (Figure 2B). Such a structural rearrangement is accompanied by a change in the ligation pattern, as the POX-cluster is coordinated by two more protein ligands than the PN-cluster.21 One of these ligands is Serβ188, which coordinates an Fe atom through an Oγ ligand together with the cysteinyl group of Cysβ153; the other ligand is Cysα88, which coordinates an Fe atom through a backbone amide nitrogen ligand and a cysteinyl group (Figure 2B). Figure 2 Crystal structures of the PN (A) and POX (B) states of the P-cluster and the M-cluster (C). The clusters are shown as ball-and-stick models. The atoms are shown as transparent balls and colored as those in Figure 1; and the ligands are shown as sticks. ... The M-cluster (also called FeMoco or cofactor) is buried within the α-subunit of NifDK, 14 A away from the P-cluster. Structurally, the M-cluster can be viewed as [Fe4S3] and [MoFe3S3] partial cubanes bridged by three μ2-sulfides (Figure 2C). In addition to its metal-sulfur core, the M-cluster also contains an organic homocitrate moiety attached through its 2-hydroxy and 2-carboxyl groups to the Mo atom and a μ6-interstitial carbide coordinated in the central cavity.11–14 The interstitial carbide cannot be exchanged upon turnover, nor can it be used as a substrate and incorporated into the products, suggesting a role of the interstitial carbide in stabilizing the structure of the M-cluster.22,23 However, a function of this atom in indirectly modulating the reactivity of the M-cluster or directly interacting with the substrate cannot be excluded.24 The M-cluster is coordinated by only two ligands in NifDK: Cysα275, which coordinates the terminal Fe atom; and Hisα442, which coordinates the opposite Mo atom. A third residue, Lysα426, provides an additional hydrogen-bonded anchor for homocitrate at the Mo end of the cluster.11–14 In addition to the covalent ligands, the M-cluster is held within NifDK through direct and water-bridged hydrogen bonds. The apparently “simple” coordination pattern of the M-cluster permits extraction of this cluster as an intact entity into organic solvents, such as N-methylformamide (NMF).25–27 The extracted M-cluster was shown to be anionic26 despite a proposed charge of +1 or +3 for the metal-sulfur core of this cluster in the resting state.28,29 The overall negative charge of the M-cluster is believed to originate from its endogenous homocitrate entity, which is −4 if the hydroxyl (-OH) group is deprotonated. The extracted M-cluster can bind CO and cyanide (CN−) at certain oxidation states.30,31 Moreover, it can catalyze the ATP-independent reduction of CO and CN− to hydrocarbons in the presence of a strong reductant, europium(II) diethylenetriaminepentaacetate [Eu(II) DTPA],32 although conditions are yet to be defined for N2 reduction by the extracted M-cluster. Both the solvent-extracted and the protein-bound M-clusters display a characteristic, S = 3/2 EPR signal at g = 4.7, 3.7 and 2.0 in the presence of excess dithionite; however, the signal displayed by the extracted M-cluster is broader in line-shape than that displayed by its protein-bound counterpart.26,33 Moreover, the M-cluster can undergo a reversible one-electron oxidation and reduction process, which is reflected by the disappearance of the S = 3/2 signal upon oxidation and the re-appearance of this signal upon re-reduction.1
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vanadium Nitrogenase a two hit wonder
Dalton Transactions, 2012Co-Authors: Chi Chung Lee, Markus W RibbeAbstract:Nitrogenase catalyzes the biological conversion of atmospheric dinitrogen to bioavailable ammonia. The molybdenum (Mo)- and vanadium (V)-dependent Nitrogenases are two homologous members of this metalloenzyme family. However, despite their similarities in structure and function, the characterization of V-Nitrogenase has taken a much longer and more winding path than that of its Mo-counterpart. From the initial discovery of this nitrogen-fixing system, to the recent finding of its CO-reducing capacity, V-Nitrogenase has proven to be a two-hit wonder in the over-a-century-long research of nitrogen fixation. This perspective provides a brief account of the catalytic function and structural basis of V-Nitrogenase, as well as a short discussion of the theoretical and practical potentials of this unique metalloenzyme.
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extending the carbon chain hydrocarbon formation catalyzed by vanadium molybdenum Nitrogenases
Science, 2011Co-Authors: Chi Chung Lee, Markus W RibbeAbstract:In a small-scale reaction, vanadium-dependent Nitrogenase has previously been shown to catalyze reductive catenation of carbon monoxide (CO) to ethylene, ethane, propylene, and propane. Here, we report the identification of additional hydrocarbon products [α-butylene, n-butane, and methane (CH4)] in a scaled-up reaction featuring 20 milligrams of vanadium-iron protein, the catalytic component of vanadium Nitrogenase. Additionally, we show that the more common molybdenum-dependent Nitrogenase can generate the same hydrocarbons from CO, although CH4 was not detected. The identification of CO as a substrate for both molybdenum- and vanadium-Nitrogenases strengthens the hypothesis that CO reduction is an evolutionary relic of the function of the Nitrogenase family. Moreover, the comparison between the CO-reducing capacities of the two Nitrogenases suggests that the identity of heterometal at the active cofactor site affects the efficiency and product distribution of this reaction.
Stefan Nordlund - One of the best experts on this subject based on the ideXlab platform.
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electron transport to Nitrogenase in rhodospirillum rubrum the role of nad p h as electron donor and the effect of fluoroacetate on Nitrogenase activity
Fems Microbiology Letters, 2006Co-Authors: Erica Brostedt, Anders Lindblad, Janet K Jansson, Stefan NordlundAbstract:The role of the reactions of the TCA cycle in the generation of reductant for Nitrogenase in Rhodospirillum rubrum has been investigated. Addition of fluoroacetate inhibited Nitrogenase activity almost completely when pyruvate or endogenous sources were used as electron donors, whereas the inhibition was incomplete when malate, succinate or fumarate were used. Addition of NAD(P)H to cells supported Nitrogenase activity, both with and without prior addition of fluoroacetate. We suggest that the role of the TCA cycle in nitrogen fixation in R. rubrum is to generate reduced pyridine nucleotides which are oxidized by the components of the electron transport pathway to Nitrogenase.
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the fixabcx genes in rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to Nitrogenase
Journal of Bacteriology, 2004Co-Authors: Tomas Edgren, Stefan NordlundAbstract:In our efforts to identify the components participating in electron transport to Nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo Nitrogenase activity compared to the wild-type values. The in vitro Nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to Nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type Nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which Nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxgenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to Nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.
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electron transport to Nitrogenase in rhodospirillum rubrum role of energization of the chromatophore membrane
Photosynthesis Research, 1997Co-Authors: Anders Lindblad, Stefan NordlundAbstract:Nitrogen fixation is dependent on a source of ATP and the generation of a reductant at low enough red-ox potential to transfer electrons to Nitrogenase. In Rhodospirillum rubrum, grown photoheterotrophically, ATP is produced by photophosphorylation, a process studied in great detail, but the source of reductant for Nitrogenase is as yet unidentified. In this report we have studied the effect on nitrogen fixation when the energization of the chromatophore membranes was changed, by decreasing the light intensity or by addition of uncouplers. When the light intensity was lowered a pronounced decrease in Nitrogenase activity was observed although there was no decrease in the ATP/ADP ratio. The inhibition observed was not due to ADP-ribosylation, as the same effect was observed in a mutant devoid of the enzymes in the metabolic regulatory cascade operating in R. rubrum and some other diazotrophs. Even at low concentrations of the uncouplers used, a drastic decrease in the ATP/ADP ratio was observed. However, this decrease in the ATP/ADP ratio did not cause a decrease in Nitrogenase activity. At higher concentrations of uncouplers, Nitrogenase activity decreased but the ATP/ADP ratio remained essentially at a constant low level. These results support a model in which reduction of the electron donor(s) to Nitrogenase in R. rubrum is coupled to the energization of the chromatophore membranes.
Caroline S Harwood - One of the best experts on this subject based on the ideXlab platform.
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influence of energy and electron availability on in vivo methane and hydrogen production by a variant molybdenum Nitrogenase
Applied and Environmental Microbiology, 2019Co-Authors: Yanning Zheng, Caroline S HarwoodAbstract:The anoxygenic phototrophic bacterium Rhodopseudomonas palustris produces methane (CH4) from carbon dioxide (CO2) and hydrogen (H2) from protons (H+) when it expresses a variant form of molybdenum (Mo) Nitrogenase that has two amino acid substitutions near its active site. We examined the influence of light energy and electron availability on in vivo production of these biofuels. Nitrogenase activity requires large amounts of ATP, and cells exposed to increasing light intensities produced increasing amounts of CH4 and H2 As expected for a phototroph, intracellular ATP increased with increasing light intensity, but there was only a loose correlation between ATP content and CH4 and H2 production. There was a much stronger correlation between decreased intracellular ADP and increased gas production with increased light intensity, suggesting that the rate-limiting step for CH4 and H2 production by R. palustris is inhibition of Nitrogenase by ADP. Increasing the amounts of electrons available to Nitrogenase by providing cells with organic alcohols, using nongrowing cells, blocking electrons from entering the Calvin cycle, or blocking H2 uptake resulted in higher yields of H2 and, in some cases, CH4 Our results provide a more complete understanding of the constraints on Nitrogenase-based production of biofuels.IMPORTANCE A variant form of Mo Nitrogenase catalyzes the conversion of CO2 and protons to the biofuels CH4 and H2 A constant supply of electrons and ATP is needed to drive these reduction reactions. The bacterium R. palustris generates ATP from light and has a versatile metabolism that makes it ideal for manipulating electron availability intracellularly. We therefore explored its potential as a biocatalyst for CH4 and H2 production. We found that intracellular ADP had a major effect on biofuel production, more pronounced than the effect caused by ATP. This is probably due to inhibition of Nitrogenase activity by ADP. In general, the amount of CH4 produced by the variant Nitrogenase in vivo was affected by electron availability much less than was the amount of H2 produced. This study shows the nature of constraints on in vivo biofuel production by variant Mo Nitrogenase.
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light driven carbon dioxide reduction to methane by Nitrogenase in a photosynthetic bacterium
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Kathryn R Fixen, Zhi-yong Yang, Lance C. Seefeldt, Yanning Zheng, Derek F Harris, Sudipta Shaw, Dennis R Dean, Caroline S HarwoodAbstract:Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled Nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO2) to methane (CH4). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO2 reduction to CH4 in vivo using this remodeled Nitrogenase. Conversion of CO2 to CH4 by R. palustris required constitutive expression of Nitrogenase, which was achieved by using a variant of the transcription factor NifA that is able to activate expression of Nitrogenase under all growth conditions. Also, light was required for generation of ATP by cyclic photophosphorylation. CH4 production by R. palustris could be controlled by manipulating the distribution of electrons and energy available to Nitrogenase. This work shows the feasibility of using microbes to generate hydrocarbons from CO2 in one enzymatic step using light energy.
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how posttranslational modification of Nitrogenase is circumvented in rhodopseudomonas palustris strains that produce hydrogen gas constitutively
Applied and Environmental Microbiology, 2012Co-Authors: Erin K Heiniger, Yasuhiro Oda, Sudip K Samanta, Caroline S HarwoodAbstract:Nitrogenase catalyzes the conversion of dinitrogen gas (N2) and protons to ammonia and hydrogen gas (H2). This is a catalytically difficult reaction that requires large amounts of ATP and reducing power. Thus, Nitrogenase is not normally expressed or active in bacteria grown with a readily utilized nitrogen source like ammonium. nifA* mutants of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris have been described that express Nitrogenase genes constitutively and produce H2 when grown with ammonium as a nitrogen source. This raised the regulatory paradox of why these mutants are apparently resistant to a known posttranslational modification system that should switch off the activity of Nitrogenase. Microarray, mutation analysis, and gene expression studies showed that posttranslational regulation of Nitrogenase activity in R. palustris depends on two proteins: DraT2, an ADP-ribosyltransferase, and GlnK2, an NtrC-regulated PII protein. GlnK2 was not well expressed in ammonium-grown NifA* cells and thus not available to activate the DraT2 Nitrogenase modification enzyme. In addition, the NifA* strain had elevated Nitrogenase activity due to overexpression of the nif genes, and this increased amount of expression overwhelmed a basal level of activity of DraT2 in ammonium-grown cells. Thus, insufficient levels of both GlnK2 and DraT2 allow H2 production by an nifA* mutant grown with ammonium. Inactivation of the Nitrogenase posttranslational modification system by mutation of draT2 resulted in increased H2 production by ammonium-grown NifA* cells.
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functional genomic analysis of three Nitrogenase isozymes in the photosynthetic bacterium rhodopseudomonas palustris
Journal of Bacteriology, 2005Co-Authors: Yasuhiro Oda, Sudip K Samanta, Federico E Rey, Xiudan Liu, Tingfen Yan, Jizhong Zhou, Caroline S HarwoodAbstract:The photosynthetic bacterium Rhodopseudomonas palustris is one of just a few prokaryotes described so far that has vnf and anf genes for alternative vanadium cofactor (V) and iron cofactor (Fe) Nitrogenases in addition to nif genes for a molybdenum cofactor (Mo) Nitrogenase. Transcriptome data indicated that the 32 genes in the nif gene cluster, but not the anf or vnf genes, were induced in wild-type and Mo Nitrogenase-expressing strains grown under nitrogen-fixing conditions in Mo-containing medium. Strains that were unable to express a functional Mo Nitrogenase due to mutations in Mo Nitrogenase structural genes synthesized functional V and Fe Nitrogenases and expressed vnf and anf genes in nitrogen-fixing growth media that contained Mo and V at concentrations far in excess of those that repress alternative Nitrogenase gene expression in other bacteria. Thus, not only does R. palustris have multiple enzymatic options for nitrogen fixation, but in contrast to reports on other nitrogen-fixing bacteria, the expression of its alternative Nitrogenases is not repressed by transition metals. Between 95 and 295 genes that are not directly associated with Nitrogenase synthesis and assembly were induced under nitrogen-fixing conditions, depending on which Nitrogenase was being used by R. palustris. Genes for nitrogen acquisition were expressed at particularly high levels during alternative Nitrogenase-dependent growth. This suggests that alternative Nitrogenase-expressing cells are relatively starved for nitrogen and raises the possibility that fixed nitrogen availability may be the primary signal that controls the synthesis of the V and Fe Nitrogenases.
Chi Chung Lee - One of the best experts on this subject based on the ideXlab platform.
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uncoupling binding of substrate co from turnover by vanadium Nitrogenase
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Chi Chung Lee, Aaron W Fay, Tsuchien Weng, Courtney M Krest, Britt Hedman, Keith O Hodgson, Markus W RibbeAbstract:Biocatalysis by Nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of Nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-Nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-Nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-Nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.
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vanadium Nitrogenase a two hit wonder
Dalton Transactions, 2012Co-Authors: Chi Chung Lee, Markus W RibbeAbstract:Nitrogenase catalyzes the biological conversion of atmospheric dinitrogen to bioavailable ammonia. The molybdenum (Mo)- and vanadium (V)-dependent Nitrogenases are two homologous members of this metalloenzyme family. However, despite their similarities in structure and function, the characterization of V-Nitrogenase has taken a much longer and more winding path than that of its Mo-counterpart. From the initial discovery of this nitrogen-fixing system, to the recent finding of its CO-reducing capacity, V-Nitrogenase has proven to be a two-hit wonder in the over-a-century-long research of nitrogen fixation. This perspective provides a brief account of the catalytic function and structural basis of V-Nitrogenase, as well as a short discussion of the theoretical and practical potentials of this unique metalloenzyme.
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extending the carbon chain hydrocarbon formation catalyzed by vanadium molybdenum Nitrogenases
Science, 2011Co-Authors: Chi Chung Lee, Markus W RibbeAbstract:In a small-scale reaction, vanadium-dependent Nitrogenase has previously been shown to catalyze reductive catenation of carbon monoxide (CO) to ethylene, ethane, propylene, and propane. Here, we report the identification of additional hydrocarbon products [α-butylene, n-butane, and methane (CH4)] in a scaled-up reaction featuring 20 milligrams of vanadium-iron protein, the catalytic component of vanadium Nitrogenase. Additionally, we show that the more common molybdenum-dependent Nitrogenase can generate the same hydrocarbons from CO, although CH4 was not detected. The identification of CO as a substrate for both molybdenum- and vanadium-Nitrogenases strengthens the hypothesis that CO reduction is an evolutionary relic of the function of the Nitrogenase family. Moreover, the comparison between the CO-reducing capacities of the two Nitrogenases suggests that the identity of heterometal at the active cofactor site affects the efficiency and product distribution of this reaction.
John W Peters - One of the best experts on this subject based on the ideXlab platform.
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excitation rate determines product stoichiometry in photochemical ammonia production by cds quantum dot Nitrogenase mofe protein complexes
ACS Catalysis, 2020Co-Authors: Katherine A Brown, Lance C. Seefeldt, John W Peters, Hayden Kallas, Jesse Ruzicka, Bryant Chica, David W Mulder, Gordana Dukovic, Paul W KingAbstract:The reduction of dinitrogen (N2) to ammonia (NH3) by Nitrogenase MoFe protein is coupled to chemically driven electron transfer by Nitrogenase Fe protein, where H2 is an obligatory side product. Di...
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control of nitrogen fixation in bacteria that associate with cereals
Nature microbiology, 2020Co-Authors: Minhyung Ryu, John W Peters, Jing Zhang, Tyler Toth, Devanshi Khokhani, Barney A Geddes, Florence Mus, Amaya GarciacostasAbstract:Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on Nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous Nitrogenase production confers an energy burden. Here, we engineer inducible Nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place Nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in Nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of Nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.
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light driven dinitrogen reduction catalyzed by a cds Nitrogenase mofe protein biohybrid
Science, 2016Co-Authors: Katherine A Brown, Derek F Harris, John W Peters, Gordana Dukovic, Molly B Wilker, Andrew J Rasmussen, Nimesh Khadka, Hayden Hamby, Stephen Keable, Lance C. SeefeldtAbstract:The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme Nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the Nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the Nitrogenase complex under optimal conditions. Inhibitors of Nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.
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a late methanogen origin for molybdenum dependent Nitrogenase
Geobiology, 2011Co-Authors: Eric S Boyd, Ariel D Anbar, Scott R Miller, Trinity L Hamilton, Matt Lavin, John W PetersAbstract:Mounting evidence indicates the presence of a near complete biological nitrogen cycle in redox-stratified oceans during the late Archean to early Proterozoic (c. 2.5-2.0 Ga). It has been suggested that the iron (Fe)- or vanadium (V)-dependent Nitrogenase rather than molybdenum (Mo)-dependent form was responsible for dinitrogen fixation during this time because oceans were depleted in Mo and rich in Fe. We evaluated this hypothesis by examining the phylogenetic relationships of proteins that are required for the biosynthesis of the active site cofactor of Mo-Nitrogenase in relation to structural proteins required for Fe-, V- and Mo-Nitrogenase. The results are highly suggestive that among extant nitrogen-fixing organisms for which genomic information exists, Mo-Nitrogenase is unlikely to have been associated with the Last Universal Common Ancestor. Rather, the origin of Mo-Nitrogenase can be traced to an ancestor of the anaerobic and hydrogenotrophic methanogens with acquisition in the bacterial domain via lateral gene transfer involving an anaerobic member of the Firmicutes. A comparison of substitution rates estimated for proteins required for the biosynthesis of the Nitrogenase active site cofactor and for a set of paralogous proteins required for the biosynthesis of bacteriochlorophyll suggests that Nif emerged from a Nitrogenase-like ancestor approximately 1.5-2.2 Ga. An origin and ensuing proliferation of Mo-Nitrogenase under anoxic conditions would likely have occurred in an environment where anaerobic methanogens and Firmicutes coexisted and where Mo was at least episodically available, such as in a redox-stratified Proterozoic ocean basin.