Health, Safety & Environment

NITROGENASE AND NITROGEN FIXATION

Exploring the alternatives of biological nitrogen fixation

Most biological nitrogen fixation (BNF) results from the activity of the molybdenum nitrogenase (Mo-nitrogenase, Nif), an oxygen-sensitive metalloenzyme complex found in all known diazotrophs. Two alternative forms of nitrogenase, the vanadium nitrogenase (V-nitrogenase, Vnf) and the iron-only nitrogenase (Fe-only nitrogenase, Anf) have also been identified in the genome of some organisms that encode for Nif. It has been suggested that alternative nitrogenases were responsible for N2-fixation on early Earth because oceans were depleted of bioavailable Mo. Results of recent phylogenetic- and structure-based studies suggest, however, that such an evolutionary path is unlikely, and favor a new model for a stepwise evolution of nitrogenase where the V-nitrogenase and the Fe-only nitrogenase are not the ancestor of the Mo-nitrogenase. Rather, Mo-nitrogenase emerged within the methanogenic archaea and then gave rise to the alternative forms suggesting they arose later in response to the availability of fixed N2 and local environmental factors that influenced metal availability. This review summarizes the current state of knowledge on (1) the biochemistry of these complex systems highlighting the common and specific structural features and catalytic activities of the enzymes, (2) the recent progress in defining the discrete set of genes associated to N2-fixation and the regulatory features that coordinate the differential expression of genes in response to metal availability, and (3) the diverse taxonomic and phylogenic distribution of nitrogenase enzymes and the evolutionary history of BNF from the perspective of metal content and metal availability.

F. Mus, A. B. Alleman, N. Pence, L. C. Seefeldt, and J. W. Peters,Exploring the alternatives of biological nitrogen fixation, Metallomics, 2018, 10, 523-538.

 

               

Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein

The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M-or V-cluster). However, the V-nitrogenase is similar to 600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an similar to 100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still similar to 6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase. IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The "normalization" of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the "weighted" contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.

J. G. Rebelein, C. C. Lee, M. Newcomb, Y. L. Hu, and M. W. Ribbe,Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein, Mbio, 2018, 9.

 

               

A VTVH MCD and EPR Spectroscopic Study of the Maturation of the "Second" Nitrogenase P-Cluster

The P-cluster of the nitrogenase MoFe protein is a [Fe8S7] cluster that mediates efficient transfer of electrons to the active site for substrate reduction. Arguably the most complex homometallic FeS cluster found in nature, the biosynthetic mechanism of the P-cluster is of considerable theoretical and synthetic interest to chemists and biochemists alike. Previous studies have revealed a biphasic assembly mechanism of the two P-clusters in the MoFe protein upon incubation with Fe protein and ATP, in which the first P-cluster is formed through fast fusion of a pair of [Fe4S4]+ clusters within 5 min and the second P-cluster is formed through slow fusion of the second pair of [Fe4S4] + clusters in a period of 2 h. Here we report a VTVH MCD and EPR spectroscopic study of the biosynthesis of the slow-forming, second P-cluster within the MoFe protein. Our results show that the first major step in the formation of the second P-cluster is the conversion of one of the precursor [Fe4S4] + clusters into the integer spin cluster [Fe4S3-(4)](alpha), a process aided by the assembly protein NifZ, whereas the second major biosynthetic step appears to be the formation of a diamagnetic cluster with a possible structure of [Fe4S7-8](beta), which is eventually converted into the P-cluster.

K. Rupnik, C. C. Lee, Y. L. Hu, M. W. Ribbe, and B. J. Hales,A VTVH MCD and EPR Spectroscopic Study of the Maturation of the "Second" Nitrogenase P-Cluster, Inorganic chemistry, 2018, 57, 4719-4725.

 

Phosphorus and species regulate N2fixation by herbaceous legumes in longleaf pine savannas

Longleaf pine savannas house a diverse community of herbaceous N2-fixing legume species that have the potential to replenish nitrogen (N) losses from fire. Whether legumes fill this role depends on the factors that regulate symbiotic fixation, including soil nutrients such as phosphorus (P) and molybdenum (Mo) and the growth and fixation strategies of different species. In greenhouse experiments, we determined how these factors influence fixation for seven species of legumes grown in pure field soil from two different regions of the southeastern US longleaf pine ecosystem. We first added P and Mo individually and in combination, and found that P alone constrained fixation. Phosphorus primarily influenced fixation by regulating legume growth. Second, we added N to plants and found that species either downregulated fixation (facultative strategy) or maintained fixation at a constant rate (obligate strategy). Species varied nearly fourfold in fixation rate, reflecting differences in growth rate, taxonomy and fixation strategy. However, fixation responded strongly to P addition across all species in our study, suggesting that the P cycle regulates N inputs by herbaceous legumes.

 M. R. Ament, J. A. Tierney, L. O. Hedin, E. A. Hobbie, and N. Wurzburger,Phosphorus and species regulate N-2 fixation by herbaceous legumes in longleaf pine savannas, Oecologia, 2018, 187, 281-290.

NITROGENASE

Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions

Cyanobacteria is a remarkable group of prokaryotic photosynthetic microorganisms, with several genera capable of fixing atmospheric nitrogen (N-2) and presenting a wide range of morphologies. Although the nitrogenase complex is not present in all cyanobacterial taxa, it is spread across several cyanobacterial strains. The nitrogenase complex has also a high theoretical potential for biofuel production, since H2 is a by-product produced during N2 fixation. In this review we discuss the significance of a relatively wide variety of cell morphologies and metabolic strategies that allow spatial and temporal separation of N2 fixation from photosynthesis in cyanobacteria. Phylogenetic reconstructions based on 16S rRNA and nifD gene sequences shed light on the evolutionary history of the two genes. Our results demonstrated that (i) sequences of genes involved in nitrogen fixation (nifD) from several morphologically distinct strains of cyanobacteria are grouped in similarity with their morphology classification and phylogeny, and (ii) nifD genes from heterocytous strains share a common ancestor. By using this data we also discuss the evolutionary importance of processes such as horizontal gene transfer and genetic duplication for nitrogenase evolution and diversification. Finally, we discuss the importance of H2 synthesis in cyanobacteria, as well as strategies and challenges to improve cyanobacterial H-2 production.

A. A. Esteves-Ferreira, J. H. F. Cavalcanti, M. Vaz, L. V. Alvarenga, A. Nunes-Nesi, and W. L. Araujo,Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions, Genetics and Molecular Biology, 2017, 40, 261-275.

Examining the relationship between coordination mode and reactivity of dinitrogen

Molecular nitrogen (N2) is the most abundant gas in Earth's atmosphere, but its low reactivity has hampered its use as a precursor to higher value nitrogen-containing compounds. Coordination of N2 to metal centres offers a way to overcome this intrinsic inertness and allows the discovery of new transformations. The expanding family of isolable N2 coordination complexes exhibits various bonding modes that, in particular cases, facilitate catalytic or stoichiometric transformations of the N2 unit. In this Review, we survey metal complexes of N2 in order to correlate bonding mode with functionalization propensity. Although many factors influence the functionalization of N2, we propose that coordination mode could be more important than previously recognized.

R. J. Burford, and M. D. Fryzuk,Examining the relationship between coordination mode and reactivity of dinitrogen, Nature Reviews Chemistry, 2017, 1.

Nitrogenase Cofactor Assembly: An Elemental Inventory

Nitrogenase is known for its remarkable ability to catalyze the reduction of N2 to NH3, and C-1 substrates to short-chain hydrocarbon products, under ambient conditions. The best-studied Mo-nitrogenase utilizes a complex metallocofactor as the site of substrate binding and reduction. Designated the M-cluster, this [MoFe7S9C(R-homocitrate)] cluster can be viewed as [MoFe3S3] and [Fe4S3] subclusters bridged by three mu(2)-sulfides and one mu(6)-interstitial carbide, with its Mo end further coordinated by an R-homocitrate moiety. The unique cofactor has attracted considerable attention ever since its discovery; however, the complexity of its structure has hindered mechanistic understanding and chemical synthesis of this cofactor. Motivated by the pressing questions related to the structure and function of the nitrogenase cofactor, one major thrust of our research has been to unravel the key biosynthetic steps of this metallocluster to cultivate a deeper understanding of these reactions and their effects on functionalizing the cofactor. In this Account, we will discuss our recent work that provides insights into how simple Fe and S atoms, along with a single C atom, a heterometallic Mo atom and an organic homocitrate entity, are assembled into one of the most complex metalloclusters known in Nature. Combined biochemical, spectroscopic and structural studies have led us to a working model of M-cluster assembly, which starts with the sequential synthesis of small [Fe2S2] and [Fe4S4] units by NifS/U, followed by the coupling and rearrangement of two [Fe4S4] clusters on NifB concomitant with the insertion of an interstitial carbide and a "9th sulfur" that give rise to a [Fe8S9C] core that is nearly indistinguishable in structure to the M-cluster except for the absence of Mo and homocitrate. This 8Fe core is then matured into an M-cluster on NifEN upon substitution of a Mo-homocitrate conjugate for one terminal Fe atom of the cluster prior to transfer of the M-cluster to its target binding site in the catalytic component of Mo-nitrogenase. Taking stock of the elemental inventory during the cofactor assembly process, the core Fe and S atoms are derived from modular fusion of FeS building blocks, going through 2Fe, 4Fe and 8Fe stages to generate an 8Fe core of the cofactor. However, such a flow of Fe/S along the biosynthetic pathway of the M-cluster is "intervened" by the insertion of C and Mo, which renders the cofactor unique in structure and reactivity. Insertion of C occurs through a novel, radical SAM-dependent mechanism, which involves SN2-type methyl transfer from SAM to a [Fe4S4] cluster pair, hydrogen abstraction of the transferred methyl group by a SAM-derived 5'-dA radical, and further deprotonation of the resultant methylene radical concomitant with radical chemistry-based coupling and rearrangement of the [Fe4S4] cluster pair into an [Fe8S9C] core. Insertion of Mo, on the other hand, employs an ATPase-dependent mechanism that parallels metal trafficking in the biosynthesis of molybdopterin and CO dehydrogenase cofactors. These findings provide a nice framework for further exploration of the "black box" of nitrogenase cofactor assembly and function.

N. S. Sickerman, M. W. Ribbe, and Y. L. Hu,Nitrogenase Cofactor Assembly: An Elemental Inventory, Accounts of Chemical Research, 2017, 50, 2834-2841.

 

Nitrogenase

Synthetic analogues of nitrogenase metallocofactors: challenges and developments

Nitrogenase is the only known biological system capable of reducing N2 to NH3, which is a critical component of bioavailable nitrogen fixation. Since the discovery of discrete iron-sulfur metalloclusters within the nitrogenase MoFe protein, synthetic inorganic chemists have sought to reproduce the structural features of these clusters in order to understand how they facilitate the binding, activation and hydrogenation of N2. Through the decades following the initial identification of these clusters, significant progress has been made to synthetically replicate certain compositional and functional aspects of the biogenic clusters. Although much work remains to generate synthetic iron-sulfur clusters that can reduce N2 to NH3, the insights borne from past and recent developments are discussed in this concept article.

Sickerman, N. S., Tanifuji, K., Hu, Y. L., and Ribbe, M. W.,Synthetic Analogues of Nitrogenase Metallocofactors: Challenges and Developments, Chemistry-a European Journal, 2017, 23, 12425-12432.

Is there computational support for an unprotonated carbon in the E4 state of nitrogenase?

In the key enzyme for nitrogen fixation in nature, nitrogenase, the active site has a metal cluster with seven irons and one molybdenum bound by bridging sulfurs. Surprisingly, there is also a carbon in the center of the cluster, with a role that is not known. A mechanism has been suggested experimentally, where two hydrides leave as a hydrogen molecule in the critical E4 state. A structure with two hydrides, two protonated sulfurs and an unprotonated carbon has been suggested for this state. Rather recently, DFT calculations supported the experimental mechanism but found an active state where the central carbon is protonated all the way to CH3 . Even more recently, another DFT study was made that instead supported the experimentally suggested structure. To sort out the origin of these quite different computational results, additional calculations have here been performed using different DFT functionals. The conclusion from these calculations is very clear and shows no computational support for an unprotonated carbon in E4 . (c) 2017 Wiley Periodicals, Inc.

P. E. M. Siegbahn,Is there computational support for an unprotonated carbon in the E4 state of nitrogenase?, Journal of computational chemistry, 2017.

Medicago truncatula Molybdate Transporter type 1 (MtMOT1.3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency

Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporter family Molybdate Transporter type 1 (MOT1) were identified in the model legume Medicago truncatula and their expression in nodules was determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Transposable Element from Nicotiana tabacum (Tnt1) insertional mutant line were carried out in the one M. truncatula MOT1 family member specifically expressed in nodules. Among the five MOT1 members present in the M. truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed impaired growth concomitant with a reduction of nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild-type-like phenotype. These data are consistent with a model in which MtMOT1.3 is responsible for introducing molybdate into nodule cells, which is later used to synthesize functional nitrogenase.

M. Tejada-Jimenez, P. Gil-Diez, J. Leon-Mediavilla, J. Wen, K. S. Mysore, J. Imperial, and M. Gonzalez-Guerrero,Medicago truncatula Molybdate Transporter type 1 (MtMOT1.3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency, The New phytologist, 2017, 216, 1223-1235.

Structural characterization of the nitrogenase molybdenum-iron protein with the substrate acetylene trapped near the active site

The biological reduction of dinitrogen (N2) to ammonia is catalyzed by the complex metalloenzyme nitrogenase. Structures of the nitrogenase component proteins, Iron (Fe) protein and Molybdenumiron (MoFe) protein, and the stabilized complexes these component proteins, have been determined, providing a foundation for a number of fundamental aspects of the complicated catalytic mechanism. The reduction of dinitrogen to ammonia is a complex process that involves the binding of N2 followed by reduction with multiple electrons and protons. Electron transfer into nitrogenase is typically constrained to the unique electron donor, the Fe protein. These constraints have prevented structural characterization of the active site with bound substrate. Recently it has been realized that selected amino acid substitutions in the environment of the active site metal cluster (Ironmolybdenum cofactor, FeMo-co) allow substrates to persist even in the resting state. Reported here is a 1.70A crystal structure of a nitrogenase MoFe protein alpha-96(ArgGln) variant with the alternative substrate acetylene trapped in a channel in close proximity to FeMo-co. Complementary theoretical calculations support the validity of the acetylene interaction at this site and is also consistent with more favorable interactions in the variant MoFe protein compared to the native MoFe protein. This work represents the first structural evidence of a substrate trapped in the nitrogenase MoFe protein and is consistent with earlier assignments of proposed substrate pathways and substrate binding sites deduced from biochemical, spectroscopic, and theoretical studies.

S. M. Keable, J. Vertemara, O. A. Zadvornyy, B. J. Eilers, K. Danyal, A. J. Rasmussen, L. De Gioia, G. Zampella, L. C. Seefeldt, and J. W. Peters,Structural characterization of the nitrogenase molybdenum-iron protein with the substrate acetylene trapped near the active site, J Inorg Biochem, 2017, 180, 129-134.

 

Revisiting the Mossbauer Isomer Shifts of the FeMoco Cluster of Nitrogenase and the Cofactor Charge

Despite decades of research, the structure-activity relationship of nitrogenase is still not understood. Only recently was the full molecular structure of the FeMo cofactor (FeMoco) revealed, but the charge and metal oxidation states of FeMoco have been controversial. With the recent identification of the interstitial atom as a carbide and the more recent revised oxidation-state assignment of the molybdenum atom as Mo(III), here we revisit the Mossbauer properties of FeMoco. By a detailed error analysis of density functional theory-computed isomer shifts and computing isomer shifts relative to the P-cluster, we find that only the charge of [MoFe7S9C]1- fits the experimental data. In view of the recent Mo(III) identification, the charge of [MoFe7S9C]1- corresponds to a formal oxidation-state assignment of Mo(III)3Fe(II)4Fe(III), although due to spin delocalization, the physical oxidation state distribution might also be interpreted as Mo(III)1Fe(II)4Fe(2.5)2Fe(III), according to a localized orbital analysis of the MS = 3/2 broken symmetry solution. These results can be reconciled with the recent spatially resolved anomalous dispersion study by Einsle et al. that suggests the Mo(III)3Fe(II)4Fe(III) distribution, if some spin localization (either through interactions with the protein environment or through vibronic coupling) were to take place.

Bjornsson, R., Neese, F., and DeBeer, S.,Revisiting the Mossbauer Isomer Shifts of the FeMoco Cluster of Nitrogenase and the Cofactor Charge, Inorg Chem, 2017, 56, 1470-1477.

Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.

Yang, J., Xie, X., Yang, M., Dixon, R., and Wang, Y. P.,Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity, Proceedings of the National Academy of Sciences of the United States of America, 2017.

Carbon dioxide reduction

Activation and reduction of carbon dioxide by nitrogenase iron proteins

The iron (Fe) proteins of molybdenum (Mo) and vanadium (V) nitrogenases mimic carbon monoxide (CO) dehydrogenase in catalyzing the interconversion between CO2 and CO under ambient conditions. Catalytic reduction of CO2 to CO is achieved in vitro and in vivo upon redox changes of the Fe-protein-associated [Fe4S4] clusters. These observations establish the Fe protein as a model for investigation of CO2 activation while suggesting its biotechnological adaptability for recycling the greenhouse gas into useful products.

Rebelein, J. G., Stiebritz, M. T., Lee, C. C., and Hu, Y.,Activation and reduction of carbon dioxide by nitrogenase iron proteins, Nature chemical biology, 2017, 13, 147-149.

Nitrogen fixation

Iron controls over di-nitrogen fixation in karst tropical forest

Limestone tropical forests represent a meaningful fraction of the land area in Central America (25%) and Southeast Asia (40%). These ecosystems are marked by high biological diversity, CO2 uptake capacity, and high pH soils, the latter making them fundamentally different from the majority of lowland tropical forest areas in the Amazon and Congo basins. Here, we examine the role of bedrock geology in determining biological nitrogen fixation (BNF) rates in volcanic (low pH) vs. limestone (high pH) tropical forests located in the Maya Mountains of Belize. We experimentally test how BNF in the leaf-litter responds to nitrogen, phosphorus, molybdenum, and iron additions across different parent materials. We find evidence for iron limitation of BNF rates in limestone forests during the wet but not dry season (response ratio 3.2 +/- 0.2; P = 0.03). In contrast, BNF in low pH volcanic forest soil was stimulated by the trace-metal molybdenum during the dry season. The parent-material induced patterns of limitation track changes in siderophore activity and iron bioavailability among parent materials. These findings point to a new role for iron in regulating BNF in karst tropical soils, consistent with observations for other high pH systems such as the open ocean and calcareous agricultural ecosystems.

Winbourne, J. B., Brewer, S. W., and Houlton, B. Z.,Iron controls over di-nitrogen fixation in karst tropical forest, Ecology, 2017, 98, 773-781.

Activation and reduction of carbon dioxide by nitrogenase iron proteins

The iron (Fe) proteins of molybdenum (Mo) and vanadium (V) nitrogenases mimic carbon monoxide (CO) dehydrogenase in catalyzing the interconversion between CO2 and CO under ambient conditions. Catalytic reduction of CO2 to CO is achieved in vitro and in vivo upon redox changes of the Fe-protein-associated [Fe4S4] clusters. These observations establish the Fe protein as a model for investigation of CO2 activation while suggesting its biotechnological adaptability for recycling the greenhouse gas into useful products.

Rebelein, J. G., Stiebritz, M. T., Lee, C. C., and Hu, Y.,Activation and reduction of carbon dioxide by nitrogenase iron proteins, Nature chemical biology, 2017, 13, 147-149.

Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a DeltanifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

Sippel, D., Schlesier, J., Rohde, M., Trncik, C., Decamps, L., Djurdjevic, I., Spatzal, T., Andrade, S. L., and Einsle, O.,Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion, Journal of biological inorganic chemistry: JBIC: a publication of the Society of Biological Inorganic Chemistry, 2017, 22, 161-168.

The nitrogenase FeMo-cofactor precursor pormed by NifB protein: A diamagnetic cluster containing eight iron atoms

The biological activation of N2 occurs at the FeMo-cofactor, a 7Fe-9S-Mo-C-homocitrate cluster. FeMo-cofactor formation involves assembly of a Fe6-8 -SX -C core precursor, NifB-co, which occurs on the NifB protein. Characterization of NifB-co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe-4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB-co and deliver it to the NifEN protein, upon which FeMo-cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB-co-loaded form provided an opportunity for Mossbauer analysis of NifB-co. The results indicate that NifB-co is a diamagnetic (S=0) 8-Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the (57) Fe electric hyperfine interactions deduced from the Mossbauer analysis suggests that NifB-co is either a 4Fe(2+) -4Fe(3+) or 6Fe(2+) -2Fe(3+) cluster having valence-delocalized states.

Guo, Y., Echavarri-Erasun, C., Demuez, M., Jimenez-Vicente, E., Bominaar, E. L., and Rubio, L. M.,The Nitrogenase FeMo-Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms, Angewandte Chemie (International ed. in English), 2016, 55, 12764-7.

Nitrogenase vanadium
Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates

Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized. Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l. We confirmed the use of the alternative vanadium-based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum. These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum-limited habitats.

Darnajoux, R., Zhang, X., McRose, D. L., Miadlikowska, J., Lutzoni, F., Kraepiel, A. M., and Bellenger, J. P.,Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates, The New phytologist, 2016.

Proteome profiling of the rhodobacter capsulatus molybdenum response reveals a role of IscN in nitrogen fixation by Fe-nitrogenase

Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed. IMPORTANCE Biological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, including Rhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome in R. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions.

Hoffmann, M. C., Wagner, E., Langklotz, S., Pfander, Y., Hott, S., Bandow, J. E., and Masepohl, B.,Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase, Journal of Bacteriology, 2016, 198, 633-643.

Biosynthesis of the metalloclusters of nitrogenases

Nitrogenase is a versatile metalloenzyme that is capable of catalyzing two important reactions under ambient conditions: the reduction of nitrogen (N2) to ammonia (NH3), a key step in the global nitrogen cycle; and the reduction of carbon monoxide (CO) and carbon dioxide (CO2) to hydrocarbons, two reactions useful for recycling carbon waste into carbon fuel. The molybdenum (Mo)- and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Each of them contains a P-cluster and a cofactor, two high-nuclearity metalloclusters that have crucial roles in catalysis.

This review summarizes the progress that has been made in elucidating the biosynthetic mechanisms of the P-cluster and cofactor species of nitrogenase, focusing on what is known about the assembly mechanisms of the two metalloclusters in Mo-nitrogenase and giving a brief account of the possible assembly schemes of their counterparts in V-nitrogenase, which are derived from the homology between the two nitrogenases. Expected final online publication date for the Annual Review of Biochemistry Volume 85 is June 02, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Hu, Y., and Ribbe, M. W.,Biosynthesis of the Metalloclusters of Nitrogenases, Annual review of biochemistry, 2016.

Leaf-residing Methylobacterium species fix nitrogen and promote biomass and seed production in Jatropha curcas

Background: Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated nitrogen-fixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective of plant-microbe interactions.

Results: An analysis of 1017 endophytic bacterial isolates derived from different parts of Jatropha revealed that diazotrophs were abundant and diversely distributed into five classes belonging to alpha, beta, gamma-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and surprisingly, 30.2 % which were able to fix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and significantly improved plant height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and 96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an increase in female-male flower ratio.

Conclusion: The ability of Methylobacterium to fix nitrogen and colonize leaf tissues serves as an important trait for Jatropha. This bacteria-plant interaction may significantly contribute to Jatropha's tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant's nitrogen supply from the leaves and may be exploited to significantly improve the productivity and Green Index of Jatropha biofuel.

Madhaiyan, M., Alex, T. H. H., Ngoh, S. T., Prithiviraj, B., and Ji, L. H.,Leaf-residing Methylobacterium species fix nitrogen and promote biomass and seed production in Jatropha curcas, Biotechnology for Biofuels, 2015, 8.

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - observation of a candidate promoting vibration

We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a similar to 12 Ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at similar to 84, 116, 189, and 226 cm(-1). Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N(2)ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226 cm-1 band is assigned as a potential 'promoting vibration' in the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group. (C) 2015 Elsevier Inc All rights reserved.

Maiuri, M., Delfino, I., Cerullo, G., Manzoni, C., Pelmenschikov, V., Guo, Y. S., Wang, H. X., Gee, L. B., Dapper, C. H., Newton, W. E., and Cramer, S. P.,Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - Observation of a candidate promoting vibration, Journal of inorganic biochemistry, 2015, 153, 128-135.

Combining a nitrogenase scaffold and a synthetic compound into an artificial enzyme

Nitrogenase catalyzes substrate reduction at its cofactor center ([(Cit)MoFe7S9C](n) ; designated M-cluster). Here, we report the formation of an artificial, nitrogenase-mimicking enzyme upon insertion of a synthetic model complex ([Fe6S9(SEt) 2]4-; designated Fe-6(RHH)) into the catalytic component of nitrogenase (designated NifDK(apo)). Two Fe-6(RHH) clusters were inserted into NifDK(apo), rendering the conformation of the resultant protein (designated NifDK(Fe)) similar to the one upon insertion of native M-clusters. NifDK(Fe) can work together with the reductase component of nitrogenase to reduce C2H2 in an ATP-dependent reaction. It can also act as an enzyme on its own in the presence of Eu(II)DTPA, displaying a strong activity in C2H2 reduction while demonstrating an ability to reduce CN- to C-1-C-3 hydrocarbons in an ATP-independent manner. The successful outcome of this work provides the proof of concept and underlying principles for continued search of novel enzymatic activities based on this approach.

Tanifuji, K., Lee, C. C., Ohki, Y., Tatsumi, K., Hu, Y. L., and Ribbe, M. W.,Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme, Angewandte Chemie-International Edition, 2015, 54, 14022-14025.

Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions

Biological nitrogen fixation can be catalysed by three isozymes of nitrogenase: molybdenum (Mo)-nitrogenase, vanadium (V)-nitrogenase and iron-only (Fe)-nitrogenase. The activity of these isozymes strongly depends on their metal cofactors, molybdenum, vanadium and iron, and their bioavailability in ecosystems.

Here, we show how metal bioavailability can be affected by the presence of tannic acid (organic matter), and the subsequent consequences on diazotrophic growth of the soil bacterium Azotobacter vinelandii.

In the presence of tannic acids, A. vinelandii produces a higher amount of metallophores, which coincides with an active, regulated and concomitant acquisition of molybdenum and vanadium under cellular conditions that are usually considered not molybdenum limiting. The associated nitrogenase genes exhibit decreased nifD expression and increased vnfD expression. Thus, in limiting bioavailable metal conditions, A. vinelandii takes advantage of its nitrogenase diversity to ensure optimal diazotrophic growth.

Jouogo Noumsi, C., Pourhassan, N., Darnajoux, R., Deicke, M., Wichard, T., Burrus, V., and Bellenger, J. P.,Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions, Environmental microbiology reports, 2016, 8, 76-84.

Proteome profiling of the rhodobacter capsulatus molybdenum response reveals a role of IscN in nitrogen fixation by Fe-nitrogenase

Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed. IMPORTANCE Biological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, including Rhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome in R. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions.

Hoffmann, M. C., Wagner, E., Langklotz, S., Pfander, Y., Hott, S., Bandow, J. E., and Masepohl, B.,Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase, Journal of Bacteriology, 2016, 198, 633-643.

Biosynthesis of the metalloclusters of nitrogenases

Nitrogenase is a versatile metalloenzyme that is capable of catalyzing two important reactions under ambient conditions: the reduction of nitrogen (N2) to ammonia (NH3), a key step in the global nitrogen cycle; and the reduction of carbon monoxide (CO) and carbon dioxide (CO2) to hydrocarbons, two reactions useful for recycling carbon waste into carbon fuel. The molybdenum (Mo)- and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Each of them contains a P-cluster and a cofactor, two high-nuclearity metalloclusters that have crucial roles in catalysis.

This review summarizes the progress that has been made in elucidating the biosynthetic mechanisms of the P-cluster and cofactor species of nitrogenase, focusing on what is known about the assembly mechanisms of the two metalloclusters in Mo-nitrogenase and giving a brief account of the possible assembly schemes of their counterparts in V-nitrogenase, which are derived from the homology between the two nitrogenases. Expected final online publication date for the Annual Review of Biochemistry Volume 85 is June 02, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Hu, Y., and Ribbe, M. W.,Biosynthesis of the Metalloclusters of Nitrogenases, Annual review of biochemistry, 2016.

Leaf-residing methylobacterium species fix nitrogen and promote biomass and seed production in Jatropha curcas

Background: Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated nitrogen-fixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective of plant-microbe interactions.

Results: An analysis of 1017 endophytic bacterial isolates derived from different parts of Jatropha revealed that diazotrophs were abundant and diversely distributed into five classes belonging to alpha, beta, gamma-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and surprisingly, 30.2 % which were able to fix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and significantly improved plant height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and 96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an increase in female-male flower ratio.

Conclusion: The ability of Methylobacterium to fix nitrogen and colonize leaf tissues serves as an important trait for Jatropha. This bacteria-plant interaction may significantly contribute to Jatropha's tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant's nitrogen supply from the leaves and may be exploited to significantly improve the productivity and Green Index of Jatropha biofuel.

Madhaiyan, M., Alex, T. H. H., Ngoh, S. T., Prithiviraj, B., and Ji, L. H.,Leaf-residing Methylobacterium species fix nitrogen and promote biomass and seed production in Jatropha curcas, Biotechnology for Biofuels, 2015, 8.

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - Observation of a candidate promoting vibration

We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a similar to 12 Ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at similar to 84, 116, 189, and 226 cm(-1). Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N(2)ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226 cm-1 band is assigned as a potential 'promoting vibration' in the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group. (C) 2015 Elsevier Inc All rights reserved.

Maiuri, M., Delfino, I., Cerullo, G., Manzoni, C., Pelmenschikov, V., Guo, Y. S., Wang, H. X., Gee, L. B., Dapper, C. H., Newton, W. E., and Cramer, S. P.,Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - Observation of a candidate promoting vibration, Journal of inorganic biochemistry, 2015, 153, 128-135.

Combining a nitrogenase scaffold and a synthetic compound into an artificial enzyme

Nitrogenase catalyzes substrate reduction at its cofactor center ([(Cit)MoFe7S9C](n) ; designated M-cluster). Here, we report the formation of an artificial, nitrogenase-mimicking enzyme upon insertion of a synthetic model complex ([Fe6S9(SEt) 2]4-; designated Fe-6(RHH)) into the catalytic component of nitrogenase (designated NifDK(apo)). Two Fe-6(RHH) clusters were inserted into NifDK(apo), rendering the conformation of the resultant protein (designated NifDK(Fe)) similar to the one upon insertion of native M-clusters. NifDK(Fe) can work together with the reductase component of nitrogenase to reduce C2H2 in an ATP-dependent reaction. It can also act as an enzyme on its own in the presence of Eu(II)DTPA, displaying a strong activity in C2H2 reduction while demonstrating an ability to reduce CN- to C-1-C-3 hydrocarbons in an ATP-independent manner. The successful outcome of this work provides the proof of concept and underlying principles for continued search of novel enzymatic activities based on this approach.

Tanifuji, K., Lee, C. C., Ohki, Y., Tatsumi, K., Hu, Y. L., and Ribbe, M. W.,Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme, Angewandte Chemie-International Edition, 2015, 54, 14022-14025.

Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions

Biological nitrogen fixation can be catalysed by three isozymes of nitrogenase: molybdenum (Mo)-nitrogenase, vanadium (V)-nitrogenase and iron-only (Fe)-nitrogenase. The activity of these isozymes strongly depends on their metal cofactors, molybdenum, vanadium and iron, and their bioavailability in ecosystems.

Here, we show how metal bioavailability can be affected by the presence of tannic acid (organic matter), and the subsequent consequences on diazotrophic growth of the soil bacterium Azotobacter vinelandii.

In the presence of tannic acids, A. vinelandii produces a higher amount of metallophores, which coincides with an active, regulated and concomitant acquisition of molybdenum and vanadium under cellular conditions that are usually considered not molybdenum limiting. The associated nitrogenase genes exhibit decreased nifD expression and increased vnfD expression. Thus, in limiting bioavailable metal conditions, A. vinelandii takes advantage of its nitrogenase diversity to ensure optimal diazotrophic growth.

Jouogo Noumsi, C., Pourhassan, N., Darnajoux, R., Deicke, M., Wichard, T., Burrus, V., and Bellenger, J. P.,Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions, Environmental microbiology reports, 2016, 8, 76-84.

Recent progress in transition-metal-catalyzed reduction of molecular dinitrogen under ambient reaction conditions

This paper describes our recent progress in catalytic nitrogen fixation by using transition-metal dinitrogen complexes as catalysts. Two reaction systems for the catalytic transformation of molecular dinitrogen into ammonia and its equivalent such as silylamine under ambient reaction conditions have been achieved by the molybdenum-, iron-, and cobalt-dinitrogen complexes as catalysts. Many new findings presented here may provide new access to the development of economical nitrogen fixation in place of the Haber Bosch process.

Nishibayashi, Y.,Recent Progress in Transition-Metal-Catalyzed Reduction of Molecular Dinitrogen under Ambient Reaction Conditions, Inorganic Chemistry, 2015, 54, 9234-9247.

Significant differences of monooxotungsten(IV) and dioxotungsten(VI) benzenedithiolates containing two intramolecular NH center dot center dot center dot S hydrogen bonds from molybdenum analogues

A monooxotungsten(IV) benzenedithiolate complex containing two intramolecular NH center dot center dot center dot S hydrogen bonds, (NEt4)(2)[(WO)-O-IV(1,2-S-2-3-t-BuNHCOC6H3)(2)] (1-W), was synthesized via a ligand-exchange reaction between a new starting complex, (NEt4)(2)[(WO)-O-IV(SC6F5)(4)], and a partially deprotonated dithiol. When dithiol was used in solution, the oxo ligand was protonated and removed to afford (NEt4)(2)[W-IV(1,2-S-2-3-t-BuNH-COC6H3)(3)]. The trans isomer, trans-1-W, was crystallized, and the molecular structure was determined via X-ray analysis. Trans-1-W was gradually isomerized by heating it in solution and it eventually achieved an approximately 1 : 1 mixture of trans/cis isomers after 48 days. However, a slightly excess amount of trans isomer remained, so the isomerization rate was considerably slower than that of the molybdenum analogue. In the presence of NEt4BH4, deuteration of the NH protons was observed in acetonitrile-d(3). The oxidation of both trans-and cis-1-W by Me3NO afforded the corresponding dioxotungsten(VI) complex, (NEt4)(2)[(WO2)-O-VI(1,2-S-2-3-t-BuNHCOC6H3)(2)] (2-W), as a single isomer. The contributions of the NH center dot center dot center dot S hydrogen bonds to the bond distances, vibrational data, and electrochemical properties are described via comparisons with their molybdenum analogues. The results of this comparative study yielded insights into both tungsten and molybdenum enzymes.

Okamura, A. T., Omi, Y., Fujii, M., Tatsumi, M., and Onitsuka, K.,Significant differences of monooxotungsten(IV) and dioxotungsten(VI) benzenedithiolates containing two intramolecular NH center dot center dot center dot S hydrogen bonds from molybdenum analogues, Dalton Transactions, 2015, 44, 18090-18100.

Correction: significant differences of monooxotungsten(iv) and dioxotungsten(vi) benzenedithiolates containing two intramolecular NHS hydrogen bonds from molybdenum analogues

Correction for 'Significant differences of monooxotungsten(iv) and dioxotungsten(vi) benzenedithiolates containing two intramolecular NHS hydrogen bonds from molybdenum analogues' by Taka-aki Okamura et al., Dalton Trans., 2015, 44, 18090-18100.

Okamura, T. A., Omi, Y., Fujii, M., Tatsumi, M., and Onitsuka, K.,Correction: Significant differences of monooxotungsten(iv) and dioxotungsten(vi) benzenedithiolates containing two intramolecular NHS hydrogen bonds from molybdenum analogues, Dalton transactions (Cambridge, England : 2003), 2015.

Evidence for functionally relevant encounter complexes in nitrogenase catalysis

Nitrogenase is the only enzyme that can convert atmospheric dinitrogen (N2) into biologically usable ammonia (NH3). To achieve this multielectron redox process, the nitrogenase component proteins, MoFe-protein (MoFeP) and Fe-protein (FeP), repeatedly associate and dissociate in an ATP-dependent manner, where one electron is transferred from FeP to MoFeP per association. Here, we provide experimental evidence that encounter complexes between Fe? and MoFeP play a functional role in nitrogenase catalysis. The encounter complexes are stabilized by electrostatic interactions involving a positively charged patch on the beta-subunit of MoFeP. Three single mutations (beta Asn399Glu, beta Lys400Glu, and beta Arg401Glu) in this patch were generated in Azotobacter vinelandii MoFeP. All of the resulting variants displayed decreases in specific catalytic activity, with the beta K400E mutation showing the largest effect. As simulated by the Thomeley-Lowe kinetic scheme, this single mutation lowered the rate constant for FeP-MoFeP association S-fold. We also found that the beta K400E mutation did not affect the coupling of ATP hydrolysis with electron transfer (ET) between FeP and MoFeP. These data suggest a mechanism where FeP initially forms encounter complexes on the MoFeP beta-subunit surface en route to the ATP-activated, ET-competent complex over the alpha beta-interface.

Owens, C. P., Katz, F. E. H., Carter, C. H., Luca, M. A., and Tezcan, F. A.,Evidence for Functionally Relevant Encounter Complexes in Nitrogenase Catalysis, Journal of the American Chemical Society, 2015, 137, 12704-12712.

Response of the nitrogen-fixing lichen Lobaria pulmonaria to phosphorus, molybdenum, and vanadium

Nitrogen-fixing lichens (cyanolichens) are an important source of nitrogen (N) in Pacific Northwest forests, but limitation of lichen growth by elements essential for N fixation is poorly understood. To investigate how nutrient limitation may affect cyanolichen growth rates, we fertilized a tripartite cyanobacterial lichen (Lobaria pulmonaria) and a green algal non-nitrogen fixing lichen (Usnea longissima) with the micronutrients molybdenum (Mo) and vanadium (V), both known cofactors for enzymes involved in N fixation, and the macronutrient phosphorus (P). We then grew treated lichens in the field for one year in western Oregon, USA. Lichen growth was very rapid for both species and did not differ across treatments, despite a previous demonstration of P-limitation in L. pulmonaria at a nearby location. To reconcile these disparate findings, we analyzed P, Mo, and V concentrations, natural abundance delta N-15 isotopes, %N and change in thallus N in Lobaria pulmonaria from both growth experiments. Nitrogen levels in deposition and in lichens could not explain the large difference in growth or P limitation observed between the two studies. Instead, we provide evidence that local differences in P availability may have caused site-specific responses of Lobaria to P fertilization. In the previous experiment, Lobaria had low background levels of P, and treatment with P more than doubled growth. In contrast, Lobaria from the current experiment had much higher background P concentrations, similar to P-treated lichens in the previous experiment, consistent with the idea that ambient variation in P availability influences the degree of P limitation in cyanolichens. We conclude that insufficient P, Mo, and V did not limit the growth of either cyanolichens or chlorolichens at the site of the current experiment. Our findings point to the need to understand landscape-scale variation in P availability to cyanolichens, and its effect on spatial patterns of cyanolichen nutrient limitation and N fixation.

Marks, J. A., Pett-Ridge, J. C., Perakis, S. S., Allen, J. L., and McCune, B.,Response of the nitrogen-fixing lichen Lobaria pulmonaria to phosphorus, molybdenum, and vanadium, Ecosphere, 2015, 6.

The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism

Nitrogenase contains a well defined and conserved chain of water molecules leading to the FeMo cofactor (FeMo-co, an [Fe7MoCS9] cluster with bidentate chelation of Mo by homocitrate) that is the active site where N2 and other substrates are sequentially hydrogenated using multiple protons and electrons. The function of this chain is proposed to be a proton wire, serially translocating protons to triply-bridging S3B of FeMo-co, where, concomitant with electron transfer to FeMo-co, an H atom is generated on S3B. Density functional simulations of this proton translocation mechanism are reported here, using a large 269-atom model that includes all residues hydrogen bonded to and surrounding the water chain, and likely to influence proton transfer: three carboxylate O atoms of obligatory homocitrate are essential. The mechanism involves the standard two components of the Grotthuss mechanism, namely H atom slides that shift H3O+ from one water site to the next, and HOH molecular rotations that convert backward (posterior) OH bonds in the water chain to forward (anterior) OH bonds. The topography of the potential energy surface for each of these steps has been mapped. H atom slides pass through very short (ca. 2.5 angstrom) O-H-O hydrogen bonds, while HOH rotations involve the breaking of O-H center dot center dot center dot O hydrogen bonds, and the occurrence of long (up to 3.6 angstrom) separations between contiguous water molecules. Both steps involve low potential energy barriers, -1. During operation of the Grotthuss mechanism in nitrogenase there are substantial displacements of water molecules along the chain, occurring as ripples. These characteristics of the 'Grotthuss two-step', coupled with a buffering ability of two carboxylate O atoms of homocitrate, and combined with density functional characterisation of the final proton slide from the ultimate water molecule to S3B (including electron addition), have been choreographed into a complete mechanism for serial hydrogenation of FeMo-co. The largest potential barrier is estimated to be 14 kcal mol-1. These results are discussed in the context of reactivity data for nitrogenase, and the occurrence of a comparable water chain in cytochrome-c oxidase. Further investigation of the low frequency conformational dynamics of the nitrogenase proteins, coupling proton transfer with other events in the nitrogenase cycle, is briefly canvassed.

Dance, I.,The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism, Dalton Transactions, 2015, 44, 18167-18186.

Binding of dinitrogen to an iron-sulfur-carbon site

Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe- N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

Coric, I., Mercado, B. Q., Bill, E., Vinyard, D. J., and Holland, P. L.,Binding of dinitrogen to an iron-sulfur-carbon site, Nature, 2015, 526, 96-9.

Binding of dinitrogen to an iron-sulfur-carbon site

Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe-N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

Coric, I., Mercado, B. Q., Bill, E., Vinyard, D. J., and Holland, P. L.,Binding of dinitrogen to an iron-sulfur-carbon site, Nature, 2015.

Light-enhanced bioaccumulation of molybdenum by nitrogen-deprived recombinant anoxygenic photosynthetic bacterium Rhodopseudomonas palustris

As molybdenum (Mo) is an indispensable metal for plant nitrogen metabolisms, accumulation of dissolved Mo into bacterial cells may connect to the development of bacterial fertilizers that promote plant growth. In order to enhance Mo bioaccumulation, nitrogen removal and light illumination were examined in anoxygenic photosynthetic bacteria (APB) because APB possess Mo nitrogenase whose synthesis is strictly regulated by ammonium ion concentration. In addition, an APB, Rhodopseudomonas palustris, transformed with a gene encoding Mo-responsive transcriptional regulator ModE was constructed. Mo content was most markedly enhanced by the removal of ammonium ion from medium and light illumination while their effects on other metal contents were limited. Increases in contents of trace metals including Mo by the genetic modification were observed. Thus, these results demonstrated an effective way to enrich Mo in the bacterial cells by the culture conditions and genetic modification.

Naito, T., Sachuronggui, Ueki, M., and Maeda, I.,Light-enhanced bioaccumulation of molybdenum by nitrogen-deprived recombinant anoxygenic photosynthetic bacterium Rhodopseudomonas palustris, Bioscience, biotechnology, and biochemistry, 2015, 1-7.

Nitrogenase

Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Phylogenetic evidence indicates that oxygen (O2)-sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. Aerobic bacteria that fix N2 have adapted a number of strategies to protect Nif from inactivation by O2, including spatial and temporal segregation of Nif from O2 and respiratory consumption of O2. Here we report the complement of Nif-encoding genes in 189 diazotrophic genomes. We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles. IMPORTANCE: Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Nif emerged in an anaerobe and later diversified into aerobes. Here we show that the transition of Nif from anaerobic to aerobic metabolism was accompanied by both gene recruitment and gene loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes is strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protective mechanisms. Rather, gene recruitment was likely a response to more efficiently regulate Nif under oxic conditions that favor protein turnover.

Boyd, E. S., Costas, A. M., Hamilton, T. L., Mus, F., and Peters, J. W.,Evolution of Molybdenum Nitrogenase during the Transition from Anaerobic to Aerobic Metabolism, J Bacteriol, 2015, 197, 1690.

X-ray absorption (XAS) and X-ray emission spectroscopy (XES) provide element specific probes of the geometric and electronic structures of metalloprotein active sites. As such, these methods have played an integral role in nitrogenase research beginning with the first EXAFS studies on nitrogenase in the late 1970s. Herein, we briefly explain the information that can be extracted from XAS and XES. We then highlight the recent applications of these methods in nitrogenase research. The influence of X-ray spectroscopy on our current understanding of the atomic structure and electronic structure of iron molybdenum cofactor (FeMoco) is emphasized. Contributions of X-ray spectroscopy to understanding substrate interactions and cluster biosynthesis are also discussed. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. (C) 2014 Elsevier B.V. All rights reserved.

Kowalska, J., and DeBeer, S.,The role of X-ray spectroscopy in understanding the geometric and electronic structure of nitrogenase, Biochimica Et Biophysica Acta-Molecular Cell Research, 2015, 1853, 1406.

Mechanism

Nitrogenase converts gaseous dinitrogen into biologically accessible ammonia. The binding of N2 to a reduced and protonated form of the FeMo-cofactor of nitrogenase including its central carbon ligand was investigated by means of density functional calculations. It was found the central ligand to stabilize the cluster N2 can associate to iron or molybdenum with iron being the preferred binding site. While endo and exo binding modes were investigated the exo modes are more stable. Implications on the mechanism of N2 reduction are discussed.

Hallmen, P. P. and Kastner, J., N2 Binding to the FeMo-Cofactor of Nitrogenase, Zeitschrift fur Anorganische und Allgemeine Chemie, 2015, 641, 118-122.

Active site

Biological nitrogen fixation is enabled by molybdenum-dependent nitrogenase enzymes, which effect the reduction of dinitrogen to ammonia using an Fe7MoS9C active site, referred to as the iron molybdenum cofactor or FeMoco. In this mini-review, we summarize the current understanding of the molecular and electronic structure of FeMoco. The advances in our understanding of the active site structure are placed in context with the parallel evolution of synthetic model studies. The recent discovery of Mo(III) in the FeMoco active site is highlighted with an emphasis placed on the important role that model studies have played in this finding. In addition, the reactivities of synthetic models are discussed in terms of their relevance to the enzymatic system

Bjornsson, R., Neese, F., Schrock, R. R., Einsle, O., and DeBeer, S., The discovery of Mo(III) in FeMoco: reuniting enzyme and model chemistry, Journal of Biological Inorganic Chemistry, 2015, 20, 447-460.

Influence of nano nutrients on heterocyst-forming cyanobacterium Anabaena ambigua Rao [Nitrogenase]

The present study deals with the influence of nanoparticles on the growth of heterocyst-forming cyanobacterium Anabaena ambigua Rao [A100].

The nano molybdenum and iron particles have been synthesized respectively from molybdenum trioxide (MoO3) and iron chloride (FeCl3) employing sodium borohydride (NaBH4) as a reducing agent and characterized comprehensively.

BG11 (N-) media containing micronutrients like molybdenum [as sodium molybdate] and iron [ammonium ferric citrate]were replaced respectively by different concentrations of nano molybdenum [BG11 (N-Mo- + nano Mo)] (0, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) and nano iron [BG11 (N-Fe- + nano Fe)] (0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) particles for the cultivation.

The effect of nano nutrients on cyanobacterial growth parameters like heterocyst formation, chlorophyll content, and protein content was analyzed.

The obtained results showed that the high concentrations of nano molybdenum induce heterocyst formation, increase protein content, and decrease chlorophyll content when compared to the control, whereas the nano iron suppresses the growth of cyanobacteria Anabaena ambigua Rao [A 100].

Yuvakkumar, R., Elango, V., Venkatachalam, R., Kannan, N., and Prabu, P., Influence of Nano Nutrients on Heterocyst-Forming Cyanobacterium Anabaena ambigua Rao, Synthesis and Reactivity in Inorganic Metal-Organic and Nano-Metal Chemistry, 2011, 41, 1234-1239.

[Heterocysts: specialized nitrogen-fixing cells formed by some filamentous cyanobacteria (blue-green algae). They fix nitrogen from dinitrogen (N2) in the air using the enzyme nitrogenase.]

Nitrogenase is the complex metalloenzyme responsible for biological dinitrogen reduction. The mechanism of nitrogen fixation in enzymes and in model systems in vitro has been extensively investigated.

Burris, R. H. and Roberts, G. P., Ann. Rev. Nutrition, 1993, 13, 317.
Sellman, D., Agnew. Chem. Int. Ed., 1993, 105, 64.

The microorganisms which fix molecular nitrogen fall into two classes: (a) the symbiotic microorganisms which fix nitrogen in association with plants, e.g., Rhizobium; (b) asymbiotic microorganisms which are free-living and include Azotobacter vinelandii and Clostridium Pasteurianum. From cell-free extracts of C. Pasteurianum, two metalloproteins have been obtained. The hydrogen donating system, azoferredoxin, which contains iron and sulfide and the nitrogen activating system, molybdoferredoxin, which contains molybdenum, iron, and sulfide. The structure of the active centre has been shown by X-ray crystallography to be a Fe-Mo-S cluster.

Kim, J., Woo, D. and Rees, D. C., Biochemistry,1993, 32 ,7104.
Chen, J., Christiansen, J., Campbasso, N., Bolin, J. T., Tittsworth, B. C., Hales, B. J., Rehr, J. J. and Cramer, S. P., Angew. Chem. Int. Edn.,1993, 32,1592.

The mechanism of the N2 fixing enzyme nitrogenase has been reviewed including a discussion of why low molecular weight complexes have not yet been able to copy nitrogenase catalyzed reactions or to act as competitive catalysts with nitrogenase-like activity.

Sellmann, D., Utz, J., Blum, N., Heinemann, F.W., On the function of nitrogenase FeMo cofactors and competitive catalysts: chemical principles, structural blue-prints, and the relevance of iron sulfur complexes for N-2 fixation, Coordination Chemistry Reviews, 1999, 192, 607-627.

Recent developments in understanding the mechanism of the Mo-based nitrogenase are reviewed: how nucleotide binding and hydrolysis are coupled to electron transfer and substrate reduction, how electrons are accumulated and transferred within the MoFe-protein, and how substrates bind and are reduced at the active site metal cluster.

Christiansen, J., Dean, D. R., and Seefeldt, L. C., Mechanistic features of the Mo-containing nitrogenase, Annual Review of Plant Physiology and Plant Molecular Biology, 2001, 52, 269.

The Mo-site and its ligand environment of the FeMo-cofactor (FeMo-co) were studied using the hybrid density functional method B3LYP.

Szilagyi, R.K., Musaev, D. G., and Morokuma, K., Theoretical studies of biological nitrogen fixation. I. Density functional modeling of the Mo-site of the FeMo-cofactor, Inorganic Chemistry, 2001, 40, 766-775

The MoFe protein of the nitrogenase enzyme from Azotobacter vinelandii was altered by substituting the alpha Gly(69) residue by serine. Then both CO and acetylene become competitive inhibitors of dinitrogen reduction. CO is also converted from a non-competitive inhibitor of the wild type enzyme to a competitive inhibitor of acetylene, nitrous oxide, and azide reduction. These results are interpreted in terms of a two-site model with both sites located in close proximity within a specific 4Fe-4S face of FeMo cofactor. Site 1 is a high affinity acetylene-binding site to which CO also binds; but dinitrogen, azide, and nitrous oxide do not bind. Site 2 is a low affinity acetylene-binding site to which CO, dinitrogen, azide, and nitrous oxide also bind.

Christiansen, J., Seefeldt, L. C., and Dean, D. R., Competitive substrate and inhibitor interactions at the physiologically relevant active site of nitrogenase, Journal of Biological Chemistry, 2000, 275, 36104-36107.

The role of molybdenum in nitrogenase has been reviewed with particular reference to theoretical modeling of the molybdenum centre.

Barriere, F., Modeling of the molybdenum center in the nitrogenase FeMo- cofactor, Coordination Chemistry Reviews, 2003, 236, 71-89.

See also

Bell, J., Dunford, A. J., Hollis, E., and Henderson, R. A., The role of Mo atoms in nitrogen fixation: Balancing substrate reduction and dihydrogen production, Angewandte Chemie-International Edition, 2003, 42 , 1149-1152.
Benton, P.M.C., Laryukhin, M., Mayer, S. M., Hoffman, B. M., Dean, D. R., and Seefeldt, L. C., Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: Trapping propargyl alcohol with an alpha-70- substituted MoFe protein, Biochemistry, 2003, 42, 9102-9109.
Hinnemann, B. and Norskov, J. K., Modeling a central ligand in the nitrogenase FeMo cofactor, Journal of the American Chemical Society, 2003, 125, 1466-1467

Barney, B. M., Lee, H. I., Dos Santos, P. C., Hoffmann, B. M., Dean, D. R., and Seefeldt, L. C., Breaking the N-2 triple bond: insights into the nitrogenase mechanism, Dalton Transactions, 2006, 2277-2284.

Nitrogenase mechanism possible involvement of molybdenum in substrate interactions during catalytic turnover

Molybdenum does not participate in binding a hydride of the catalytically central E-4 intermediate and only Fe ions are involved. Nonetheless, the response of the molybdenum coupling to subtle conformational changes in E-0 and to the formation of E-4 suggests that molybdenum is intimately involved in tuning the geometric and electronic properties of FeMo-co in these states

Lukoyanov, D., Yang, Z. Y., Dean, D. R., Seefeldt, L. C., and Hoffman, B. M., Is Mo Involved in Hydride Binding by the Four-Electron Reduced (E-4) Intermediate of the Nitrogenase MoFe Protein, Journal of the American Chemical Society, 2010, 132, 2526.

Nitrogenase ― tungstate toxicity and effect of catechol

Tungsten is toxic to N2-fixing bacteria, partly through substituting for Mo in nitrogenase. Catechol siderophores produced by A. vinelandii (essential for iron acquisition) modulate the relative uptake of molybdenum and tungsten. At high tungsten, concentrations of these catechol siderophores (particularly protochelin) in the growth medium increase sharply; they complex all the tungstate along with molybdate and some of the iron. The molybdenum-catechol complex is taken up much more rapidly than the tungsten complex, allowing A. vinelandii to satisfy its molybdenum requirement and avoid tungsten toxicity. Mutants deficient in the production of catechol siderophores are more sensitive to tungstate and have higher cellular tungsten quotas than the wild type. The binding of metals by excreted catechol siderophores allows A. vinelandii to discriminate in its uptake of essential metals, iron and molybdenum, over that of the toxic metal, tungsten, and to sustain high growth rates under adverse environmental conditions.

Wichard, T., Bellenger, J. P., Loison, A., and Kraepiel, A. M. L., Catecholsiderophores control tungsten uptake and toxicity in the nitrogen-fixing bacterium Azotobacter vinelandii, Environmental Science & Technology, 2008, 42, 2408-2413.

What determines the efficiency of N2-fixing rhizobium-legume symbioses?

Biological nitrogen fixation is vital to nutrient cycling in the biosphere and is the major route by which atmospheric dinitrogen (N2) is reduced to ammonia. The largest single contribution to biological N2 fixation is carried out by rhizobia, which include a large group of both alpha and beta-proteobacteria, almost exclusively in association with legumes. Rhizobia must compete to infect roots of legumes and initiate a signalling dialog with host plants that leads to nodule formation. The most common form of infection involves the growth of rhizobia down infection threads which are laid down by the host plant. Legumes form either indeterminate or determinate types of nodules, with these groups differing widely in nodule morphology and often in the developmental program by which rhizobia form N2fixing bacteroids. In particular, indeterminate legumes from the inverted repeat-lacking clade (IRLC) (e.g., peas, vetch, alfalfa, medics) produce a cocktail of antimicrobial peptides which cause endoreduplication of the bacterial genome and force rhizobia into a nongrowing state. Bacteroids often become dependent on the plant for provision of key cofactors, such as homocitrate needed for nitrogenase activity or for branched chain amino acids. This has led to the suggestion that bacteroids at least from the IRLC can be considered as ammoniaplasts, where they are effectively facultative plant organelles. A low O2 tension is critical both to induction of genes needed for N2 fixation and to the subsequent exchange of nutrient between plants and bacteroids. To achieve high rates of N2 fixation, the legume host and Rhizobium must be closely matched not only for infection, but for optimum development, nutrient exchange, and N2 fixation. In this review, we consider the multiple steps of selection and bacteroid development and how these alter the overall efficiency of N2 fixation.

Terpolilli, Jason J., Hood, Graham A., and Poole, Philip S., What Determines the Efficiency of N2-Fixing Rhizobium-Legume Symbioses?, Advances in Microbial Physiology, Vol 60, 2012, 60, 325-389.

Nitrogenase – assembly of MoFe protein

Recent advances in elucidation of the mechanism of FeMoco biosynthesis in four aspects: (1) the ex situ assembly of FeMoco on NifEN, (2) the incorporation of FeMoco into MoFe protein, (3) the in situ assembly of P-cluster on MoFe protein, and (4) the stepwise assembly of MoFe protein are reviewed.

Hu, Y.L., Fay, A. W., Lee, C. C., Yoshizawa, J., and Ribbe, M. W., Assembly of nitrogenase MoFe protein, Biochemistry, 2008, 47, 3973-3981.

IR-monitored photolysis of CO-inhibited nitrogenase: a major EPR-silent species with coupled terminal CO ligands

Fourier transform infrared spectroscopy (FTIR) was used to observe the photolysis and recombination of a new EPR-silent CO-inhibited form of a-H195Q nitrogenase from Azotobacter vinelandii. Photolysis at 4 K reveals a strong negative IR difference band at 1938 cm-1, along with a weaker negative feature at 1911 cm-1. These bands and the associated chemical species have both been assigned the label Hi-3. A positive band at 1921 cm-1 was assigned to the Lo-3 photoproduct. By using an isotopic mixture of 12C16O and 13C18O, we show that the Hi-3 bands arise from coupling of two similar CO oscillators with one uncoupled frequency at approximately 1917 cm-1. Although in previous studies Lo-3 was not observed to recombine, by extending the observation range to 200240 K, we found that recombination to Hi-3 does indeed occur, with an activation energy of approximately 6.5 kJmol-1. The frequencies of the Hi-3 bands suggest terminal CO ligation. This hypothesis was tested with DFT calculations on models with terminal CO ligands on Fe2 and Fe6 of the FeMo-cofactor. An S=0 model with both CO ligands in exo positions predicts symmetric and asymmetric stretches at 1938 and 1909 cm-1, respectively, with relative band intensities of about 3.5:1, which is in good agreement with experiment. From the observed IR intensities, Hi-3 was found to be present at a concentration about equal to that of the EPR-active Hi-1 species. The relevance of Hi-3 to the nitrogenase catalytic mechanism and its recently discovered FischerTropsch chemistry is discussed.

Yan, L. F., Pelmenschikov, V., Dapper, C. H., Scott, A. D., Newton, W. E., and Cramer, S. P., IR-Monitored Photolysis of CO-Inhibited Nitrogenase: A Major EPR-Silent Species with Coupled Terminal CO Ligands, Chemistry-A European Journal, 2012, 18, 16349-16357.

Nitrogenase biosynthesis

Recent developments in the understanding of the biosynthesis of the active site of the nitrogenase enzyme, the structure of the iron centre of [Fe]-hydrogenase and the structure and biomimetic chemistry of the [FeFe] hydrogenase H-cluster as deduced by application of X-ray spectroscopy are reviewed.

Best, S. P. and Cheah, M. H., Applications of X-ray absorption spectroscopy to biologically relevant metal-based chemistry, Radiation Physics and Chemistry, 2010, 79, 185-194.

Vanadium nitrogenase

Vanadium is a cofactor in the alternative V-nitrogenase that is expressed by some N2-fixing bacteria when Mo is not available. The V requirements, the kinetics of V uptake, and the production of catechol compounds across a range of concentrations of vanadium in diazotrophic cultures of the soil bacterium Azotobacter vinelandii were investigated. In strain CA11.70, a mutant that expresses only the V-nitrogenase, V concentrations in the medium between 10-8 and 10-6 M sustain maximum growth rates; they are limiting below this range and toxic above. A. vinelandii excretes in its growth medium micromolar concentrations of the catechol siderophores azotochelin and protochelin, which bind the vanadate oxoanion. The production of catechols increases when V concentrations become toxic. Short-term uptake experiments with the radioactive isotope V-49 show that bacteria take up the V-catechol complexes through a regulated transport system, which shuts down at high V concentrations. The modulation of the excretion of catechols and of the uptake of the V-catechol complexes allows A. vinelandii to precisely manage its V homeostasis over a range of V concentrations, from limiting to toxic.

Bellenger, J. P., Wichard, T., and Kraepiel, A. M. L., Vanadium requirements and uptake kinetics in the dinitrogen-fixing bacterium Azotobacter vinelandii, Applied and Environmental Microbiology, 2008, 74, 1478-1484.

Nitrogen fixation: nitrogenase

Molecular nitrogen is the source of all of the nitrogen necessary to sustain life on earth. Biological nitrogen fixation takes N2 and converts it into ammonia using nitrogenase enzymes, whereas industrial nitrogen fixation converts N2 and H2 to NH3 using heterogeneous iron or ruthenium surfaces. In both cases, the processes are energy-intensive. Is it possible to discover a homogeneous catalyst that can convert molecular nitrogen into higher-value organonitrogen compounds using a less energy-intensive pathway? If this could be achieved, it would be considered a major breakthrough.

In contrast to carbon monoxide, which is reactive and an important feedstock in many homogeneous catalytic reactions, the ischelectronic but inert N2 molecule is a very poor ligand and not a common industrial feedstock, except for the industrial production of NH3 .

Because N2 is available from the atmosphere and because nitrogen is an essential element for the biosphere, attempts to discover new processes involving this simple small molecule have occupied chemists for over a century. Since the first discovery of a dinitrogen complex in 1965, inorganic chemists have been key players in this area and have contributed much fundamental knowledge on structures, binding modes, and reactivity patterns. For the most part, the synthesis of dinitrogen complexes relies on the use of reducing agents to generate an electron-rich intermediate that can interact with this rather inert molecule. In this Account, a facile reaction of dinitrogen with a ditantalum tetrahydride species to generate the unusual side-on end-on bound N2 moiety is described. This particular process is one of a growing number of new, milder ways to generate dinitrogen complexes. Furthermore, the resulting dinitrogen complex undergoes a number of reactions that expand the known patterns of reactivity for coordinated N2. This Account reviews the reactions of ([NPN]Ta)(2)(mu-H)(2)(mu-eta(1):eta(2)-N-2), 2 (where NPN = PhP(CH2SiMe2NPh)(2)), with a variety of simple hydride reagents, E-H (where E-H = R2BH, R2AlH, RSiH3, and Cp2ZrCl(H)), each of which results in the cleavage of the N-N bond to form various functionalized imide and nitride moieties. This work is described in the context of a possible catalytic cycle that in principle could generate higher-value nitrogen-containing materials and regenerate the starting ditantalum tetrahydride. How this fails for each particular reagent is discussed and evaluated.

Fryzuk, M. D., Side-on End-on Bound Dinitrogen: An Activated Bonding Mode That Facilitates Functionalizing Molecular Nitrogen, Accounts of Chemical Research, 2009, 42, 127-133.

Nitrogenase mechanism - homologue approach

The (Mo)-nitrogenase is a complex metalloenzyme that catalyzes the key step in the global nitrogen cycle, the reduction of atmospheric dinitrogen (N-2) to bioavailable ammonia (NH3), at the iron-molybdenum cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. One major challenge for the mechanistic study of nitrogenase is the redox versatility of its FeMoco center. The ability of FeMoco to shuttle between oxidation states in a rapid and unsynchronized manner results in a mixed oxidation state of the cofactor population during turnover. The substrate and the various intermediates can only interact with the FeMoco site in a transient manner, so it is extremely difficult to capture any substrate- or intermediate-bound form of nitrogenase for the direct examination of substrate-enzyme interactions during catalysis.

In this Account, we describe the approach of identifying a partially "defective" nitrogenase homologue, one with a slower turnover rate, as a means of overcoming this problem.

The NifEN protein complex serves as an ideal candidate for this purpose. It is an alpha(2)beta(2)-heterotetramer that contains cluster-binding sites homologous to those found in the MoFe protein: the "P-cluster site" at the interface of the alpha beta-subunit dimer, which accommodates a [Fe4S4]-type cluster; and the "FeMoco site" within the alpha-subunit, which houses an all-iron homologue to the FeMoco. Moreover, NifEN mimics the MoFe protein in catalysis: it is capable of reducing acetylene (C2H2) and azide (N-3(-)) in an ATP- and iron (Fe) protein-dependent manner. However, NifEN is unable to reduce proton (H+) and N-2, and it is an inefficient enzyme with a restricted electron flux during the turnover.

The extremely slow turnover rate of NifEN and the possible "synchronization" of its FeMoco homologue at a certain oxidation level permit the observation of a new S = 1/2 EPR signal upon turnover of C2H2 by NifEN, which is analogous to the signal reported for a MoFe protein variant upon turnover of the same substrate.

This result is exciting, because it suggests the possibility of naturally enriching a C2H2-bound form of NifEN for the successful crystallization of the first intermediate-bound nitrogenase homologue.

On the other hand, the fact that NifEN represents a partially "defective" homologue of the MoFe protein makes it a promising mutational platform on which a functional MoFe protein equivalent may be reconstructed by introducing the missing features of MoFe protein step-by-step into NifEN.

Such a strategy allows us to define the function of each feature and address questions such as the following: What is the function of P-cluster in catalysis? Are Mo and homocitrate the essential constituents of the cofactor in N-2 reduction? How does substrate accessibility affect the reactivity of the enzyme? This homologue approach could complement the mechanistic analysis of the nitrogenase MoFe protein, and information derived from both approaches will help achieve the ultimate goal of solving the riddle of biological nitrogen fixation.

Hu, Y. L. and Ribbe, M. W., Decoding the Nitrogenase Mechanism: The Homologue Approach, Accounts of Chemical Research, 2010, 43, 475-484.

Nitrogenase mechanism - protons
The controlled relay of multiple protons required at the active site of nitrogenase

The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described.

The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay.

The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed.

This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase.

Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested.

The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

Dance, Ian, The controlled relay of multiple protons required at the active site of nitrogenase, Dalton Transactions, 2012, 41, 7647-7659.

Nitrogenase: New insights into the biological and synthetic fixation of nitrogen

Scheibel, Markus G. and Schneider, Sven, New Insights into the Biological and Synthetic Fixation of Nitrogen, Angewandte Chemie-International Edition, 2012, 51, 4529-4531.

Nitrogenase: Emerging paradigms for complex iron-sulfur cofactor assembly and insertion

[FeFe]-hydrogenses and molybdenum (Mo)-nitrogenase are evolutionarily unrelated enzymes with unique complex iron-sulfur cofactors at their active sites.

The H cluster of [FeFe]-hydrogenases and the FeMo cofactor of Mo-nitrogenase require specific maturation machinery for their proper synthesis and insertion into the structural enzymes. Recent insights reveal striking similarities in the biosynthetic pathways of these complex cofactors.

For both systems, simple iron-sulfur cluster precursors are modified on assembly scaffolds by the activity of radical S-adenosylmethionine (SAM) enzymes. Radical SAM enzymes are responsible for the synthesis and insertion of the unique nonprotein ligands presumed to be key structural determinants for their respective catalytic activities. Maturation culminates in the transfer of the intact cluster assemblies to a cofactor-less structural protein recipient.

Required roles for nucleotide binding and hydrolysis have been implicated in both systems, but the specific role for these requirements remain unclear. In this review, we highlight the progress on [FeFe]-hydrogenase H cluster and nitrogenase FeMo-cofactor assembly in the context of these emerging paradigms.

Peters, John W. and Broderick, Joan B., Emerging paradigms for complex iron-sulfur cofactor assembly and insertion, Annual review of biochemistry, 2012, 81, 429-450.

Nitrogenase: Electron transfer in nitrogenase catalysis

Nitrogenase is a two-component enzyme that catalyzes the nucleotide-dependent reduction of N2 to 2NH3. This process involves three redox-active metal-containing cofactors including a [4Fe-4S] cluster, an eight-iron P cluster and a seven-iron plus molybdenum FeMo-cofactor, the site of substrate reduction.

A deficit-spending model for electron transfer has recently been proposed that incorporates protein conformational gating that favors uni-directional electron transfer among the metalloclusters for the activation of the substrate-binding site.

Also reviewed is a proposal that each of the metal clusters cycles through only two redox states of the metal-sulfur core as the system accumulates the multiple electrons required for substrate binding and reduction. In particular, it was suggested that as FeMo-cofactor acquires the four electrons necessary for optimal binding of N2, each successive pair of electrons is stored as an Fe-H--Fe bridging hydride, with the FeMo-cofactor metal-ion core retaining its resting redox state.

We here broaden the discussion of stable intermediates that might form when FeMo-cofactor receives an odd number of electrons.

Seefeldt, Lance C., Hoffman, Brian M., and Dean, Dennis R., Electron transfer in nitrogenase catalysis, Current Opinion in Chemical Biology, 2012, 16, 19-25.

Nitrogenase: Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase

Nitrogenase catalyzes the reduction of N2 and protons to yield two NH3 and one H2. Substrate binding occurs at a complex organometallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley.

Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN=N-CH3), or hydrazine as substrate recently as shown to trap a common S = 1/2 intermediate, designated I. It was further concluded that the two N-atoms of N2 are hydrogenated alternately ("Alternating" (A) pathway).

In the present work, Q-band CW EPR and Mo-95 ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state "non-Kramers" doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of N-14,N-15 isotopologs plus H-1,H-2 isotopologs of methyldiazene. It is concluded that: H has NH2 bound to FeMo-co and corresponds to the penultimate intermediate of N2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH3; I corresponds to the final intermediate in N2 reduction, the state formed after accumulation of eight electrons/protons, with NH3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co.

A proposed unification of the Lowe-Thorneley kinetic model with the "prompt" alternating reaction pathway represents a draft mechanism for N2 reduction by nitrogenase.

Lukoyanov, Dmitriy, Yang, Zhi Yong, Barney, Brett M., Dean, Dennis R., Seefeldt, Lance C., and Hoffman, Brian M., Unification of reaction pathway and kinetic scheme for N-2 reduction catalyzed by nitrogenase, Proceedings of the National Academy of Sciences of the United States of America, 2012, 109, 5583-5587.

Nitrogenase: Molybdenum-catalyzed reduction of molecular dinitrogen under mild reaction conditions

Quite recently we have found two nitrogen fixation systems catalyzed by molybdenum-dinitrogen complexes under mild reaction conditions; one is the transformation of molecular dinitrogen into its synthetic equivalent of ammonia and the other is that into ammonia.

A molybdenum-dinitrogen complex bearing two ferrocenyl diphosphines works as a good catalyst in the transformation of molecular dinitrogen into silylamine, where up to 226 equiv are produced based on the catalyst.

A dinitrogen-bridged dimolybdenum complex bearing a PNP-type pincer ligand works as a good catalyst in the direct transformation of molecular dinitrogen into ammonia, where up to 23 equiv are produced based on the catalyst.

We believe that both systems provide a new aspect in the development of novel nitrogen fixation.

Nishibayashi, Yoshiaki, Molybdenum-catalyzed reduction of molecular dinitrogen under mild reaction conditions, Dalton transactions (Cambridge, England : 2003), 2012, 41, 7447-7453.

Nitrogen fixation pre-biotic

Nitrogen reduction by iron(II) has been suggested as an important mechanism in the formation of ammonia on pre-biotic Earth. This paper examines the effects of adsorption of Fe2+ onto a goethite (alpha-FeOOH) substrate on the thermodynamic driving force and rate of a Fe2+-mediated reduction of N2 as compared with the homogeneous aqueous reaction. The key to reduction in both cases is N2adsorption to multiple transition metal centers with competitive H2 production.

Wander, M.C.F., Kubicki, J. D., and Schoonen, M. A. A., Reduction of N2by Fe2+ via homogeneous and heterogeneous reactions Part 2: The role of metal binding in activating N2 for reduction; a requirement for both pre-biotic and biological mechanisms, Origins of Life and Evolution of Biospheres, 2008, 38, 195-209.

Lichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation system

In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the well-studied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genus Nostoc associated with four samples from different geographical regions, representing different lichen-forming fungal species in the genus Peltigera. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N-2 fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylene-reduction assay data.

Hodkinson, B. P., Allen, J. L., Forrest, L. L., Goffinet, B., Serusiaux, E., Andresson, O. S., Miao, V., Bellenger, J. P., and Lutzoni, F., Lichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation system, European Journal of Phycology, 2014, 49, 11-19.