Mechanism of nitrogen fixation by nitrogenase: the next stage.
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ABSTRACT:
SUBMITTER: Hoffman BM
PROVIDER: S-EPMC4012840 | biostudies-literature | 2014 Apr
REPOSITORIES: biostudies-literature
Publications
Mechanism of nitrogen fixation by nitrogenase: the next stage.
Hoffman Brian M BM Lukoyanov Dmitriy D Yang Zhi-Yong ZY Dean Dennis R DR Seefeldt Lance C LC
Chemical reviews 20140127 8
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Project description:"Nitrogen fixation", the reduction of dinitrogen (N2) to two ammonia (NH3) molecules, by the Mo-dependent nitrogenase is essential for all life. Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase activity make it the "Everest of enzymes". This Account describes our efforts to climb one "face" of this mountain by meeting two interdependent challenges central to determining the mechanism of biological N2 reduction. The first challenge is to determine the reaction pathway: the composition and structure of each of the substrate-derived moieties bound to the catalytic FeMo cofactor (FeMo-co) of the molybdenum-iron (MoFe) protein of nitrogenase. To overcome this challenge, it is necessary to discriminate between the two classes of potential reaction pathways: (1) a "distal" (D) pathway, in which H atoms add sequentially at a single N or (2) an "alternating" (A) pathway, in which H atoms add alternately to the two N atoms of N2. Second, it is necessary to characterize the dynamics of conversion among intermediates within the accepted Lowe-Thorneley kinetic scheme for N2 reduction. That goal requires an experimental determination of the number of electrons and protons delivered to the MoFe protein as well as their "inventory", a partition into those residing on each of the reaction components and released as H2 or NH3. The principal obstacle to this "climb" has been the inability to generate N2 reduction intermediates for characterization. A combination of genetic, biochemical, and spectroscopic approaches recently overcame this obstacle. These experiments identified one of the four-iron Fe-S faces of the active-site FeMo-co as the specific site of reactivity, indicated that the side chain of residue alpha70V controls access to this face, and supported the involvement of the side chain of residue alpha195H in proton delivery. We can now freeze-quench trap N2 reduction pathway intermediates and use electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize them. However, even successful trapping of a N2 reduction intermediate occurs without synchronous electron delivery to the MoFe protein. As a result, the number of electrons and protons, n, delivered to MoFe during its formation is unknown. To determine n and the electron inventory, we initially employed ENDOR spectroscopy to analyze the substrate moiety bound to the FeMo-co and 57Fe within the cofactor. Difficulties in using that approach led us to devise a robust kinetic protocol for determining n of a trapped intermediate. This Account describes strategies that we have formulated to bring this "face" of the nitrogenase mechanism into view and afford approaches to its climb. Although the summit remains distant, we look forward to continued progress in the ascent.
| S-EPMC2684573 | biostudies-literature
Project description:The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α2ß2(γ2) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N2 to NH3, with no significant difference in selectivity over obligatory/parasitic H2 formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H2 and NH3 formation (under Ar or N2), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein-protein communication in biological N2 fixation in Azotobacter vinelandii.
| S-EPMC10207099 | biostudies-literature
Project description:Biological fixation of dinitrogen (N2), the primary natural source of new bioavailable nitrogen (N) on Earth, is catalyzed by the enzyme nitrogenase through a complex mechanism at its active site metal cofactor. How this reaction functions in cellular environments, including its rate-limiting step, and how enzyme structure affects functioning remain unclear. Here, we investigated cellular N2 fixation through its N isotope effect (15εfix), measured as the difference between the 15N/14N ratios of diazotroph net new fixed N and N2 substrate. The value of 15εfix underpins N cycle reconstructions and differs between diazotrophs using molybdenum-containing and molybdenum-free nitrogenases. By examining 15εfix for Azotobacter vinelandii strains with natural and mutated nitrogenases, we determined if 15εfix reflects enzyme-scale isotope effects and, thus, N2 use efficiency. Distinct and relatively stable 15εfix values for wild-type molybdenum- and vanadium-nitrogenase isoforms (2.5‰ and 5.8-6.6‰, respectively), despite changing cellular growth rate and electron availability, support 15εfix as a proxy for isoform type among extant nitrogenases. Structural mutation of active site N2 access altered molybdenum-nitrogenase 15εfix (3.0-6.8‰ for α-70VI mutant). Structure-function and isotopic modeling results indicated cellular N2 reduction is rate-limited by N2 diffusion inside nitrogenase due to highly efficient catalysis by the active site cofactor, exemplifying 15εfix as a tool to probe N2 fixation mechanisms. Diffusion-constrained reactions could reflect structural tradeoffs that protect the oxygen-sensitive cofactor from oxygen inactivation. This suggests that nitrogenase function is optimized for modern oxygenated environments and that pre-Great Oxidative Event nitrogenases were less diffusion-limited and potentially exhibited larger 15εfix values.
| S-EPMC11913218 | biostudies-literature
Project description:BACKGROUND:The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. RESULTS:Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. CONCLUSIONS:Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.
| S-EPMC3464626 | biostudies-literature
Project description:Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.
| S-EPMC9209457 | biostudies-literature
Project description:Plants depend upon beneficial interactions between roots and root-associated microorganisms for growth promotion, disease suppression, and nutrient availability. This includes the ability of free-living diazotrophic bacteria to supply nitrogen, an ecological role that has been long underappreciated in modern agriculture for efficient crop production systems. Long-term ecological studies in legume-rhizobia interactions have shown that elevated nitrogen inputs can lead to the evolution of less cooperative nitrogen-fixing mutualists. Here we describe how reprogramming the genetic regulation of nitrogen fixation and assimilation in a novel root-associated diazotroph can restore ammonium production in the presence of exogenous nitrogen inputs. We isolated a strain of the plant-associated proteobacterium Kosakonia sacchari from corn roots, characterized its nitrogen regulatory network, and targeted key nodes for gene editing to optimize nitrogen fixation in corn. While the wild-type strain exhibits repression of nitrogen fixation in conditions replete with bioavailable nitrogen, such as fertilized greenhouse and field experiments, remodeled strains show elevated levels in the rhizosphere of corn in the greenhouse and field even in the presence of exogenous nitrogen. Such strains could be used in commercial applications to supply fixed nitrogen to cereal crops.
| S-EPMC7382387 | biostudies-literature
Project description:Expression of nifH in 28 surface water samples collected during fall 2007 from six stations in the vicinity of the Cape Verde Islands (north-east Atlantic) was examined using reverse transcription-polymerase chain reaction (RT-PCR)-based clone libraries and quantitative RT-PCR (RT-qPCR) analysis of seven diazotrophic phylotypes. Biological nitrogen fixation (BNF) rates and nutrient concentrations were determined for these stations, which were selected based on a range in surface chlorophyll concentrations to target a gradient of primary productivity. BNF rates greater than 6?nmolN?l(-1)?h(-1) were measured at two of the near-shore stations where high concentrations of Fe and PO(4)(3-) were also measured. Six hundred and five nifH transcripts were amplified by RT-PCR, of which 76% are described by six operational taxonomic units, including Trichodesmium and the uncultivated UCYN-A, and four non-cyanobacterial diazotrophs that clustered with uncultivated Proteobacteria. Although all five cyanobacterial phylotypes quantified in RT-qPCR assays were detected at different stations in this study, UCYN-A contributed most significantly to the pool of nifH transcripts in both coastal and oligotrophic waters. A comparison of results from RT-PCR clone libraries and RT-qPCR indicated that a ?-proteobacterial phylotype was preferentially amplified in clone libraries, which underscores the need to use caution interpreting clone-library-based nifH studies, especially when considering the importance of uncultivated proteobacterial diazotrophs.
| S-EPMC3146282 | biostudies-literature
Project description:Nitrogenase is the sole enzyme responsible for the ATP-dependent conversion of atmospheric dinitrogen into the bioavailable form of ammonia (NH3 ), making this protein essential for the maintenance of the nitrogen cycle and thus life itself. Despite the widespread use of the Haber-Bosch process to industrially produce NH3 , biological nitrogen fixation still accounts for half of the bioavailable nitrogen on Earth. An important feature of nitrogenase is that it operates under physiological conditions, where the equilibrium strongly favours ammonia production. This biological, multielectron reduction is a complex catalytic reaction that has perplexed scientists for decades. In this review, we explore the current understanding of the molybdenum nitrogenase system based on experimental and computational research, as well as the limitations of the crystallographic, spectroscopic, and computational techniques employed. Finally, essential outstanding questions regarding the nitrogenase system will be highlighted alongside suggestions for future experimental and computational work to elucidate this essential yet elusive process.
| S-EPMC10100503 | biostudies-literature
Project description:Cyanobacteria are widely distributed in marine, aquatic, and terrestrial ecosystems, and play an important role in the global nitrogen cycle. In the present study, we examined the genome sequence of the thermophilic non-heterocystous N2-fixing cyanobacterium, Thermoleptolyngbya sp. O-77 (formerly known as Leptolyngbya sp. O-77) and characterized its nitrogenase activity. The genome of this cyanobacterial strain O-77 consists of a single chromosome containing a nitrogen fixation gene cluster. A phylogenetic analysis indicated that the NifH amino acid sequence from strain O-77 was clustered with those from a group of mesophilic species: the highest identity was found in Leptolyngbya sp. KIOST-1 (97.9% sequence identity). The nitrogenase activity of O-77 cells was dependent on illumination, whereas a high intensity of light of 40 μmol m-2 s-1 suppressed the effects of illumination.
| S-EPMC5745016 | biostudies-literature
Project description:Nitrogenase is a fascinating enzyme in biology that reduces dinitrogen from air to ammonia through stepwise reduction and protonation. Despite it being studied in detail by experimental and computational groups, there are still many unknown factors in the catalytic cycle of nitrogenase, especially related to the addition of protons and electrons and their order. A recent biomimetic study characterized a potential dinitrogen-bridged diiron cluster as a synthetic model of nitrogenase. Using strong acid and reductants, the dinitrogen was converted into ammonia molecules, but details of the mechanism remains unknown. In particular, it was unclear from the experimental studies whether the proton and electron transfer steps are sequential or alternating. Moreover, the work failed to establish what the function of the diiron core is and whether it split into mononuclear iron fragments during the reaction. To understand the structure and reactivity of the biomimetic dinitrogen-bridged diiron complex [(P2P'PhFeH)2(μ-N2)] with triphenylphosphine ligands, we performed a density functional theory study. Our computational methods were validated against experimental crystal structure coordinates, Mössbauer parameters, and vibrational frequencies and show excellent agreement. Subsequently, we investigated the alternating and consecutive addition of electrons and protons to the system. The calculations identify a number of possible reaction channels, namely, same-site protonation, alternating protonation, and complex dissociation into mononuclear iron centers. The calculations show that the overall mechanism is not a pure sequential set of electron and proton transfers but a mixture of alternating and consecutive steps. In particular, the first reaction steps will start with double proton transfer followed by an electron transfer, while thereafter, there is another proton transfer and a second electron transfer to give a complex whereby ammonia can split off with a low energetic barrier. The second channel starts with alternating protonation of the two nitrogen atoms, whereafter the initial double proton transfer, electrons and protons are added sequentially to form a hydrazine-bound complex. The latter split off ammonia spontaneously after further protonation. The various reaction channels are analyzed with valence bond and orbital diagrams. We anticipate the nitrogenase enzyme to operate with mixed alternating and consecutive protonation and electron transfer steps.
| S-EPMC10498488 | biostudies-literature