RESUMEN
Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2 . These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.
Asunto(s)
Azotobacter vinelandii , Nitrogenasa , Azotobacter vinelandii/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Molibdoferredoxina/metabolismo , Nitrogenasa/genética , Nitrogenasa/metabolismoRESUMEN
Nitrogenase harbors three distinct metal prosthetic groups that are required for its activity. The simplest one is a [4Fe-4S] cluster located at the Fe protein nitrogenase component. The MoFe protein component carries an [8Fe-7S] group called P-cluster and a [7Fe-9S-C-Mo-R-homocitrate] group called FeMo-co. Formation of nitrogenase metalloclusters requires the participation of the structural nitrogenase components and many accessory proteins, and occurs both in situ, for the P-cluster, and in external assembly sites for FeMo-co. The biosynthesis of FeMo-co is performed stepwise and involves molecular scaffolds, metallochaperones, radical chemistry, and novel and unique biosynthetic intermediates. This review provides a critical overview of discoveries on nitrogenase cofactor structure, function, and activity over the last four decades.
Asunto(s)
Molibdoferredoxina/biosíntesis , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , Molibdoferredoxina/químicaRESUMEN
The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Compuestos de Hierro/metabolismo , Saccharomyces cerevisiae/metabolismo , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Redes y Vías Metabólicas , Methanocaldococcus , Mitocondrias/metabolismo , Fijación del Nitrógeno/fisiología , Nitrogenasa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Biología SintéticaRESUMEN
The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α2ß2 heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N2-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity-based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αß-unit with a FeMo-cofactor and mature P-cluster and the other αß-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.
Asunto(s)
Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/metabolismo , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Molibdoferredoxina/genética , Nitrogenasa/genéticaRESUMEN
Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as "assembly factors." Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein-assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein-NafY, MoFe protein-NifY, and MoFe protein-NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.
Asunto(s)
Azotobacter vinelandii/enzimología , Molibdoferredoxina/metabolismo , Nitrogenasa/química , Nitrogenasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Transporte de Electrón , Conformación ProteicaRESUMEN
NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed.
Asunto(s)
Proteínas Bacterianas/química , Compuestos de Hierro/química , Methanocaldococcus/enzimología , Nitrogenasa/química , S-Adenosilmetionina/química , Espectroscopía de Resonancia por Spin del Electrón , Molibdoferredoxina/química , Especificidad por SustratoRESUMEN
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 Mössbauer 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 Mössbauer analysis suggests that NifB-co is either a 4Fe(2+) -4Fe(3+) or 6Fe(2+) -2Fe(3+) cluster having valence-delocalized states.
Asunto(s)
Compuestos de Hierro/metabolismo , Hierro/metabolismo , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Hierro/química , Compuestos de Hierro/química , Campos Magnéticos , Molibdoferredoxina/química , Nitrogenasa/química , Teoría CuánticaRESUMEN
Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs.
Asunto(s)
Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Fijación del Nitrógeno/fisiología , Amoníaco , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Eliminación de Gen , Genoma Bacteriano , Cinética , Transcripción Genética , TranscriptomaRESUMEN
Nitrogenase-dependent H2 production by photosynthetic bacteria, such as Rhodobacter capsulatus, has been extensively investigated. An important limitation to increase H2 production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutants. Previously, we engineered R. capsulatus strains that emitted fluorescence in response to H2 and used them to identify mutations in the nitrogenase Fe protein leading to H2 overproduction. Here, we used ultraviolet light to induce random mutations in the genome of the engineered H2-sensing strain, and fluorescent-activated cell sorting to detect and isolate the H2-overproducing cells from libraries containing 5 × 105 mutants. Three rounds of mutagenesis and strain selection gradually increased H2 production up to 3-fold. The whole genomes of five H2 overproducing strains were sequenced and compared to that of the parental sensor strain to determine the basis for H2 overproduction. No mutations were present in well-characterized functions related to nitrogen fixation, except for the transcriptional activator nifA2. However, several mutations mapped to energy-generating systems and to carbon metabolism-related functions, which could feed reducing power or ATP to nitrogenase. Time-course experiments of nitrogenase depression in batch cultures exposed mismatches between nitrogenase protein levels and their H2 and ethylene production activities that suggested energy limitation. Consistently, cultivating in a chemostat produced up to 19-fold more H2 than the corresponding batch cultures, revealing the potential of selected H2 overproducing strains.
RESUMEN
Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.
RESUMEN
The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.
Asunto(s)
Azotobacter vinelandii/metabolismo , Dominio Catalítico , Coenzimas/metabolismo , Nitrogenasa/química , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Coenzimas/genética , Molibdoferredoxina/metabolismo , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Nitrogenasa/metabolismoRESUMEN
The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of â¼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V3+, 3Fe3+, 4Fe2+]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined 51V (I = 7/2) hyperfine splitting, a iso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V3+, 4Fe3+, 3Fe2+]. Our findings suggest that the V3+ does not change valency throughout the catalytic cycle.
RESUMEN
Three known nitrogenase isozymes, Mo-, V-, and Fe-, catalyze biological reduction of dinitrogen (N2) to ammonia (NH3). All three utilize the same reductive elimination mechanism: an intermediate with two metal-bound hydrides reductively-eliminates hydrogen gas (H2) in a reaction coupled to binding and activation of N2. Nonetheless, the three isozymes show dramatically different relative rates of H2 formation and N2 reduction, revealing important differences in reactivity with substrates. Carbon monoxide (CO) has been characterized as both an inhibitor and substrate for Mo- and Vnitrogenases, but not for the Fenitrogenase. Here, we present a comparative study of the reactivity of the three isozymes with CO, examining CO both as a substrate and as an inhibitor of proton (H+) reduction under steady-state conditions. For Monitrogenase, there is neither detectable reduction of CO nor inhibition of H+ reduction. Fe- and Vnitrogenase show CO reduction and inhibition of H+ reduction that depends on the CO partial pressure. For Vnitrogenase, ethylene (C2H4) is the major reduction product with a maximum specific activity of ~7.5 nmol C2H4/nmol VFe protein/min at 1 atm CO. The major product of CO reduction for Fenitrogenase is methane (CH4) with a maximum specific activity of ~4.8 nmol CH4/nmol FeFe protein/min at 0.05 atm CO. The rate of CH4 production by Fenitrogenase progressively increases to a maximum at 0.05 atm CO and then declines by ~90% with increasing CO partial pressure up to 1 atm. CO does not inhibit proton reduction in Monitrogenase but shows 16% inhibition for Vnitrogenase and 35% for Fenitrogenase.
Asunto(s)
Monóxido de Carbono/química , Hidrógeno/química , Hierro/química , Molibdeno/química , Nitrogenasa/química , Vanadio/química , Catálisis , Oxidación-ReducciónRESUMEN
Nitrogenases are complex two-component metalloenzymes that catalyze biological nitrogen fixation. Three different nitrogenase types are found in the model nitrogen-fixing microbe Azotobacter vinelandii. In the case of the Mo-dependent enzyme, the two catalytic partners are referred to as the Fe protein and MoFe protein. In addition to genes encoding the catalytic components, there are a total of 68 other gene products known to be variously involved in producing, activating, protecting, sustaining, and regulating formation of the Mo-dependent nitrogenase. In order to support experiments designed to gain insight into the catalytic mechanism and assembly of nitrogenase, four different affinity-based purification protocols have been developed. These include an improved Co2+-based Immobilized Metal Affinity Chromatography (IMAC) method for the purification of MoFe protein, a newly developed StrepTactin Affinity Chromatography (STAC) method for the purification of MoFe protein and its assembly intermediates, a combined IMAC and STAC method for isolation of highly pure MoFe protein, and a STAC-based bait-prey method for isolation of complexes variously involved in the maturation process.
Asunto(s)
Azotobacter vinelandii/enzimología , Cromatografía de Afinidad/métodos , Nitrogenasa/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Catálisis , Molibdoferredoxina/aislamiento & purificación , Molibdoferredoxina/metabolismo , Nitrogenasa/aislamiento & purificaciónRESUMEN
One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (single-domain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (ΔnifB) mutant restoring the Nif+ phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.
RESUMEN
When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Hidrógeno/metabolismo , Hidrogenasas/genética , Nitrogenasa/genética , Oxidorreductasas/genética , Rhodobacter capsulatus/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Biocombustibles , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Citometría de Flujo , Ingeniería Genética/métodos , Ensayos Analíticos de Alto Rendimiento , Hidrogenasas/metabolismo , Operón Lac , Modelos Moleculares , Mutagénesis , Nitrogenasa/metabolismo , Oxidorreductasas/metabolismo , Fotosíntesis/genética , Regiones Promotoras Genéticas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Rhodobacter capsulatus/enzimologíaRESUMEN
Mo and Fe K-edge EXAFS analysis of NifQ shows the presence of a [MoFe3S4] cluster and a second independent Mo environment that includes Mo-O bonds and Mo-S bonds. Both environments are relevant to FeMo-co biosynthesis and may represent different stages of Mo biochemical transformations catalyzed by NifQ.
Asunto(s)
Proteínas Bacterianas/metabolismo , Coenzimas/química , Metaloproteínas/química , Nitrogenasa/metabolismo , Pteridinas/química , Factores de Transcripción/metabolismo , 2,2'-Dipiridil/química , Azotobacter vinelandii/enzimología , Proteínas Bacterianas/química , Cobre/química , Hierro/química , Cofactores de Molibdeno , Factores de Transcripción/química , Espectroscopía de Absorción de Rayos XRESUMEN
The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct nif gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe-S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe-S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N2-fixing cereals.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Hierro-Azufre/genética , Mitocondrias/genética , Oxidorreductasas/genética , Saccharomyces cerevisiae/genética , Aerobiosis , Proteínas Bacterianas/metabolismo , Ingeniería Celular/métodos , Expresión Génica , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fijación del Nitrógeno/genética , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Oxígeno/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , TransgenesRESUMEN
Biosynthesis of metal clusters for the nitrogenase component proteins NifH and NifDK involves electron donation events. Yet, electron donors specific to the biosynthetic pathways of the [4Fe-4S] cluster of NifH, or the P-cluster and the FeMo-co of NifDK, have not been identified. Here we show that an Azotobacter vinelandii mutant lacking fdxN was specifically impaired in FeMo-co biosynthesis. The ΔfdxN mutant produced 5-fold less NifB-co, an early FeMo-co biosynthetic intermediate, than wild type. As a consequence, it accumulated FeMo-co-deficient apo-NifDK and was impaired in NifDK activity. We conclude that FdxN plays a role in FeMo-co biosynthesis, presumably by donating electrons to support NifB-co synthesis by NifB. This is the first role in nitrogenase biosynthesis unequivocally assigned to any A. vinelandii ferredoxin.