
Luis M. RubioSpanish National Research Council | CSIC
Luis M. Rubio
PhD
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Publications (96)
The free-living diazotroph Azotobacter vinelandii produces three genetically distinct but functionally and mechanistically similar nitrogenase isozymes, designated as Mo-dependent, V-dependent, and Fe-only. They respectively harbor nearly identical catalytic cofactors that are distinguished by a heterometal site occupied by Mo (FeMo-cofactor), V (F...
The maturation and installation of the active site metal cluster (FeMo-co, Fe 7 S 9 CMo- R -homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe 8 S 9 C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. H...
The Azotobacter vinelandii molybdenum nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires an existing [Fe-S] cluster to form a [Mo-Fe3-S4] group, which acts as a specific molybdenum donor during nitrogenase FeMo-co biosynthesis. Here, we show biochemical evidence supporting the role of NifU as the...
Biological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo- R -homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereal...
Simple Summary
Azotobacter vinelandii is a model organism used to study biological nitrogen fixation, a process by which nitrogen gas is transformed into ammonia, a form of nitrogen that can be assimilated by most organisms. This requires the synthesis and transfer of specific iron-containing cofactors to enzymes involved in nitrogen fixation. Ther...
Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as...
Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N 2 . Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the acc...
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...
The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe−9S−C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron−molybdenum cofactor (FeMo-co) found...
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 ag...
Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first co...
Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] c...
In nitrogenase biosynthesis, the iron-molybdenum cofactor (FeMo-co) is externally assembled at scaffold proteins and delivered to the NifDK nitrogenase component by the NafY metallochaperone. Here we have used nuclear magnetic resonance, molecular dynamics, and functional analysis to elucidate the environment and coordination of FeMo-co in NafY. H1...
Integration of prokaryotic nitrogen fixation ( nif ) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N 2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting...
The nitrogenase MoFe protein contains two different FeS centers, the P-cluster and the iron-molybdenum cofactor (FeMo-co). The former is a [Fe8S7] center responsible for conveying electrons to the latter, a [MoFe7S9C-(R)-homocitrate] species, where N2 reduction takes place. NifB is arguably the key enzyme in FeMo-co assembly as it catalyzes the fus...
Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe‐only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show...
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco t...
Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abunda...
Nitrogenase is a key player in the global nitrogen cycle as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF...
The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural “green” revolution. Here we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes nifH, M, U...
Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria t...
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...
Significance
Nitrogen is a constituent of many essential biomolecules and plentiful on earth as inert N 2 gas. For its assimilation by eukaryotes, N 2 must be converted to a metabolically tractable form such as ammonium. Such conversion is catalyzed by nitrogenase, an enzyme produced by a select group of microorganisms called diazotrophs. Crop yiel...
The N2 fixing bacterium Azotobacter vinelandii carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron–molybden...
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 catalys...
Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of rec...
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 bios...
Background
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of...
Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic org...
Corrigendum to data in Figure 1 of the corresponding paper
Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle's high O2 consumption and the presence of bacterial-type iron-sulfur cluster biosynthetic ma...
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...
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.
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-4...
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]...
NifB utilizes two equivalents of 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 SA...
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 mitochondria...
Supplementary Figures 1-5, Supplementary Table 1, Supplementary Methods and Supplementary References.
Oleaginous microalgae have a great potential as a feedstock for biodiesel and other biofuels. However, the current cost of producing biofuels from microalgae biomass is still high to envision massive and profitable commercialization in the near future. One of the drawbacks of implementing large-scale cultivation of these organisms is the unsustaina...
Nitrogen fixation is a highly regulated trait. In the free-living diazotrophic bacterium Azotobacter vinelandii the nitrogen fixation (nif) regulon consists of at least 50 genes distributed in two chromosomal regions. Analysis of polar mutations, Northern blots, lacZ transcriptional fusions and, more recently, quantitative real-time PCR and transcr...
Nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMo-co) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex pathway involving proteins acting as molecular scaffolds, as metallocluster carriers, or enzymes...
The most dependable factor to perform successful biochemical experiments in an O2-free environment is the experience required to set up an efficient laboratory, to properly manipulate samples, to anticipate potential O2-related problems, and to maintain the complex laboratory setup operative. There is a long list of O2-related issues that may ruin...
NifB is the key protein in the biosynthesis of nitrogenase iron-molybdenum cofactor. Due to its extreme sensitivity to O2 and inherent protein instability, NifB proteins must be purified under strict anaerobic conditions by using affinity chromatography methods. We describe here the methods for NifB purification from cells of the strict aerobic nit...
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 expressi...
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 s...
The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities and spectroscopic properties of the en...
The major part of biological nitrogen fixation is catalysed by the molybdenum nitrogenase that carries at its active site the iron and molybdenum cofactor (FeMo-co). The nitrogen fixation (nif) genes required for the biosynthesis of FeMo-co are derepressed in the absence of a source of fixed nitrogen. The nifB gene product is remarkable because it...
NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier
and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function
(n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that...
The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron-molybdenum cofactor (FeMo-co) is replaced by NifB-co. The NifDK/NifB-co enzyme was unable to reduce N(2) to NH(3), while exhibiting residual C(2)H(4) and considerable H(2) production activities. Production of H(2) by Nif...
The molybdenum nitrogenase is responsible for most biological nitrogen fixation, a prokaryotic metabolic process that determines the global biogeochemical cycles of nitrogen and carbon. Here we describe the trafficking of molybdenum for nitrogen fixation in the model diazotrophic bacterium Azotobacter vinelandii. The genes and proteins involved in...
Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response
to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought
and other physical and chemi...
The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron–molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of mol...
The iron-molybdenum cofactor (FeMo-co), located at the active site of the molybdenum nitrogenase, is one of the most complex metal cofactors known to date. During the past several years, an intensive effort has been made to purify the proteins involved in FeMo-co synthesis and incorporation into nitrogenase. This effort is starting to provide insig...
NifB-co, an Fe-S cluster produced by the enzyme NifB, is an intermediate on the biosynthetic pathway to the iron molybdenum cofactor (FeMo-co) of nitrogenase. We have used Fe K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy together with (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the structure of NifB-co w...
The major part of biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which carries at its active site
the most complex metallocluster known in biology, the iron-molybdenum cofactor (FeMo-co). It is composed of 7 Fe, 9 S, 1 Mo,
1 homocitrate, and 1 unidentified central atom. The genetic and biochemical analysis of nitrogen-fixa...
The majority of the N2 fixed biologically is catalyzed by the molybdenum nitrogenase, which is composed of two O2-labile metalloproteins; dinitrogenase (also referred to as MoFe protein) and dinitrogenase reductase (also referred to as
Fe protein). Dinitrogenase is a 230-kDa α2β2 tetramer of the nifD and nifK gene products (Kim and Rees, 1992). Eac...
The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] clu...
Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the i...
The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. Purified NifEN protein contains multiple metal-sulfur clusters, where the exact types, structures, or functions are still unclear. Recent