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 insights into the structures of the FeMo-co biosynthetic intermediates and into the biochemical details of FeMo-co synthesis. 93

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 chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

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. CONTENTS 1. Introduction 4922 2. Structure of Mo−Nitrogenase Complex 4924 3. Organization of Mo−Nitrogenase Genes and Proposed Functions of Their Products 4925 3.1. Genomic Organization of A. vinelandii Mo− Nitrogenase Genes 4925 3.2. Proposed Functions of nif Gene Products 4926 3.3. Essential and Ancillary Proteins for Mo− Nitrogenase 4926 3.4. Biosynthesis of Genetically Simpler Mo− Nitrogenases 4927 4. Biosynthesis of Simple [Fe-S] Clusters for Nitrogenase: Roles of NifU and NifS 4928 4.1. Information from nif U and nif S Mutagenesis 4928 4.2. NifS Is a Cysteine Desulfurase Involved in Metallocluster Biosynthesis 4928 4.3. NifU Is a Molecular Scaffold for Assembly of Nitrogenase-Destined [4Fe-4S] Clusters 4929 4.4. NifS-Mediated Assembly of Transient [Fe-S] Clusters at NifU 4930 4.5. NifS and NifU Transfer of [4Fe-4S] Cluster to Fe Protein 4930 5. Fe Protein Maturation 4930 5.1. Role of NifM 4930 5.2. Proposed Function of NifM in Fe Protein Maturation 4931 6. Interaction of Maturation Factors with Cofactor Deficient MoFe Protein 4932 7. Formation of MoFe Protein P-Clusters 4933 7.1. NifU and NifS 7.2. Fe Protein Is Required for P-Cluster Formation 7.3. NifZ Is Involved in P-Cluster Formation 7.3.1. Model 1: NifZ Is Only Required for Maturation of Second P-Cluster in Each Apo-MoFe Protein Molecule 7.3.2. Model 2: NifZ Is Involved in Maturation of Both P-Clusters 8. FeMo-co: Description of the Cofactor and Methods to Measure Its Biosynthesis 8.1. Discovery and Isolation of FeMo-co 8.2. In Vitro Systems for FeMo-Cofactor Synthesis and Insertion 9. Model for FeMo-co Biosynthesis 10. Biosynthesis of FeMo-co Fe-S Core: Roles of NifU, NifS, NifB, and FdxN 10.1. NifS and NifU Assembly of Precursor [Fe-S] Clusters for FeMo-co 10.2. NifB and NifB-co 10.2.1. Information from nif B Mutagenesis 10.2.2. Identification and Isolation of NifB-co, the Product of NifB Activity 10.2.3. Interstitial Atom of FeMo-co Is Present at NifB-co

Nitrate uptake and reduction to nitrite and ammonium are driven in cyanobacteria by photosynthetically generated assimilatory power, i.e., ATP and reduced ferredoxin. High-affinity nitrate and nitrite uptake takes place in different cyanobacteria through either an ABC-type transporter or a permease from the major facilitator superfamily (MFS). Nitrate reductase and nitrite reductase are ferredoxin-dependent metalloenzymes that carry as prosthetic groups a [4Fe-4S] center and Mo-bis-molybdopterin guanine dinucleotide (nitrate reductase) and [4Fe-4S] and siroheme centers (nitrite reductase). Nitrate assimilation genes are commonly found forming an operon with the structure: nir (nitrite reductase)-permease gene(s)-narB (nitrate reductase). When the cells perceive a high C to N ratio, this operon is transcribed from a complex promoter that includes binding sites for NtcA, a global nitrogen-control regulator that belongs to the CAP family of bacterial transcription factors, and NtcB, a pathway-specific regulator that belongs to the LysR family of bacterial transcription factors. Transcription is also affected by other factors such as CnaT, a putative glycosyl transferase, and the signal transduction protein P(II). The latter is also a key factor for regulation of the activity of the ABC-type nitrate/nitrite transporter, which is inhibited when the cells are incubated in the presence of ammonium or in the absence of CO(2). Notwithstanding significant advance in understanding the regulation of nitrate assimilation in cyanobacteria, further post-transcriptional regulatory mechanisms are likely to be discovered.

No abstract

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.

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 iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe 2؉ , S 2؊ , MoO4 2؊ , and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe 2؉ , S 2؊ , MoO4 2؊ , R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.FeMo-co ͉ nitrogen fixation ͉ nif ͉ Fe-S proteins B iological nitrogen fixation, the conversion of atmospheric N 2 to NH 3 , is an essential process of the global biogeochemical cycle of nitrogen that supports life on Earth (1). Biological nitrogen fixation provides an alternative solution to the heavy use of chemically synthesized nitrogen fertilizers and, thus, is important to achieve sustainable agricultural practices (2). The major part of biological nitrogen fixation is catalyzed by the molybdenum nitrogenase enzyme according to the following reaction: N 2 ϩ 8e Ϫ ϩ 16 MgATP ϩ 8 H ϩ 3 2 NH 3 ϩ H 2 ϩ 16 MgADP ϩ 16 Pi (3). Remarkably, this reaction produces H 2 as a by-product and has regained attention over the last few years as a promising source of clean energy (4). The molybdenum nitrogenase has two component proteins: dinitrogenase (NifDK) and dinitrogenase reductase (NifH). Dinitrogenase carries at its active site one of the most complex biological metalloclusters known to date, the iron-molybdenum cofactor (FeMo-co), composed of seven Fe, nine S, one Mo, one R-homocitrate, and one light unidentified atom (5, 6).The systematic phenotypic analyses of Azotobacter vinelandii and Klebsiella pneumoniae strains with mutations in nitrogen fixation (nif ) genes and the subsequent biochemical analyses have led to the identification of at least 11 genes (nifUS-BQ-ENX-V-H-Y and nafY) encoding proteins involved in FeMo-co biosynthesis and its insertion into a FeMo-co-deficient apoNifDK protein (7-9). Fig. 1 depicts an integrative model of the current understanding of the pathway for FeMo-co biosynthesis. It is important to emphasize that this model is mostly based on the ability to isolate nif muta...

SummaryThe iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.