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

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.

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.

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.

SignificanceNitrogen is a constituent of many essential biomolecules and plentiful on earth as inert N2 gas. For its assimilation by eukaryotes, N2 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 yields necessary to feed the world's population have critically depended on applying nitrogenous fertilizers. Incorporation of prokaryotic determinates required to produce active nitrogenase into crop plants would have enormous economic and environmental benefits. The active-site cofactors of all nitrogenases have a common metallocluster precursor synthesized by NifB. Here, we identify the genetic determinants for NifB function in mitochondria of Saccharomyces cerevisiae, thereby advancing prospects to generate N2-fixing crops.

Nitrogen fixation is a tightly regulated trait. Switching from N 2 fixation-repressing conditions to the N 2 -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 negativefeedback 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 N 2 fixation in nondiazotrophs.

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.

The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α 2 β 2 heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N 2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N 2 -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 αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-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.