The conversion of dinitrogen gas (N2) to ammonia (NH3) is called nitrogen fixation. Because ammonia is necessary for the formation of biologically essential, nitrogen-containing compounds, such as amino acids and nucleic acids, a fixed nitrogen source is necessary to sustain life on earth. Furthermore, the ammonia necessary to support essential biosynthetic reactions is continually sequestered into sediments or reconverted to N2 through the combined biological processes of nitrification and denitrification (Fig. 1). So the pool of fixed nitrogen within the biosphere must constantly be replenished. Nitrogen fixation is necessary to maintain the diversity of life on earth because most organisms cannot metabolize the abundant but relatively inert N2 molecule and must assimilate nitrogen in a "fixed" form, such as ammonia or nitrate. The three ways that nitrogen fixation occurs in the biosphere include (1) lightning and other natural combustion processes, (2) the industrial Haber-Bosch process, and (3) biological nitrogen fixation. Of these three, biological nitrogen fixation, the most significant contributor, accounts for about 65% of the total (1, 2). In addition to its pivotal role in the global nitrogen cycle, nitrogen fixation has agronomic importance because the availability of fixed nitrogen—commonly referred to as fertilizer nitrogen—usually limits to crop production. Consequently, the need to grow sufficient crops to feed the world’s population requires the application of nitrogen fertilizers as a common agricultural practice. Such application of nitrogen fertilizers is expensive for several reasons: First, the Haber-Bosch process used to produce fertilizer nitrogen requires high consumption of nonrenewable resources in the form of fossil fuels. Secondly, significant transportation costs are incurred in shipping industrially produced fertilizer nitrogen to the field. Third, the application of nitrogen fertilizers often results in run-off contamination of local water systems. An alternative approach to the Haber-Bosch process for nitrogen fertilizers is to exploit the biological process, and toward this end a considerable effort has been directed at understanding the molecular mechanism of biological nitrogen fixation.
Figure 1. Simplified diagram of the biological nitrogen cycle.
1. Diazotrophs and nodulation
Although most organisms cannot fix nitrogen, a select group of microorganisms can. These organisms are called diazotrophs, which means "nitrogen eaters," and they are widely distributed among and restricted to the Archae and Bacterial kingdoms. Examples of bacterial species that fix nitrogen and have been extensively studied include Azotobacter vinelandii (an obligate aerobe), Clostridium pasteurianum (an obligate anaerobe), Klebsiella pneumoniae (a facultative anaerobe), Rhodospirillum rubrum (a photosynthetic bacteria), Anabaena sp. 7120 (a heterocyst-forming cyanobacterium), and Bradyrhizobium japonicum (a symbiotic bacterium). From the agronomic perspective, symbiotic nitrogen fixers are the most important. These organisms invade leguminous plants, such as pea and alfalfa, and induce the formation of specialized structures, called nodules, on their roots. In this symbiotic association, the root nodule contains differentiated bacteria that specialize in nitrogen fixation (3). In this symbiosis, the plant provides the microbe a carbon energy source through the process of photosynthesis whereas the microbe supplies the plant host a fixed nitrogen source through the process of nitrogen fixation. Because the enzyme that catalyzes biological nitrogen fixation, nitrogenase , is extremely oxygen sensitive, another important functional feature of the root nodule is to provide an anoxic environment for nitrogen fixation. Protection of nitrogenase within the root nodule occurs by separating the oxygen-evolving process of photosynthesis and the oxygen-sensitive process of nitrogen fixation. This separation is accomplished in two primary ways: First, the plant-derived, outer cortical layer of the nodule provides a relatively strong barrier against free oxygen diffusion into the central core of the nodule, where the nitrogen-fixing bacteria are located and where photosynthesis does not occur. Secondly, the oxygen needed for bacteroidal respiration, which in turn is necessary to provide the energy and reducing equivalents to drive nitrogen fixation within the bacteroid, is delivered by an oxygen-binding plant protein called leghemoglobin.
Establishing of a symbiotic relationship between a leguminous plant host and an associated rhizobium is a complicated process that is specific between a particular host and a particular symbiont (4). During initiation of the infection process, bacteria induce the formation of curls at the tip of plant root hairs (5, 6), which then envelop the invading bacteria. Then infection threads are formed within the root hair. Infection threads are tubular structures of plant origin that penetrate the root hairs and the root cortex through which the invading bacterium traverse (7). At or about the time infection thread formation occurs, cell division is also induced within the root cortex, resulting in the formation of a nodule primordium (8). When the infection thread contacts a newly divided primordial cell within the root cortex, the bacteria are released from the infection thread tip. Subsequent penetration of the released bacterial cell into the cytoplasm of the primordial cell occurs through a process of endocytosis resulting in the formation of a plant cell membrane (bacteroidal membrane) that surrounds the bacterium (9). Bacterial cell division and bacteroid membrane proliferation then occur, resulting in the host plant cell cytoplasm becoming filled with bacteria. These bacteria then differentiate into cells specialized for nitrogen fixation that are called bacteroids.
To establish an effective symbiosis, developmental and metabolic cooperation between the plant and microbe are necessary. This cooperation is accomplished through the reciprocal communication and control of gene expression between the two partners. During nodule development, signaling occurs through the action of nodulation (nod) gene expression by the bacterium and expression of nodulins (ENOD sequences) by the plant. The first of these signals is provided by flavanoid molecules, three-ring aromatic compounds derived from phenylpropanoid metabolism, which are produced by the plant and found in the root exudate (10). Flavanoids act as specific inducers of bacterial nodD gene expression (11). The nodD gene encodes a regulatory protein that controls the expression of other nod genes. The concerted action of other nod gene products, whose synthesis is induced by nodD gene expression, results in forming and releasing bacterial signal molecules called Nod factors (12) . Nod factors are responsible for signaling the initial root hair curling event. Their core structures are composed of b1-4 linked #-acetylglucosamine residues of various length (usually one to four molecules). The specificity of a particular Nod factor is determined by its level of oligomerization and by certain chemical modifications of the oligosaccharide core, such as O-acylation or N-acylation at the nonreducing end of the oligosaccharide and sulfation at the reducing end. The nodABC gene products are common among all the rhizobia, and they are required for catalyzing formation of the oligosaccharide core, whereas other "host-specific" nod gene products are responsible for oligomerizing and modifying the oligosaccharide core.
1.1. Nitrogenase
Nitrogenase is the enzyme that catalyzes biological nitrogen fixation. The reaction is usually depicted as follows:
All diazotrophs studied so far contain a nitrogenase that is a complex, two-component metalloprotein (13-15). The individual nitrogenase component proteins have been designated as the Fe protein and the MoFe protein , which were derived from the respective compositions of their associated metal centers. The Fe protein is a homodimer (Mr 64,000) that contains two MgATP binding sites and a single [4Fe-4S] cluster (see Iron-Sulfur Proteins). The MoFe protein is an a2b2 heterotetramer (Mr 250,000) that contains two pairs of metalloclusters, called P-clusters, and iron molybdenum cofactors (FeMo cofactors). Ribbon diagrams for the three dimensional structures of the Fe protein and MoFe protein are shown in Fig. 2, and structures of the associated nitrogenase metal clusters are shown in Fig. 3. Because the Fe protein is a specific reductant of the MoFe protein, which in turn provides the site of substrate reduction, some investigators refer to the Fe protein as dinitrogenase reductase and the MoFe protein as dinitrogenase (16).
Figure 2. Ribbon diagrams of the three-dimensional structures of the nitrogenase Fe protein and MoFe protein. The view interaction with a single ab-unit of the MoFe protein (bottom).
Figure 3. Organization and structures of the nitrogenase metalloclusters. The path of electrons is from the [4Fe-4S] clusl cluster and then to the FeMo cofactor. Transfer of an electron from the Fe protein to the MoFe protein is coupled to MgA provides the N2 binding and reduction site.
![Organization and structures of the nitrogenase metalloclusters. The path of electrons is from the [4Fe-4S] clusl cluster and then to the FeMo cofactor. Transfer of an electron from the Fe protein to the MoFe protein is coupled to MgA provides the N2 binding and reduction site.](http://what-when-how.com/wp-content/uploads/2011/05/tmp2E28_thumb1.jpg "Organization and structures of the nitrogenase metalloclusters. The path of electrons is from the [4Fe-4S] clusl cluster and then to the FeMo cofactor. Transfer of an electron from the Fe protein to the MoFe protein is coupled to MgA provides the N2 binding and reduction site.")
1.1.1. Nitrogenase Mechanism and Role of the Metal Centers
During catalysis, the Fe protein delivers electrons, one at a time, to the MoFe protein in a process that couples MgATP binding and hydrolysis to the association and dissociation of the two component proteins and concomitant electron transfer. Both component proteins are required for MgATP hydrolysis, and neither component protein reduces any substrate in the absence of its catalytic partner. The process by which electrons are sequentially delivered to the MoFe protein and subsequently to substrate has been described by a kinetic model that involves two interlocking cycles called the Fe protein cycle and the MoFe protein cycle (17). The Fe protein cycle involves oxidizing and reducing the Fe protein’s [4Fe-4S] cluster between the 1+ and 2+ redox states as it sequentially delivers electrons to the MoFe protein and is re-reduced by other electron transfer proteins (usually a ferredoxin or flavodoxin ). The MoFe protein cycle involves the progressive reduction of the MoFe protein, which ultimately leads to N2 binding and reduction. Because eight electrons and eight protons are required for N2 reduction and H2 evolution, each MoFe protein cycle requires eight Fe protein cycles and storage of the electrons. Kinetic studies have also shown that N2 does not become bound to the active site until at least two, and probably more, electrons have been accumulated within the MoFe protein (18). It is not yet known where and how the electrons delivered to the MoFe protein are stored before to the binding and reduction of the substrate. However, current structural and biochemical data indicates that cluster-to-cluster electron transfer occurs as shown in Fig. 3 (13, 14, 19).
In addition to the individual structural models for the Fe protein and MoFe protein (Fig. 2), an X-ray crystallograpic model for the docked complex has also been determined (19). In this model, docking occurs so that the two-fold symmetry axis surrounding the Fe protein’s [4Fe-4S] cluster becomes paired with the surface of the MoFe protein’s pseudosymmetrical ab-interface. This places the [4Fe-4S] cluster close to the P-cluster of the MoFe protein and places the P-cluster between the Fe protein [4Fe-4S] cluster and the FeMo cofactor. This arrangement is consistent with the electron transfer scheme shown in Fig. 3 and indicates that the P-cluster’s role is to broker electron transfer between the Fe protein and the substrate reduction site provided by the FeMo cofactor. The P-cluster is located at the ab interface of the MoFe protein subunits and, in its fully reduced form, is constructed from two [4Fe-4S] subclusters that share a central sulfide (Fig. 3). Upon oxidation of the P-cluster, a structural rearrangement occurs involving movement of two Fe atoms and a change in the ligand arrangement around the cluster (20). Such redox-dependent structural changes within the P-cluster might be mechanistically related to its role in accepting electrons from the Fe protein and delivering them to the FeMo cofactor.
The FeMo cofactor consists of a metal sulfur framework (MoFe7S9) and one molecule of (R)- homocitrate (Fig. 3). The framework is constructed from S-bridged MoFe3S3 and Fe4S3 cluster subfragments. Homocitrate is coordinated to the Mo atom through its b-hydroxy and b-carboxy groups. Several lines of evidence indicate that the FeMo-cofactor provides the substrate binding and reduction site: First, mutant strains that cannot biosynthesize the FeMo cofactor also cannot catalyze nitrogen fixation (21). When the isolated FeMo cofactor is added to crude extracts prepared from such mutant strains, the in vitro ability to fix nitrogen is restored. Secondly, a MoFe protein that contains an altered form of the FeMo cofactor, where citrate replaces homocitrate as its organic constituent, exhibits altered catalytic properties (22, 23). Thirdly, altered MoFe proteins that have amino acid substitutions located within the FeMo cofactor’s polypeptide environment also exhibit altered catalytic properties (24). Although it is not yet known how substrates interact with the FeMo cofactor during turnover, the presence of six coordinately unsaturated Fe atoms and the attachment of homocitrate to the Mo atom has invited speculation about the nature of substrate binding (25). For example, in one model it has been proposed that the carboxylate group coordinated to the Mo atom might serve as a leaving group in a mechanism that activates Mo to provide a substrate coordination site (26).
1.1.2. The Role of MgATP in Nitrogenase Catalysis
The reduction of N2 to yield 2 NH3 is thermodynamically favorable. Thus, the need for MgATP binding and hydrolysis during nitrogenase catalysis is kinetic so that electron transfer toward substrate reduction is favored and the flow of electrons back toward the Fe protein is prevented. One way of envisioning this process is to consider that the energy released through MgATP binding and hydrolysis could be used to open and close electron gates to ensure that multiple electrons are accumulated within the MoFe protein before they are donated to substrates. In support of this model, primary sequence and structural comparisons have revealed that the Fe protein is a member of a large class of signal transduction proteins that undergo conformational changes upon MgATP binding and hydrolysis. A consensus view (27) of the events that occur during a single turn of the Fe protein cycle and which accounts for the role of MgATP in nitrogenase catalysis is as follows. Intercomponent interaction is initiated when the reduced Fe protein binds two MgATP molecules. This elicits a conformational change in the Fe protein that makes it competent to interact with the MoFe protein. Upon complex formation, changes occur in the midpoint potentials of the respective clusters such that electron flow toward the FeMo cofactor is energetically favorable. For example, in the complexed form, the Fe protein’s [4Fe-4S] cluster has a redox potential of about -620mV, the P-cluster a potential of about -390mV, and the FeMo cofactor a potential of about -40mV (28). In addition to eliciting redox changes that lead to electron transfer, the component docking event also triggers MgATP hydrolysis at about the same time as, or shortly after, electron transfer. Conversion of the Fe protein from the MgATP-bound state to the MgADP-bound state subsequently causes complex dissociation, which is believed to be the rate-limiting step in nitrogenase catalysis (29). Thus, the accumulation of electrons within the MoFe protein is a dynamic process that involves transmitting signals back and forth between the Fe protein and MoFe protein. Then the role of MgATP is to synchronize these events through sequential conformational changes induced by MgATP binding, component protein interaction, and nucleotide hydrolysis.
1.1.3. Alternative Nitrogenases
All organisms capable of nitrogen fixation that have been examined at the biochemical level have a Mo-containing nitrogenase as described previously. All such nitrogenases exhibit a high level of primary structural identity compared to each other. Particularly high sequential conservation is found in the respective MgATP- and metallocluster-binding sites (30). There is, however, another class of nitrogenases that do not contain Mo, and these have been designated alternative nitrogenases (31). Two types of alternative nitrogenases have been identified so far (32, 33). One of these contains a cofactor whose Mo atom is substituted by vanadium, V (FeV cofactor), and the other contains a cofactor whose Mo atom is substituted by Fe (FeFe cofactor). The conservation in primary sequences recognized among the Mo-dependent nitrogenases also extends to the alternative nitrogenases. Thus, all nitrogenases have common structural and mechanistic features. All three nitrogenase types are found in A. vinelandii, and expression of each of them is under a hierarchical control that depends on the availability of Mo or V in the growth medium (34, 35). When Mo is present in the growth medium, expression of the Mo-dependent nitrogenase is stimulated, and the expression of the alternative nitrogenases is repressed. Similarly, when Mo is absent, but V is available, only expression of the V-dependent enzyme occurs. When neither Mo nor V is available, only the alternative nitrogenase that contains FeFe cofactor is expressed. Such hierarchical control by the availability of metals makes physiological sense because the Mo-dependent nitrogenase has an intrinsically higher capacity to reduce nitrogen than either of the alternative nitrogenases, and the V-dependent nitrogenase is more efficient than the Fe-only nitrogenase (36).
1.2. Nif-Genes and Maturation of Nitrogenase
In addition to the structural genes for nitrogenase, other genes are required for (1) coupling the reduction of the Fe protein to intermediary metabolism, (2) maturation of the nitrogenase components, and (3) regulating the expression of the nitrogenase genes. The organism that has the simplest organization of nitrogen fixation specific (nif) genes and is best studied at the molecular genetic level is the facultative anaerobe Klebsiella pneumoniae. There are 20 nif genes in this organism, organized into seven transcriptional units (Fig. 4). The specific designations for individual Klebsiella pneumoniae nif genes are also used to denote genes whose products have homologous functions in other organisms. For example, the nif structural genes from all diazotrophs are designated nifH, nifD, and nifK, and they respectively encode the Fe protein and the a- and b-subunits of the MoFe protein. A complication in the genetic nomenclature of genes, whose products are involved in nitrogen fixation, is that some organisms do not have homologues to all of the Klebsiella pneumoniae n/f-specific genes, but have other genes related to nitrogen fixation that are not present in K. pneumoniae. The general convention used is that the "nif" designation is reserved only for those genes that have functional counterparts in K. pneumoniae. Genes in other organisms, whose products are involved in the process of nitrogen fixation but do not have functional counterparts in K. pneumoniae, have been given various other designations. One example is the designation "fix" which designates such genes from the rhizobia.
Figure 4. Organization of the nitrogen fixation specific ( nif) genes of Klebsiella pneumoniae. Black arrows indicate transcription units and the direction of their transcription. Structural genes encoding the nitrogenase Fe protein ( nifH) and MoFe protein subunits (nifDK) are shaded. Functions of the nif gene products are described in the text.
1.2.1. Electron Transport to Nitrogenase
A source of reducing equivalents of sufficiently low potential is required to regenerate reduced Fe protein after it has donated an electron to the MoFe protein. Both flavodoxins and ferredoxins serve this function in vitro. In K. pneumoniae, two genes, nifF and nifJ, are involved in coupling the reduction of Fe protein to intermediary metabolism (37, 38). The nifF gene product is a flavodoxin that, in its reduced hydroquinone form, donates an electron to the oxidized Fe protein, thereby generating the semiquinone form of the flavin moiety. The re-reduction of flavodoxin is accomplished through the catalytic activity of the nifJ gene product (pyruvate-flavodoxin oxidoreductase), which couples the oxidation of pyruvate, yielding acetyl-CoA and CO2, to the reduction of the semiquinone form of flavodoxin to the hydroquinone form.
1.2.2. Maturation of the Fe Protein and MoFe Protein
The primary translation products of the nitrogenase structural genes are not active. Instead, a consortium of other n//-specific genes are required to activate the immature structural components. The function of the n/f-specific maturation gene products is to catalyze the formation and insertion of the metalloclusters into apo-forms of the Fe protein and MoFe protein. In the Fe protein, only the nifMgene product is specifically required for its maturation (39). The nifMgene product has not yet been isolated in an active form, but it is a member of a family of peptidyl prolyl cis/trans isomerases (40). Such enzymes are thought to assist in protein folding by catalyzing the cis/trans isomerization of certain prolyl peptide bonds in some proteins. The requirement for such an activity in maturation of the Fe protein is not obvious but could be related to the appropriate organization of the cysteine ligands needed for properly inserting the [4Fe-4S] cluster that is located at the Fe protein dimer interface.
Maturation of the MoFe protein, particularly the formation and insertion of the FeMo cofactor into the MoFe protein, is much more complicated. This process involves the products of the nifH, nifE, nifN, nifB, nifV, and nifQ genes (41). The nitrogenase Fe protein, a product of nifH, is required for both forming and inserting the FeMo cofactor (42, 43). Although the specific function of Fe protein in these processes is not known, neither its MgATP-binding or MgATP-hydrolytic properties nor its ability to transfer electrons are necessary (44, 45). Biochemical complementation experiments have shown that the FeMo-cofactor is synthesized separately and then inserted into an apo-form of the MoFe protein that contains intact P-clusters but lacks the FeMo cofactor (46). At least a portion of the biosynthetic process occurs within a complex of the nifEN products. The nifEN gene products bear primary sequential similarity to the products of the nifDK structural genes (47), and an a2b2 complex of the nifEN products is a molecular scaffold for FeMo cofactor assembly (48). The product of the nifB gene catalyzes the formation of a FeMo cofactor precursor called the B-cofactor (49). The B-cofactor probably provides the Fe-S cage necessary for FeMo cofactor construction and is inserted into the NifEN complex at an intermediate stage in FeMo cofactor formation (50). The nifV gene catalyzes the condensation of acetyl-CoA and a-ketoglutarate to form homocitrate, the organic constituent of the FeMo cofactor (51, 52). The nifQ gene product has a role in inserting the Mo atom into the FeMo cofactor, but its exact role in this process is not known (53). The products of the nifW and nifZ genes might also have some role in FeMo cofactor biosynthesis because mutations in either or both or them result in lowered MoFe protein activity (54). In K. pneumoniae, apo-MoFe protein produced by strains lacking nifB or nifEN activity contain a low molecular weight protein encoded by nifY. The nifY gene product has a role in stabilizing a conformation of the apo-MoFe protein that is amenable to FeMo cofactor insertion (55, 56). In A. vinelandii, a different low molecular weight protein (called gamma), not encoded by nifY, serves the same function (57).
In addition to the gene products that are specifically involved in nitrogenase metallocluster biosynthesis, there are two other nif-genes, nifS and nifU, whose products catalyze reactions that are generally involved in mobilizing Fe and S for metallocluster assembly (58, 59). The nifS gene product is a cysteine desulfurase that activates S for [Fe-S] cluster formation, whereas the nifU gene product probably has a complementary role as an Fe carrier. Homologues to nifU and nifS, whose expression is not under nzf-specific control, are present in many organisms. The products of these genes probably have general functions in [Fe-S] cluster formation and repair (60).
1.3. Regulation of Nitrogenase Expression
For most free-living, nitrogen-fixing organisms, such as K. pneumoniae, nif-gene expression should be responsive to three environmental conditions. These parameters include (1) the availability of a fixed nitrogen source, (2) whether or not oxygen is present, and (3) the energy charge status of the cell (see Adenylate Charge). The reasons that these conditions are important is that nitrogenase activity is both oxygen-sensitive and requires the intense consumption of metabolic energy. Thus, if oxygen is present, if a fixed nitrogen source is already available, and if cellular growth is limited by the availability of a carbon source rather than a nitrogen source, the expression of nif-genes would represent a waste of metabolic energy. In free-living nitrogen fixers that have alternative nitrogenases, the expression of nif-genes is additionally controlled by the availability of Mo or V.
The organism that has been most thoroughly studied in terms of nif-gene expression is K. pneumoniae. The 20 nif-specific genes from K. pneumoniae are organized into seven transcriptional units (Fig. 4). Six of these transcription units include the nif structural genes, electron transport genes, and nitrogenase maturation genes, whereas the seventh transcription unit contains the nzf-specific regulatory genes, nifL and nifA (61). Expression of the nifLA genes is controlled by the global regulatory elements, products of the ntrC, ntrB, and ntrA genes. The expression of the other nif transcriptional units is controlled by the products of nifL, nifA, and ntrA . The ntrA gene product (NTRA) is an alternative sigma factor that controls the expression of a wide variety of gene families. The nif and ntr genes are only two examples (62). NTRA (also called a54) imparts a specificity to RNA polymerase so that it recognizes the consensus promoter sequence CTGG-N8-TTGCA. This consensus sequence spans a region located 24 to 12 base pairs preceding the transcription initiation site. In contrast, normal RNA polymerase contains the abundant sigma factor, called a , and this form of RNA polymerase recognizes the typical prokaryotic promoter sequence TTGACA-N17-TATACA. The so-called housekeeping RNA polymerase binding site is located in the -35, -10 regions preceding the transcription initiation site. Expression of ntrA is not tightly controlled. Instead, the presence of NTRA is a way for the cell to reserve a certain portion of the RNA polymerase for specialized functions, such as nitrogen fixation.
Specific regulation of the expression of the nifLA genes is controlled by the products of the global nitrogen regulatory genes ntrC (NTRC) and ntrB (NTRB). NTRB is a phosphatase/kinase sensor protein that controls the phosphorylation of NTRC in response to the ratio of a-ketoglutarate to ammonia in the cell (63). When this ratio is high, NTRC is phosphorylated. When the ratio is low, NTRC is dephosphorylated. The former represents a condition of fixed nitrogen limitation and high energy charge, which signals the initiation of a two-tiered regulatory cascade that first leads to activation of the nifLA promoter and then results in activation of the other nif promoters. In its phosphorylated form, NTRC recognizes a DNA consensus sequence located upstream from the nifA a54-RNA polymerase binding site. Once bound at this upstream activator site, phosphorylated NTRC catalyzes an ATP-dependent conformational isomerization at the promoter site, which ultimately results in transcriptional initiation of the nifLA promoter (64). Then accumulation of the products of nifL (NIFL) and nifA (NIFA) permits specific control of the other nf-promoters. NIFA is structurally and functionally similar to NTRC (65, 66). Like NTRC, it binds to an upstream activator sequence, although one that differs in consensus sequence to the NTRC binding site. The NIFA binding site has a consensus sequence motif (TGT-N^-ACA) located approximately 100 bp preceding each of the nif promoters, except the nifLA promoter (67). NIFA probably activates expression of the nif promoters in an ATP-dependent manner analogous to the function of the phosphorylated form of NTRC.
Unlike NTRC, NIFA does not undergo phosphorylation and dephosphorylation in response to environmental signals. Instead, whether or not NIFA activates nif gene expression is controlled by NIFL, which acts as an antiactivator. When the oxygen level or the level of fixed nitrogen is sufficiently high, so that nitrogen fixation is either futile or not necessary, NIFL interacts with NIFA to prevent activation of the nif promoters. Although the details of how the complexation of NIFA and NIFL occurs are not known, it has been shown that NIFL is a flavoprotein which senses the redox status of the cell by conformational changes controlled by the oxidation state of its FAD moiety (68).
The mechanism by which K. pneumoniae controls nif gene expression is by no means universal.
For example, very little is known about how nif gene expression is controlled in the clostridia or the Archae, although control does not appear to be through the two-tiered NTR-dependent mechanism described previously. In certain organisms another layer of nitrogenase control activity also occurs at the posttranslational level. The best described example of this type of control is the reversible ADP-ribosylation of the nitrogenase Fe protein that occurs in Rhodobacter rubrum. In this organism, nitrogenase activity is controlled at the transcriptional level and also through the antagonistic activity of ADP-ribosylation and glycohydrolase enzymes whose activities are controlled by various environmental conditions (69, 70). Another example of the control of the expression of nitrogenase activity is found in the cyanobacterium Anabaena 7120 (71). This organism restricts nitrogen fixation to specialized cells called heterocysts . The formation of heterocysts, and nitrogenase expression are controlled both temporally and spatially by terminal differentiation events involving rearrangements of the genome.
Another specialized case of the control of nitrogenase gene expression occurs in the root nodule. It does not make sense to control nitrogenase gene expression and activity in response to the level of fixed nitrogen in the root nodule because the bacteroid functions to supply its host with fixed nitrogen. Thus, in the root nodule, nif gene expression is controlled by a two-tiered cascade that responds to the cellular oxygen tension. Similar to the NTRC/NTRB system, oxygen control of nif gene expression in rhizobia occurs through the concerted activity of an environmental sensor (FIXL) and a positive regulator of gene expression (FIXJ) (72). The sensor protein FIXL is a hemeprotein located within the cell membrane, and like NTRB, it has phosphatase/kinase activities. At low oxygen tension (microaerobicity), FIXL promotes the phosphorylation of FIXJ, which then activates the expression of nifA analogously to NTRC activation in K. pneumoniae. The rhizobia do not contain a protein analogous to NIFL, so control of nif gene expression in this case does not involve an antiactivator. Instead of the involvement of an antiactivator in the posttranslational control of certain rhizobial NIFA proteins, NIFA activity is directly sensitive to the presence of oxygen (73).
