COMPLEXITY OF NITRATE REDUCTION PATHWAYS

Nitrogen is a basic element for life because it is a component of the two preeminent biological macromolecules: proteins and nucleic acids. Nitrogen exists in the biosphere in several oxidation states, from N(V) to N(III). Interconversions of these nitrogen species constitute the global biogeochemical nitrogen cycle, which is sustained by biological processes, with bacteria playing a predominant role (74). Briefly, inorganic nitrogen is converted to a biologically useful form by dinitrogen fixation or nitrate assimilation and the further incorporation of ammonia into C skeletons. Nitrogen is removed from the environment by both nitrification, the oxidative conversion of ammonia to nitrate, and denitrification, a respiratory process whereby nitrate is successively reduced to nitrite, N oxides (NO and N₂O), and dinitrogen (N₂). Nitrate reduction plays a key role in the nitrogen cycle and has important agricultural, environmental, and public health implications. Assimilatory nitrate reduction, performed by bacteria, fungi, algae, and higher plants, is one of the most fundamental biological processes, accounting for more than 10⁴ megatons of inorganic nitrogen transformed each year (38). However, there is worldwide concern over the excessive use of fertilizers in agricultural activities, leading to nitrate accumulation in groundwater. Consumption of drinking water with high nitrate levels has been associated with methemoglobinemia and gastric cancer due to endogenous formation of genotoxic N-nitroso compounds by bacteria in the gastrointestinal tract (93). The main threat to the environment comes from eutrophication of aquatic ecosystems. Nitrogen oxides generated by denitrification are also associated with the greenhouse effect and the depletion of stratospheric ozone (100). Therefore, nitrate reduction has become an important focus for research in the last several years, generating a vast literature. The aim of this minireview is to summarize recent advances in the physiology, biochemistry, and genetics of prokaryotic nitrate reduction, emphasizing the different molecular characteristics of the bacterial nitrate reductases. Comprehensive reviews covering nitrate assimilation or denitrification have been published elsewhere (7, 17, 31, 38, 43, 49, 86, 100).

Nitrate reduction can be performed with three different purposes: the utilization of nitrate as a nitrogen source for growth (nitrate assimilation), the generation of metabolic energy by using nitrate as a terminal electron acceptor (nitrate respiration), and the dissipation of excess reducing power for redox balancing (nitrate dissimilation). Four types of nitrate reductases catalyze the two-electron reduction of nitrate to nitrite: the eukaryotic assimilatory nitrate reductases and three distinct bacterial enzymes, comprising the cytoplasmic assimilatory (Nas), membrane-bound respiratory (Nar), and periplasmic dissimilatory (Nap) nitrate reductases. All eukaryotic and bacterial nitrate reductases contain a molybdenum cofactor at their active sites. The basic structure of the eukaryotic cofactor is molybdopterin, a 6-alkyl pterin derivative with a phosphorylated C₄ chain with two thiol groups binding the Mo atom. By contrast, the cofactor found in bacterial nitrate reductases and some molybdoenzymes is the bis-molybdopterin guanine dinucleotide (MGD) form (7, 24, 69, 100). Nitrite oxidase of nitrifying bacteria also shows nitrate reductase activity. This membrane-bound enzyme, which contains MGD and shows a high sequence similarity to the membrane-bound Nar, catalyzes nitrite oxidation to nitrate to allow chemoautotrophic growth, but it can also catalyze the reverse reaction (89). As nitrite oxidase is not a proper nitrate reductase, we will not consider it further.

Eukaryotic assimilatory nitrate reductases are cytosolic homodimeric enzymes that use pyridine nucleotides as electron donors. Each monomer is composed of a 100- to 120-kDa polypeptide with three prosthetic groups, flavin adenine dinucleotide (FAD), cytochrome b₅₅₇, and Mo cofactor, which are located in three functional domains highly conserved among eukaryotic species. The Mo cofactor domain is located at the N-terminal end, the heme region corresponds to the middle domain, and the FAD-NAD(P)H domain is present at the C-terminal end. Structural genes coding for nitrate and nitrite reductases and for high-affinity nitrate and nitrite transporters have been cloned in several eukaryotes (Fig. 1). Biochemistry and molecular genetics of eukaryotic nitrate reduction have been investigated intensively during the last decades (17, 31, 38, 86). However, eukaryotic and prokaryotic assimilatory nitrate reductases share no sequence similarity and have little in common beyond their physiological function.

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Nitrate assimilation pathway in the eukaryotic green alga Chlamydomonas reinhardtii. The components of the high-affinity nitrate and nitrite transport systems, the NAD(P)H-dependent nitrate reductase, and the ferredoxin-dependent nitrite reductase, are drawn in the same color as the corresponding nuclear genes coding for them. The functions of the products of the white genes are unknown. Cytb, cytochrome b; Fdred, reduced ferredoxin; Fdox, oxidized ferredoxin; MoCo, molybdenum cofactor.

In addition to the assimilatory enzyme, two types of dissimilatory nitrate reductases are present in bacteria: the respiratory membrane-bound Nar, which generates a transmembrane proton motive force (PMF) allowing ATP synthesis, and the periplasmic Nap of some gram-negative bacteria (Table 1). Nap seems to be a dissimilatory enzyme sensu stricto, because quinol oxidation by Nap is not directly coupled to the generation of a PMF and because it is independent of the cytochrome bc₁ complex. Nap could still generate a PMF if a proton-translocating NADH dehydrogenase were involved in reducing the quinone pool, but the resulting PMF seems to be insufficient to support ATP synthesis in some bacteria [see "Dissimilatory Periplasmic Nitrate Reductases (Nap)" below] (60). However, depending on the metabolic fate of nitrite, a certain type of nitrate reductase can have different functions under various conditions. Thus, Escherichia coli assimilates nitrite generated by anaerobic nitrate respiration (88), and some denitrifiers use the nitrite formed by Nap to perform anaerobic nitrite respiration or aerobic denitrification (2-4).

Nitrite formed by nitrate reduction can be reduced to ammonium or to nitric oxide by different types of nitrite reductases (Table 2) (15). In nitrate-assimilating bacteria, ammonium is generated in the cytoplasm by a NADH-dependent (the nasB gene product in Klebsiella oxytoca [50]) or a ferredoxin-dependent assimilatory nitrite reductase (the nirA gene product in cyanobacteria [52, 90]). In E. coli, a cytoplasmic NADH-dependent enzyme encoded by the nirB gene (42) catalyzes the reduction of nitrite to ammonium to detoxify the nitrite that accumulates in anaerobic nitrate-respiring cells and to regenerate NAD⁺. Although ammonium generated by this enzyme can be assimilated, the process is termed nitrite dissimilation (88). All these cytoplasmic nitrite reductases contain a single siroheme and a [4Fe-4S] center. The NADH-dependent enzymes also contain FAD (15, 17, 38). It is worth noting that the ferredoxin-nitrite reductase structure is very similar in cyanobacteria, eukaryotic algae, and vascular plants; these organisms all have conserved Cys residues for binding the Fe-S and siroheme cofactors (31). Alternatively, nitrite can be excreted to the periplasm where, depending on the bacteria, three classes of respiratory enzymes can couple its reduction to energy-conserving electron transport pathways. One of these enzymes is the E. coli multiheme cytochrome c nitrite reductase, encoded by the nrf operon, which catalyzes the formate-dependent nitrite reduction to ammonium (28, 44). This enzyme is also known as hexaheme nitrite reductase in some bacteria (15), although the E. coli enzyme binds only five heme c groups (28). Finally, two different respiratory enzymes reduce nitrite to nitric oxide in the periplasm of denitrifying bacteria: the nirS-encoded cytochrome cd₁ nitrite reductase, which is found in Pseudomonas and most denitrifiers, and the nirK-encoded copper nitrite reductase, which is present in some bacteria (7, 15, 43, 100).

	BACTERIAL ASSIMILATORY NITRATE REDUCTASES (NAS)

Structure and biochemical properties of assimilatory nitrate reductases. Nitrate assimilation has been studied at the biochemical or genetic level in several phototrophic and heterotrophic bacteria. Two classes of assimilatory nitrate reductases are found in bacteria: the ferredoxin- or flavodoxin-dependent Nas and the NADH-dependent enzyme (Fig. 2). Both types of Nas contain MGD cofactor and one N-terminal iron-sulfur cluster but are devoid of heme groups, in contrast to eukaryotic and other bacterial nitrate reductases. The cyanobacterial ferredoxin-Nas is a single subunit of 75 to 85 kDa (59, 80), whereas the flavodoxin-Nas of Azotobacter vinelandii is a polypeptide of 105 kDa (34, 35). The purified Nas proteins of A. vinelandii and Plectonema boryanum contain one Mo, four Fe, and four acid-labile S atoms per molecule (35, 59). Amino acid sequence analysis reveals the presence of a Cys motif in the N-terminal end of the proteins, probably binding one [4Fe-4S] or [3Fe-4S] center. Ferredoxin-Nas is also present in Azotobacter chroococcum, Clostridium perfringens, and Ectothiorhodospira shaposhnikovii (38). On the other hand, the NADH-Nas proteins of Klebsiella pneumoniae (50) and Rhodobacter capsulatus (12) are heterodimers of a 45-kDa FAD-containing diaphorase and a 95-kDa catalytic subunit with MGD cofactor and a putative N-terminal [4Fe-4S] center. This NADH-Nas, as deduced by the Klebsiella nasA gene sequence, probably contains an additional [2Fe-2S] center linked to a C-terminal Cys cluster that is similar to a sequence of the NifU protein (49). This region is absent from the ferredoxin-Nas and could act as a ferredoxin-like electron transfer domain. The Bacillus subtilis NADH-Nas does not contain the NifU-like domain in the catalytic subunit but has two tandem NifU-like modules in a central region of the FAD-containing diaphorase (65).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Nitrate assimilation and comparison of the organization of the nitrate assimilation gene clusters in bacteria. The scheme shows the cyanobacterial ferredoxin (Fd)-dependent assimilatory nitrate and nitrite reductases (right) and the NADH-dependent nitrate and nitrite reductases from Klebsiella and Rhodobacter (left). The organization of the Synechococcus, Synechocystis, and K. oxytoca (pneumoniae) nitrate assimilation gene clusters is shown beneath the corresponding proteins. Genes are drawn approximately to scale, and arrows show the direction of transcription. The genes and their products are shown in the same color. Regulatory genes are indicated by black arrows with white vertical lines. The product of the white gene is not shown.

Organization of the genes coding for the assimilatory nitrate reductases. The genes coding for the assimilatory nitrate-reducing system are normally clustered and have been cloned in several bacterial species. These gene clusters include regulatory and structural genes coding for proteins required for uptake and reduction of both nitrate and nitrite (Fig. 2). Nomenclature of these genes is confusing because different names have been given to homologous genes in different bacteria. In our opinion, the nas gene designation in K. pneumoniae (49, 50) is more appropriate. In this bacterium, the nasR gene encoding a transcription antiterminator is linked to the nasFEDCBA operon (36, 48-50, 98). The nasFED genes code for a multicomponent nitrate or nitrite transport system, the nasB gene encodes a siroheme-dependent assimilatory nitrite reductase, and the NADH-nitrate reductase is encoded by the nasC (diaphorase) and nasA (catalytic subunit) genes.

Nitrate transport in bacterial Nas systems. Due to the cytoplasmic location of the Nas enzyme, nitrate reduction is preceded by nitrate transport into the cells (Fig. 2). In most bacterial Nas systems, nitrate seems to be transported by an ABC-type transporter requiring a periplasmic binding protein. Nitrate and nitrite transport systems (and genes coding for their components) have been thoroughly studied in cyanobacteria and Klebsiella. In Synechococcus, nitrate transport is mediated by a periplasmic binding protein (the nrtA gene product), an integral membrane protein (encoded by the nrtB gene), and two homologous ATP-binding proteins, the nrtC and nrtD gene products (53, 66). Similar nrt clusters have been reported for Synechocystis (45), Anabaena (16, 33), and Phormidium laminosarum (56). In Klebsiella, the nasFED genes encode a typical ABC transporter for both nitrate and nitrite: a 46-kDa periplasmic binding protein (NasF) homologous to NrtA, a homodimeric membrane protein (NasE) related to NrtB, and a homodimeric ATP-binding protein (NasD) similar to NrtD (49, 50, 98). A similar nitrate permease has been found in A. chroococcum (62) and a 47-kDa periplasmic protein is involved in the ATP-dependent nitrate transport system of R. capsulatus (18, 25). However, an electrogenic nitrate uptake mediated by a different transporter, the nasA gene product, is present in B. subtilis (65). NasA protein, a member of the major facilitator superfamily, shows sequence similarity to the E. coli narK gene product, a membrane potential-dependent nitrite extrusion protein (79). A narK-homologous gene encoding the putative nitrite efflux porter is also found in B. subtilis.

Regulation of bacterial assimilatory nitrate reductases. Expression of Klebsiella nas genes is subjected to dual control: ammonia repression by the general nitrogen regulatory system (Ntr) and specific nitrate or nitrite induction (36, 49). The Ntr system regulates the synthesis of most enzymes required for utilizing alternate nitrogen sources. During nitrogen-limited growth, the NtrC protein is activated by phosphorylation mediated by NtrB and binds to upstream sequences of promoters recognized by the alternate rpoN-encoded ^N (⁵⁴) factor, activating transcription of the Ntr-regulated genes (57). The Nac protein, a member of the LysR family, also activates expression of several nitrogen-regulated operons (49, 57). Specific nitrate or nitrite induction of nas gene expression in Klebsiella is mediated by NasR, a positive regulator that acts by a transcription attenuation mechanism. In the absence of nitrate or nitrite, a factor-independent transcription terminator present in the nasF leader region prevents nas gene transcription. When nitrate or nitrite is present, NasR promotes transcription antitermination in the leader region, increasing nasF operon expression. Thus, nitrate regulation does not act by controlling transcription initiation but by controlling transcription termination (36, 48, 88).

	RESPITATORY MEMBRANE-BOUND NITRATE REDUCTASES (NAR)

Structure and biochemical properties of membrane-bound nitrate reductases. Membrane-bound nitrate reductases are associated with denitrification and anaerobic nitrate respiration (Fig. 3). Although the most exhaustive biochemistry and genetic studies have been performed in E. coli and Paracoccus denitrificans, Nar enzymes have been purified from several denitrifying and nitrate-respiring bacteria (100). A thermophilic Nar protein with an optimal temperature of 80°C has also been found in Thermus thermophilus (70). In E. coli, there are two different membrane-bound isoenzymes: NRA, which is expressed under anaerobiosis in the presence of nitrate and represents 90% of total activity, and NRZ, which is expressed constitutively (9, 13).

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Nitrate respiration and denitrification pathways in bacteria. The organization of the anaerobic electron transport chains for nitrate respiration and denitrification is shown at the top. The E. coli respiratory nitrate reductase (nar) gene clusters and the Pseudomonas stutzeri denitrification nos, nir, and nor gene clusters are also shown. Genes are drawn approximately to scale, and arrows show the direction of transcription. The genes and their products are drawn in the same color. Regulatory genes are indicated by black arrows with white vertical lines. The products of the white genes are not shown. The quinone pool is indicated by the white oval labeled QH2Q. cyt, cytochrome.

Organization of the genes coding for the membrane-bound nitrate reductases. In E. coli, NRA is encoded by the narGHJI operon located in the chlC locus at 27 min on the chromosome, and NRZ is encoded by the narZYWV operon in the chlZ locus at 32.5 min (Fig. 3) (13). The presence of narGHJI-homologous genes has also been reported for other bacteria. In E. coli, NRA and NRZ show a high similarity: 76% identity for the catalytic subunits (NarG and NarZ), 75% identity for the  subunits (NarH and NarY), and 87% similarity for the NarI and NarV proteins (9). The E. coli chlC locus also includes the narK gene encoding a nitrite efflux porter (79) and the narXL genes encoding a nitrate response two-component regulatory system, in which NarX is the nitrate sensor and NarL is the DNA-binding regulator (88). The chlZ locus does not include regulatory genes, but a narK-homologous gene (narU) is located upstream of narZYWV operon. The narQP genes coding for a second nitrate sensor (NarQ) and a second nitrate response regulator (NarP) are located at 53 and 46 min on the E. coli genetic map, respectively (13, 88).

Nitrate transport in Nar systems. As a consequence of the cytoplasmic location of the active site of NarG, nitrate has to be transported into the cells before it is reduced, and nitrite is usually excreted to the periplasm by a specific nitrite extrusion system. The respiratory nitrate uptake is poorly understood, although it is clear that the nitrate porter is highly specific for nitrate and is inhibited by oxygen (23). Oxygen inhibition of nitrate transport seems to be caused by an indirect mechanism (i.e., the diversion of electrons to oxygen), rather than causing conformational changes in the porter system (23). In contrast to assimilatory nitrate uptake, which uses an ABC-type transporter, the nitrate transport system in nitrate-respiring bacteria has not been identified, although several mechanisms for nitrate uptake have been proposed, including passive nitrate uniport, ATP-dependent uniport, PMF-dependent NO₃/H⁺ symport, and NO₃/NO₂ antiport (7). In E. coli, the narK gene product was considered a nitrate/nitrite antiporter for several years. However, more-detailed studies have demonstrated that NarK is a nitrite exporter which mediates electrogenic nitrite excretion rather than nitrate uptake (79). A gene (narT) encoding a putative nitrate transporter involved in dissimilatory nitrate reduction has been identified in Staphylococcus carnosus (29). NarT shows homology with E. coli and B. subtilis NarK proteins and with B. subtilis NasA, suggesting a role in both nitrate import and nitrite extrusion. In addition, a putative nitrite transporter gene (nirC) has been identified in E. coli and other bacteria. However, it is unclear if NirC is a nitrite importer or exporter (42).

Regulation of respiratory membrane-bound nitrate reductases. In E. coli, Nar proteins are synthesized during anaerobic growth, via the Fnr protein, in the presence of nitrate or nitrite, by two-component regulatory systems of sensor proteins (NarX and NarQ) and DNA-binding regulators (NarL and NarP). Synthesis of Nar enzymes is unaffected by ammonium (7, 43, 88, 100). Although both NRA and NRZ show a high identity, the narZ operon is not regulated either by O₂ (Fnr) or by nitrate (9, 13). Constitutive NRZ could play a role, as proposed for the Nap system, in adaptation to anaerobic metabolism after the transition from aerobic conditions to anoxia.

	DISSIMILATORY PERIPLASMIC NITRATE REDUCTASES (NAP)

Structure and biochemical properties of periplasmic nitrate reductases. Periplasmic nitrate reductases were first reported for phototrophic and denitrifying bacteria, but they are widespread among gram-negative bacteria. Different physiological functions have been proposed for this enzyme. The Nap activity seems not to be primarily involved in nitrate assimilation or anaerobic respiration, although the nitrite generated by Nap can be used as a nitrogen source or as a substrate for anaerobic respiration depending on the organism. The Nap enzyme, as a consequence of its periplasmic location, does not directly contribute to the generation of a PMF. The Nap system is also independent of the energy-conserving cytochrome bc₁ complex, but it is likely linked to the generation of a PMF when the electrons from NADH are passed through the proton-translocating NADH dehydrogenase (7, 74). However, this seems to be insufficient to support anaerobic growth on nitrate in Rhodobacter sphaeroides in the dark (46, 60). Also, the anaerobic growth rate on nitrate of a T. pantotropha NarH mutant overexpressing Nap activity is decreased threefold on account of the reduced energy conservation by Nap relative to Nar during denitrification (4). However, in Pseudomonas sp. strain G-179, the Nap enzyme catalyzes the first step of denitrification in an energy-generating process, although the mechanism used by Nap to gain energy is unclear (2). Thus, the physiological role of the Nap system may vary in different organisms or even in the same bacterium under different metabolic conditions. There are clear evidences that Nap is a dissimilatory enzyme used for redox balancing (7, 60, 75, 77, 84). Maintenance of an appropriate redox balance can be necessary for optimal bacterial growth under some physiological conditions, particularly during fermentative processes in enteric bacteria, oxidative metabolism of highly reduced carbon substrates in aerobic heterotrophs, or anaerobic photoheterotrophic growth in photosynthetic bacteria. In addition, since oxygen primarily inhibits denitrification at the level of nitrate transport (23) and the Nap system does not require this step, some denitrifiers perform aerobic denitrification coupling the Nap enzyme to the nitrite and N-oxide reductases (3, 7). Aerobic denitrification can be a valuable feature for organisms growing under microaerobic conditions or in environments rapidly changing between aerobic conditions and anoxia. Other proposed roles for Nap are the adaptation to anaerobic metabolism after transition from aerobic conditions, the utilization of alternate reductants (85), or even a self-defense mechanism forming high nitrite levels to inhibit the growth of potential competing bacteria (46).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Periplasmic nitrate reduction and organization of the nap gene clusters in bacteria. The R. sphaeroides periplasmic nitrate-reducing system is shown at the top (the small black arrows indicate the electron flow). The comparative organization of the nap gene clusters of R. sphaeroides, T. pantotropha, E. coli, and A. eutrophus is also shown. Genes are drawn approximately to scale, and arrows show the direction of transcription. The genes and their products are drawn in the same color. The products of the white genes are not shown. The quinone pool is indicated by the white oval labeled QQH2.

Organization of genes coding for periplasmic nitrate reductases. Since the napAB structural genes of A. eutrophus were first identified (85), several nap loci have been sequenced (Fig. 4) (2, 8, 32, 37, 72, 73). It is worth noting that nap genes are located on endogenous plasmids in R. capsulatus (97), R. sphaeroides (18), A. eutrophus (85), and P. denitrificans (76). In these bacteria, nap gene expression is unaffected by O₂. However, the E. coli nap genes are clustered on the chromosome (aeg-46.5 locus) and are induced anaerobically by Fnr (21, 37). The plasmid location of most nap genes, the heterologous expression of nap genes (72), and the fact that the ability to reduce nitrate is present in only a few wild-type strains of purple bacteria suggest the possibility of horizontal transfer of nap genes within the bacterial community.

Regulation of dissimilatory periplasmic nitrate reductases. Although there are some differences in the nap gene expression depending on the organisms, the Nap systems are normally unaffected by ammonium or oxygen. In phototrophic bacteria, the Nap activity is present under aerobic and anaerobic conditions and is unaffected by ammonium or by the intracellular C and N balance. In addition, Nap activity is stimulated by nitrate, although a basal activity is also observed in the absence of nitrate (26, 72). In P. denitrificans, the Nap activity is observed in aerobically grown cells even in the absence of nitrate. Activity is maximally expressed during growth on highly reduced carbon sources, such as butyrate, suggesting a Nap regulation in response to the redox state of the bacterium (84). Similarly, the Nap system is not induced by nitrate in A. eutrophus, and maximal expression is observed under aerobic conditions at the stationary phase (85). However, expression of the E. coli nap operon (aeg-46.5 locus) is induced during anaerobic conditions, via the Fnr regulator, and by nitrate or in a lesser extent by nitrite, via the homologous regulators NarL and NarP. Both proteins compete in vivo for a common binding site in the aeg-46.5 promoter region, but only NarP activates gene expression. Thus, NarL has a negative effect on expression of the aeg-46.5 operon because it antagonizes NarP-dependent activation (21). The nap gene cluster of Pseudomonas sp. strain G-179 could also be regulated by a Fnr-like protein under anaerobic conditions (2).

Translocation of Nap to the periplasm. The periplasmic location of the Nap enzyme raises important questions about its export process. The cytochrome c subunit (NapB) contains the typical N-terminal signal sequence required for a Sec-dependent translocation (for a recent review, see reference 30). Therefore, heme binding to the NapB apoprotein can take place in the periplasm, as demonstrated for other cytochromes c (67). However, several observations suggest that the catalytic NapA subunit could be assembled in the cytoplasm and exported into the periplasm in a folded conformation by a Sec-independent pathway. First, NapA and other periplasmic molybdoenzymes contain N-terminal signal sequences that are unusually long and bear a twin-Arg motif. This presequence can be involved in an alternative translocation system similar to the PMF-dependent thylakoid import pathway (30). Second, MGD cofactor is synthesized in the cytoplasm by the action of five different loci (moa, mob, mod, moe, and mog), and no cofactor export system has been reported for any bacteria analyzed so far. In addition, the size of the cofactor and its almost completely buried environment within the protein (24) suggest that the MGD cofactor should be assembled in the cytoplasm prior to protein translocation. It has been demonstrated that cofactor insertion into the apoprotein is a prerequisite for the translocation of the E. coli trimethylamine N-oxide reductase by a Sec-independent pathway (81). Recently, the E. coli genes required for the Sec-independent export of cofactor-containing periplasmic proteins have been identified (82, 95).

	CONCLUDING REMARKS

Top Conclusion References

Recent biochemical and genetic studies of nitrate reduction have revealed an unexpected complexity for this process, which is of particular significance within the biogeochemical nitrogen cycle. Three distinct classes of bacterial nitrate-reducing systems, which are clearly different at the level of cellular location, structure, biochemical properties, regulation, and gene organization, have been described. Bacterial nitrate reductases are also different from eukaryotic nitrate reductase. The three bacterial nitrate reductases, all of which are predicted to contain a MGD cofactor, can be present in the same organism (85). Although some differences among various organisms are observed for each type of nitrate-reducing system, the properties of distinct nitrate reductases can be rationalized. Some bacteria contain a cytoplasmic assimilatory nitrate reductase that enables the utilization of nitrate as a nitrogen source. This Nas enzyme is usually induced by nitrate and repressed by ammonium but is not affected by oxygen. Depending on the organism, the enzyme uses ferredoxin, flavodoxin, or NADH as the electron donor. The ferredoxin- and flavodoxin-Nas proteins are single polypeptides with MGD and one [4Fe-4S] or [3Fe-4S] center, whereas the NADH-Nas protein is a heterodimer of a large iron-sulfur and MGD-containing catalytic subunit and a small FAD-containing diaphorase subunit. The membrane-bound respiratory nitrate reductase of some denitrifiers and nitrate-respiring bacteria allows ATP synthesis by using nitrate as an alternative electron acceptor under anaerobic conditions. This Nar system is generally induced by nitrate and repressed by oxygen, but it is insensitive to ammonium. The enzyme is a three-subunit complex of a MGD-containing catalytic subunit, an iron-sulfur subunit with one [3Fe-4S] and three [4Fe-4S] centers, and a biheme b membrane-anchoring subunit. Finally, a periplasmic dissimilatory nitrate reductase is found in many gram-negative bacteria; in most of these gram-negative bacteria, the enzyme is not repressed by either ammonium or oxygen. This Nap system seems to be involved in aerobic denitrification and/or the maintenance of an optimal redox balance. Structural differences among bacterial nitrate reductases are also revealed by comparisons of the amino acid sequences of the catalytic subunits. These comparisons show that only the enzymes of the same type are closely related (more than 60% of sequence identity), whereas Nas, Nar, and Nap are only 20 to 35% identical, which is the same degree of sequence identity found for other bacterial molybdoenzymes (72). The results of spectroscopic and sequence analyses also indicate that the soluble Nas and Nap proteins are more closely related, with essentially identical active sites, but they are distinct from the membrane-bound Nar. In addition, the crystal structure of Nap shows more resemblance to the formate dehydrogenase enzyme than to dimethylsulfoxide reductase (24). Although Nas, Nar, and Nap systems seem to perform different physiological functions, some differences are observed among the organisms. Also, the enzymes can sometimes play distinct roles under different metabolic conditions and assimilatory, respiratory, and dissimilatory pathways can be interconnected to facilitate a rapid adaptation to changing nitrogen and/or oxygen conditions, increasing the metabolic plasticity for survival in natural environments.