Respiration of Nitrate and Nitrite
JEFFREY A. COLE1* AND DAVID J. RICHARDSON2*
[SECTION EDITOR: VALLEY STEWART]
Posted August 18, 2008
School of Biosciences, University of Birmingham, Birmingham B15 2TT,1 and Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, 2 United Kingdom
*Corresponding authors. Mailing address for Jeffrey A. Cole: School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom. Phone: 44 121 414 5440, fax: 44 121 414 5925, E-mail:
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. Mailing address for David J. Richardson: Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. Phone: 44 1603 593250, fax: 44 1603 592250, E-mail:
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Nitrate reduction to ammonia via nitrite occurs widely as an anabolic process through which bacteria, archaea, and plants can assimilate nitrate into cellular biomass. Since the primary role of such a reaction is to assimilate nitrogen from the environment, the process is not energy conserving: indeed, it is energetically costly and occurs in both aerobic and anaerobic environments. By contrast, Escherichia coli and related enteric bacteria can couple the eight-electron reduction of nitrate to ammonium to growth by coupling the nitrate and nitrite reductases involved to energy-conserving respiratory electron transport systems. Like nitrate assimilation, this respiratory reduction of nitrate to ammonium is a two-step process involving nitrate reduction to nitrite, followed by nitrite reduction to ammonia. However, unlike nitrate assimilation, which is always a cytoplasmic process, it can occur in the cytoplasm, in the periplasm, or both, depending on the bacterial species and growth conditions. Furthermore, unlike nitrate assimilation, in enteric bacteria it is strictly an anaerobic process in which the expression of the nitrate and nitrite reductase genes is tightly repressed in the presence of oxygen, induced during anaerobic growth, and further regulated by the availability of nitrate and nitrite. In global terms, the respiratory reduction of nitrate to ammonium dominates nitrate and nitrite reduction in many electron-rich environments such as anoxic marine sediments and sulfide-rich thermal vents, the human gastrointestinal tract, and the bodies of warm-blooded animals (28, 45, 115, 137). In this chapter, we will review the regulation and enzymology of this process in E. coli and, where relevant detail is available, also in Salmonella and draw comparisons with and implications for the process in other bacteria where it is pertinent to do so.
In both E. coli and Salmonella, two major nitrate reductases, one located in the cytoplasm and the other in the periplasm (Fig. 1) (99), are coupled to two nitrite reductases to provide independent pathways for nitrate reduction to ammonia in the two cellular compartments. That these two major pathways are complementary, rather than mutually redundant, is revealed by an analysis of how each pathway is regulated and by a consideration of the quinol pool to which each of the nitrate reductases is preferentially coupled. Although formate was shown more than 50 years ago to be an effective electron donor to the cytoplasmic nitrate reductase, NarGHI, there is surprisingly little information about electron transfer from other physiological electron donors, such as NADH and glycerol, via the quinol pool to NarGHI. In "Regulation of the cytoplasmic and periplasmic pathways for nitrate reduction to ammonia," below, it will be argued that transcription control circuits prevent dihydrogen from serving as an effective electron donor to the cytoplasmic nitrate reductase. The soluble, energy-dissipating nitrite reductase (NirBD) can reduce nitrite generated during nitrate reduction in the cytoplasm by using NADH as the only electron donor.
The genes for the cytoplasmic pathway for the respiratory reduction of nitrate to ammonia are present in all strains of E. coli and species of Salmonella and Shigella for which genome sequences are available. Nitrate is reduced to nitrite by a respiratory nitrate reductase, nitrate reductase A, and the nitrite produced can then be further reduced by a cytoplasmic, siroheme-containing nitrite reductase, the expression of which is regulated almost coordinately with the expression of the narGHJI operon encoding the nitrate reductase, although there are also important differences. For example, unlike the expression of narG, that of nirB is induced by nitrite but repressed by Cra (123, 124), and although both are activated by FNR (regulator of fumarate and nitrate reduction) in response to anaerobiosis and by the substrates of the gene products, the degree of activation of narG expression by nitrate is far greater than the degree of activation of nirB expression by nitrite (compare data in references 97 and 124). However, this situation immediately raises the first of many unsolved but fascinating puzzles: given that the nitrate and nitrite reductases are expressed under essentially the same conditions, why does up to 70% or even more (120) of the nitrite generated during nitrate reduction accumulate in the external environment rather then being reduced in the cytoplasm to ammonia by the fully induced NirB nitrite reductase (26, 37, 104, 129, 130)?
Nitrate reductase A (NarA) consists of three structural components: a catalytic molybdoprotein, NarG; a nonheme iron-sulfur protein, NarH, that mediates electron transfer to NarG; and a b-type cytochrome, NarI, that transfers electrons from the quinol pool to NarH in the NarGH complex (Fig. 1 and Fig. 2). These components are encoded in a four-gene operon, narGHJI, which encodes a fourth component, NarJ, that is a pathway-specific chaperone or assembly factor required for the posttranslational assembly of the functional complex. A second copy of this operon, the narUZYWV operon, in which narU encodes a nitrate-nitrite transport protein, is also present in all E. coli and Salmonella genomes. The four proteins NarZ, NarY, NarW, and NarV are isozymes of NarGHJI. They accumulate at very low levels in E. coli and Salmonella during both aerobic and anaerobic exponential growth, even in the absence of nitrate or nitrite. The expression of the narU operon in both genera is regulated by the stationary-phase sigma factor RpoS, consistent with a role for the corresponding proteins in a stress response (20, 27, 116). It was recently established that an E. coli strain expressing only narUZYWV survives severe nutrient starvation during anaerobic, nitrate-limited growth better than a strain expressing only narK and narGHJI (27). Furthermore, there is another formate dehydrogenase operon that is expressed preferentially at the end of exponential growth, suggesting that this operon and narUZYWV may be coregulated (88). Like the narU operon, the fdoGHI operon is expressed at a very low level during aerobic growth, conditions under which pyruvate formate-lyase is inactive and therefore no formate is produced from pyruvate, although it becomes available as oxygen becomes limiting at later stages of growth. However, ample NADH should be available to reduce ubiquinone and, hence, act as an electron donor to NarZYV. The inability of E. coli to use nitrate as a sole nitrogen source and the failure of nitrite to accumulate both indicate that neither NarZ nor either of the nitrite reductases is sufficiently active to generate ammonia even slowly during aerobic growth.
The molybdenum ion of NarG is part of a bismolybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor. This cofactor provides four thiol ligands to the Mo ion, which is further coordinated by an aspartate ligand provided by the polypeptide chain. Different X-ray crystal structures of NarG show that it is coordinated either as a bidentate dioxygen ligand or as a monodentate oxygen ligand with the second oxygen atom hydrogen bonded to an active-site histidine residue (6, 68). The catalytic relevance of either structure is uncertain, especially since the bidentate ligand state does not present an obvious vacant coordination site to bind nitrate. However, in the case of the monodentate structure, the sixth coordination position is occupied by a Mo=O/H2O (Fig. 3) group that may be lost as water upon reduction, generating a vacant coordination site for nitrate binding. It is possible that the different coordination states reflect different oxidation states, and thus, the catalytic relevance of either state requires further study. In studies in which the E. coli enzymes and the closely related Paracoccus denitrificans Nar enzymes have been examined using protein film voltammetry, it has emerged that optimum activity is observed when NarG is in a partially reduced, rather than fully reduced, state. This finding has led to proposals of models for a catalytic cycle in which nitrate binds with higher affinity to Mo(V) than to the fully reduced Mo(IV) form (2, 17, 42). The enzyme is also a chlorate reductase, but neither E. coli nor Salmonella can sustain respiratory growth on chlorate because they lack the chlorite dismutase required to detoxify the chlorite produced from chlorate reduction (2, 17, 19). There have also been suggestions from whole-cell studies that at high nitrite concentrations NarG can reduce nitrite to NO (50) or N2O (113), but this scenario has yet to be demonstrated with purified enzyme.
NarI is an integral membrane protein with five transmembrane helices. This subunit carries two hemes b that are ligated by histidine residues conserved in all known NarI proteins. One heme (low potential) is located at the periplasmic site, and the other (higher potential) is located at the cytoplasmic site. NarH contains four iron-sulfur centers: one 3Fe-4S cluster and three 4Fe-4S clusters. NarG and NarH are located in the cytoplasm and associate with NarI (6, 68). The contact between NarGH and NarI is mediated by the C-terminal part of NarH. The crystal structure of NarGHI in a complex with the quinol analogue pentachlorophenol, which is a potent inhibitor of quinol:nitrate oxidoreductase, has been determined. This structure shows the contact site between NarGH and NarI to be close to the low-potential heme, and this finding is consistent with a perturbation of the electron paramagnetic resonance (EPR) spectrum of this heme associated with quinol binding (7).
During quinol oxidation, NarI receives two electrons, one at a time, at the low-potential heme on the periplasmic, membrane-potential-positive (Δψ+) side of the membrane. The electron is then passed via the higher-potential heme, located on the cytoplasmic side of the membrane, to the 3Fe-4S cluster FS4 of NarH (Fig. 2). From there it flows via the iron-sulfur clusters in NarH to the single iron-sulfur cluster in NarG, which is the direct electron donor to the Mo-bis-MGD cofactor-containing catalytic site in NarG. Here, on the membrane-potential-negative (Δψ−) side of the cytoplasmic membrane, the nitrate is reduced to nitrite (Fig. 2). Since the oxidation of quinol occurs on the periplasmic side of NarI, the two protons released as the quinol (QH2) is oxidized are released onto the Δψ+ side of the membrane. However, since the two electrons are moved from the outside low-potential heme to the inside higher-potential heme (Fig. 1 and Fig. 2), there is a separation of positive and negative charges across the membrane. This charge separation makes the enzyme electrogenic in that it contributes to the generation of a proton electrochemical gradient across the membrane (two positive-charge separations during the transfer of two electrons to nitrate: a stoichiometry expressed hereinafter as 2q+/2e−).
In total, the NarA "electron wire" spans around 90 Å and comprises two hemes, five iron-sulfur clusters, and the Mo-bis-MGD. Each cofactor is within 14 Å of its nearest neighbor, ensuring rapid electron transfer. The wire-like arrangement of the iron-sulfur clusters (Fig. 2) can be seen in a number of different kinds of respiratory enzymes in E. coli that includes formate dehydrogenase (100) and the seven-iron-sulfur-cluster, ~8-nm electron input arm of NADH dehydrogenase, the structure of which in Thermus thermophilus has recently been resolved (106). There are some structural similarities between the arrangements of the Nar and NADH dehydrogenase iron-sulfur clusters that point to a common evolutionary origin for the two enzymes.
In E. coli, the reduction of respiratory substrates such as nitrate can theoretically be coupled to a wide range of electron donors, including NADH, formate, and hydrogen. We have seen in the previous section that NarA is an electrogenic enzyme complex (2q+/2e−). However, when NarA is coupled to electron input from formate via the nitrate-inducible formate dehydrogenase (Fdh-N), the Fdh-N-NarA respiratory chain of E. coli emerges as a paradigm for a full proton motive redox loop (Fig. 2) (4q+/2e−). E. coli synthesizes three Fdh isozymes, all selenomolybdoenzymes. They catalyze the oxidation of formate, which is produced by E. coli under anaerobic conditions by the enzyme pyruvate formate-lyase. Fdh-N, together with its close homologue Fdh-O, is involved in respiration, whereas Fdh-H (which produces hydrogen) is not thought to be energy conserving. In Fdh-N, by contrast to NarGHI, the catalytic site is in the periplasm. However, like those in the NarGHI system, the electrons generated pass down a 90-Å wire of redox centers that comprises five iron-sulfur clusters and two hemes, with each again being within 14 Å of its nearest neighbor to ensure rapid electron transfer (67, 99, 100). However, in this case, the wire proceeds in the opposite way from the NarGHI wire and electrons run in the reverse direction. The Fdh-N wire connects the Mo-bis-MGD cofactor, located in the periplasm, to a menaquinone reductase site at the cytoplasmic Δψ− face of the inner membrane. Like that by NarGHI, the electron transfer translocates two positive charges across the membrane for every two electrons extracted from formate (2q+/2e−). Thus, together the electron-carrying arms of the Fdh-N and Nar systems form a proton motive redox loop that has a ΔE of 800 mV (−420 mV to +420 mV) and a coupling stoichiometry of 4H+/2e− and spans an electron transfer distance of some 150 Å (Fig. 2).
When the whole electron transfer system from the Fdh-N Mo-bis-MGD via the quinone pool to the Nar Mo-bis-MGD is considered, it should be noted that formate oxidation, quinone/quinol reduction/oxidation, and nitrate reduction are two-electron reactions. However, the heme and iron-sulfur cluster electron carriers are one-electron transfer centers. Thus, the Mo-bis-MGD cofactors and the quinone/quinol reduction/oxidation sites at either end of the Fdh-N and NarGHI electron transfer wires are important for gating the two-electron/one-electron oxidoreductions. The very low midpoint redox potentials of some of the iron-sulfur clusters in the Fdh-Nar electron transfer systems (for example, that of the FS2 cluster of Nar) had raised the possibility, before knowledge of the molecular structures, that these clusters were not all directly involved in electron transfer between formate and quinone or quinol and nitrate. However, consideration of the Fdh-N and NarGHI structures leaves no doubt that all 10 iron-sulfur clusters are directly involved in mediating electron transfer, and thus, the system provides an example of electron transfer chains in which there are mixtures of endergonic and exergonic electron transfer steps but in which the overall ΔE favors unidirectional electron transport from formate to nitrate via the quinone pool.
Although formate is undoubtedly an effective physiological electron donor to the cytoplasmic nitrate reductase, far less is known about how electrons are transferred from other substrates to the quinone pool, or even to which quinone, menaquinone or ubiquinone, electrons are transferred. Attention has been drawn to the possibly unique ability of NarG to receive electrons from either quinol pool, though there is a preference for the ubiquinol pool (12). Results from chemostat studies suggested that NADH generated from the oxidation of pyruvate may be an electron donor to nitrate (35), but there are two NADH dehydrogenases in E. coli, one that conserves and another that dissipates the energy released during electron transfer (reference 8; see also chapter NADH as Donor). Significantly, it is the energy-conserving complex, Nuo, that is preferentially synthesized during nitrate reduction by NarG, because the expression of the gene for the alternative enzyme, Ndh, is repressed by FNR during anaerobic growth in the presence of nitrate but FNR is essential for the expression of the narGHJI operon (29, 53). This situation suggests that there may be another extended electron wire initiating with NADH bound to Nuo but terminating with nitrate bound to NarG. Note, however, that in contrast to the formate-nitrate oxidoreductase complexes in which electron transfer initiates in the periplasm, in this case both the electron source and sink are located in the cytoplasm. Assuming a 4q+/2e− coupling stoichiometry for Nuo, which is a proton pump, the NADH (Nuo)-nitrate (Nar) electron transfer process would yield 6q+/2e−, a stoichiometry greater than the 4q+/2e− associated with the Fdh-N-Nar electron transfer system.
A further question on electron transfer to NarA is why, in contrast to alpha- and betaproteobacteria, enteric bacteria have not been reported to grow prolifically in nitrate-supplemented minimal medium in which succinate is the primary source of carbon and energy. Similarly, during fermentative growth when nitrate is scarce, formate is cleaved to carbon dioxide and dihydrogen. This hydrogen can diffuse freely to adjacent environments where electron acceptors such as fumarate, nitrite, and nitrate are available. Why, then, is there so little evidence of electron transfer from hydrogen via the quinone pools to nitrate? The answers to both questions lie in understanding the regulation of transcription of the suc and hyd operons. Succinate dehydrogenase synthesis is totally repressed during anaerobic growth by the ArcB-ArcA two-component regulatory system (62, 86), and the expression of the hydrogenase operons is repressed by nitrate-activated NarL (29), which is a key activator of the narGHJI operon when nitrate is abundant (97). Effectively, therefore, electrons are transferred to NarA by two major pathways, from formate (via Fdh-N) and from NADH (via Nuo), with ubiquinone as the major intermediary electron transfer component and menaquinone playing a more minor role.
Another important and electron-rich group of organic electron donors for respiration are fatty acids. These may be present in high levels in many of the natural environments of E. coli and Salmonella in which oxygen is limited but nitrate is available to support respiration. Fatty acid breakdown generates electron donors in the form of reduced flavin adenine dinucleotide, via an acyl coenzyme A (acyl-CoA) dehydrogenase that most likely feeds these electrons directly into the quinone pool, and NADH, via the 3-hydroxyacyl-CoA dehydrogenase. Thus, given the discussion above, it is to be expected that fatty acids can serve as good electron donors for nitrate respiration. Accordingly, it has recently been shown that E. coli can indeed use fatty acids as a sole carbon and energy source under anaerobic nitrate respiratory conditions (18). Intriguingly, though, the enzymes used for the anaerobic fatty acid degradation pathway turned out to be distinct from those used in the aerobic pathway. This arrangement enables the anaerobic pathway to function with fatty acids (octanoate and decanoate) that could not be used under aerobic conditions. The genes associated with anaerobic nitrate respiratory fatty acid oxidation in E. coli are also conserved in Salmonella.
The cytoplasmic nitrite reductase consists either of a single, large polypeptide (usually designated NirB) or a two-subunit enzyme, NirB-NirD (56). It is a soluble protein that includes two nucleotide binding domains, an iron-sulfur center, and a siroheme binding site (63). Siroheme is a specialized heme group that is found in many sulfite and nitrite reductases, enzymes that catalyze six-electron transfer reactions by reducing nitrite to ammonia or sulfite to sulfide (63, 110). This soluble nitrite reductase is so far unique in being the only known enzyme involved in nitrate reduction that does not receive electrons directly from the quinol pool: in this respect, the term dissimilatory rather than respiratory nitrite reduction is more appropriate. In contrast to the efficient cytoplasmic reduction of nitrate to nitrite, there is a profligate waste of energy as nitrite is reduced to ammonia in the cytoplasm by the NADH-dependent nitrite reductase. Note, however, a point that is often overlooked: that when fermentative bacteria reduce nitrite to ammonia, acetate rather than ethanol is the final product of carbon metabolism. This outcome results in the generation of one extra ATP molecule by substrate-level phosphorylation for each molecule of acetate generated.
The location of the Nar active site on the cytoplasmic (Δψ−) side of the membrane introduces the requirement for the negatively charged nitrate anion to be transported against the membrane potential into the cell. Two polytopic membrane proteins of the major facilitator family, NarK and NarU, that transport both nitrate and nitrite across the E. coli cytoplasmic membrane have been identified, but the mechanism of transport is unknown (26). A third protein, NirC, catalyzes only nitrite transport (26). It has largely been assumed that NarK and NarU are inserted into the cytoplasmic membrane with 12 membrane-spanning helices (82), However, there are only six predicted membrane-spanning helices in the much smaller NirC (87), raising the fascinating possibility that NirC may form a single channel that is specific for nitrite transport. If so, NarK and NarU may form two channels, one for nitrate and the other for nitrite. In view of the emerging structural and mutagenesis data from studies of other polytopic membrane proteins of the major facilitator family, such a proposal may seem unlikely. For example, in E. coli, the 12 membrane helices of GlpT form a single substrate translocation pore that mediates the exchange of glycerol-3-phosphate for inorganic phosphate (52, 59). Also, the 14-helix TetL and TetK tetracycline efflux proteins from Staphylococcus aureus and Bacillus subtilis are believed to catalyze Na+, K+, and tetracycline antiport via a single channel that alternately imports one substrate and expels another (134). It is therefore possible that NarK and NarU use a single channel for nitrate uptake, nitrite export, and nitrate-H+ symport.
The narK gene is expressed at high levels during anaerobic growth in the presence of nitrate, consistent with a role for NarK in nitrate transport coupled to nitrate reduction by the most active nitrate reductase encoded by the adjacent narGHJI operon. Unlike the monocistronic narK gene, narU is cotranscribed with narZ as the first gene of a five-gene narUZYWV operon. During anaerobic growth in the presence of nitrate, far less NarU than NarK accumulates in the membrane during the exponential phase. Nevertheless, strains expressing either NarU or NarK as the only nitrate transport protein are able to support nitrate-dependent anaerobic growth at similar rates. However, NarU is more abundant than NarK in stationary-phase cultures in the absence of nitrate (27). In chemostat competition experiments, a strain expressing only narU had a selective advantage during nutrient starvation or very slow growth relative to a strain expressing only narK but NarK+ bacteria had a much greater selective advantage during rapid growth. The data suggest that NarU confers a selective advantage during severe nutrient starvation or slow growth, conditions similar to those encountered in vivo.
A critical unanswered question is whether NarK and NarU simply provide two-way channels for nitrate and nitrite to cross the cytoplasmic membrane by facilitated diffusion or whether they are nitrate-nitrite antiporters. Moir and Wood (82) noted that the members of the NarK family of nitrate transport proteins fall into two subfamilies and suggested that they may differ in the mechanism of nitrate uptake. One subgroup was suggested to catalyze nitrate-H+ symport; the other was suggested to function by nitrate-nitrite antiport (see also reference 134). Significantly, both NarU and NarK fall into the same antiporter subgroup. As the cytoplasm is negatively charged relative to the periplasm, one reason advanced to propose nitrate transport by nitrate-H+ symport is that the import of a negatively charged nitrate against a 180-mV charge gradient is energetically unfavorable. Such a mechanism would enable nitrate to enter bacteria in the absence of intracellular nitrite required by an obligatory nitrate-nitrite antiport mechanism. Furthermore, if NarK and NarU function as two-way channels, the coupling of nitrate-H+ uptake with nitrite-H+ export, like nitrate-nitrite antiport, would be energetically neutral. Note that any energy expended in nitrate-H+ symport would be more than compensated for in two ways. First, energy is conserved during nitrate reduction. Second, some of the nitrite formed during nitrate reduction is reduced directly to the positively charged NH4+ by using NADH generated either during fermentation or during the oxidation of more reduced, nonfermentable carbon sources such as glycerol and lactate. The reduction of each nitrite to ammonia results indirectly in the generation of three molecules of ATP, as acetyl-CoA is converted via acetyl phosphate to acetate rather than reduced to ethanol to achieve redox balance during glycerol oxidation (28).
Based upon the almost stoichiometric accumulation of nitrite as the product of nitrate reduction during anaerobic growth in the presence of high concentrations of nitrate, it was initially proposed that NarK is a nitrate-nitrite antiporter (37, 84). An alternative suggestion that NarK is primarily a nitrite extrusion protein is now known to be incorrect (26, 27, 104). However, the reason why nitrite formed in the cytoplasm during nitrate reduction is expelled rather than immediately reduced to ammonia by the highly active NADH-dependent nitrite reductase remains unclear. The role of NirC in nitrite transport is far less obvious than that of NarK, though NirC clearly is the major protein that accelerates nitrite uptake (W. Jia and J. A. Cole, unpublished data). The primary role of NirC may therefore be to recapture nitrite that has been expelled through NarK as nitrate becomes scarce but while nitrite is abundant. This possibility is consistent with the almost coordinate regulation of the nirBDC operon and the narK and narGHJI operons (15, 29).
In parallel with the cytoplasmic pathway for nitrate and nitrite reduction, E. coli and Salmonella can catalyze the reduction of nitrate to ammonium in the periplasmic compartment and couple this reduction to energy-conserving respiratory electron transport (Fig. 1). The process involves two enzymes: a periplasmic nitrate reductase (NapA) that converts nitrate to nitrite and a periplasmic cytochrome c nitrite reductase (NrfA) that converts nitrite to ammonia (Fig. 1). In E. coli, nitrate reduction in the periplasm involves the products of two seven-gene operons, napFDAGHBC, encoding the periplasmic nitrate reductase, and nrfABCDEFG, encoding the periplasmic nitrite reductase (Fig. 4). In Salmonella enterica serovar Typhimurium, the nrfEF genes are fused. Ideas of how electrons are transferred from physiological substrates such as hydrogen, formate, and NADH to the periplasmic nitrate reductase, Nap, remain unsubstantiated by direct experimental data, though again inferences can be drawn from how the synthesis of the various dehydrogenases and quinones and the periplasmic nitrate and nitrite reductases is regulated.
Anaerobic respiratory reactions of pathogens are important for host invasion and colonization. They provide energy for ATP synthesis and growth in microoxic environments, such as biofilms in lungs and in the intestinal-colonic tract. They also provide electrons for the reductive detoxification of reactive nitrogen species, such as nitric oxide. The Nrf system may serve both functions. It is coupled to energy-conserving electron transport systems. In addition to the role of Nrf in reducing nitrite, the nrf operon has been shown to be up-regulated in the presence of NO, while the deletion of the nrf operon results in susceptibility to NO in both E. coli (89) and Salmonella serovar Typhimurium (43, 81), suggesting a physiological role for Nrf in NO detoxification. The importance of NapA and NrfA in anaerobic metabolism in enteric bacteria has led to structure-function studies of these enzymes that have revealed their crystal structures and yielded a spectropotentiometric description of their redox centers (4, 65). This information has then allowed for the development of mechanistic models of electron transfer and catalysis using protein film voltammetry (3, 55). Most notable has been the use of this methodology to establish the kinetic parameters of NrfA for NO reduction (127). This information demonstrates that the Km for NO is around 10-fold higher than that for nitrite but that the kcats for nitrite and NO reduction are comparable. This finding suggests that Nrf will select NO over nitrite only when the former is present in a much greater concentration than the latter. NrfA is also most active at acidic pHs. At acidic pHs, nitrite dismutates in a series of reactions that leads to NO, and so the possibility that the Nrf-NO reduction reaction is important in the stomach has been raised (127).
The organization of typical nap gene clusters from bacteria that reduce nitrate to ammonia is shown in Fig. 4. Several points immediately emerge from this figure. First, eight different types of polypeptide are encoded in these nap gene clusters. Seven of these genes are organized as an operon in both E. coli and Salmonella species in the order napFDAGHBC. Three of these genes, napA, napD, and napG, are often found together in bacteria that reduce nitrate to ammonia but not in denitrifying bacteria. They encode the catalytic subunit NapA, the pathway-specific chaperone NapD (94), and the nonheme iron-sulfur cluster protein NapG.
Until recently, the diheme cytochrome c NapB was considered to be an essential component in all Nap systems in bacteria, but there is no napB gene in either the Symbiobacterium thermophilum or the Desulfovibrio desulfuricans nap gene cluster (78, 126). The Nap system of Wolinella succinogenes was the first to be described that lacks the tetraheme cytochrome NapC: neither of the only two tetraheme c-type cytochromes encoded elsewhere on the Wolinella chromosome is essential for nitrate reductase (111). An interesting point from Fig. 4 is that certain components tend to be found in bacteria of a particular physiological type. The seven-gene nap operons of facultatively anaerobic bacteria such as E. coli and the small-genome pathogen Haemophilus parainfluenzae include genes for all four components, NapB, NapC, NapG, and NapH. In E. coli, electrons are transferred preferentially from menadiol via NapC and NapB to NapA, but there is also a lower rate of electron transfer from ubiquinol via NapH and NapG to NapB-NapA, again via NapC (11, 12). While this finding implies that NapH and NapG form a ubiquinol dehydrogenase, it is clear that the homologous proteins in Wolinella succinogenes, which lacks ubiquinol, function as a menadiol dehydrogenase (111). This difference emphasizes the danger of trying to draw general conclusions from just a few examples. Although the nonheme iron-sulfur proteins NapG and NapH seem always to be present in obligate anaerobes, they do not seem to occur in nonfermentative, denitrifying species of the alpha- and betaproteobacteria, which instead always include both of the c-type cytochromes, NapB and NapC (93).
NapA, like NarG, is a Mo-bis-MGD binding enzyme, but it shows less than 15% sequence identity to its NarG cousin, and the Mo ion is coordinated by a cysteine rather than an aspartate residue. In alphaproteobacteria, Nap is around 90 kDa and forms a tight complex with its immediate electron donor, the diheme NapB. However, NapA from Desulfovibrio desulfuricans has been purified as a single subunit, and no napB gene was found in the nap gene cluster of this bacterium (78). The E. coli NapA protein has intermediate properties in that it accepts electrons from a NapB protein with which it does not form a tight complex (65).
NapA binds a 4Fe-4S cluster and the Mo-bis-MGD cofactor (Fig. 5). The coordination sphere around the Mo-bis-MGD of E. coli NapA includes four thiol ligands from the two MGD groups, one sulfur ligand from a polypetide cysteine, and one oxygen ligand (Fig. 3 and Fig. 5). The Mo―O bond length is 2.6 Å, consistent with a Mo-OH2 ligand. This arrangement creates the potential for H bond interactions with a conserved glutamine residue in the active-site pocket. It is then likely that this residue participates in oxo group extraction and, along with a conserved arginine that lies at the bottom of the substrate cleft, nitrate binding during the catalytic cycle (65) (Fig. 5). Consistent with this view, findings from mutagenesis studies on the closely related NapA from Rhodobacter sphaeroides show that the mutagenesis of the conserved residue increases the Km for nitrate by 200-fold (36). An oxotransfer mechanism that requires the involvement of a Mo(VI)=O species is widely envisaged for nitrate reductases. However, the length of 2.6 Å for the Mo-O bond in the E. coli NapA structure is more indicative of water ligation and, hence, of a des-oxo-Mo(VI) state. When the enzyme is reduced to a Mo(V) state, there are no solvent exchangeable protons detected by EPR (65). Thus, it seems likely that the water molecule is lost upon reduction to Mo(V), vacating a coordination site for nitrate binding. Based on this assertion and the measured midpoint redox potentials of the iron-sulfur cluster and the Mo(VI)-Mo(V) and Mo(V)-Mo(IV) couples, a catalytic cycle for E. coli NapA has been proposed (65). In the absence of added nitrate, only a two-electron reduced enzyme is thermodynamically accessible to physiological electron donors (e.g., those with electrons originating from reduced menaquinol in the electron transport chain) because of the very low mid-point redox potential Em of the Mo(V)-Mo(IV) couple (<−400 mV). This reduced enzyme will thus have the redox state (4Fe-4S)1+/Mo(V). The binding of nitrate to the Mo(V) may raise the potential such that the Em of the Mo(V)-nitrate/Mo(IV)-nitrate couple is accessible to reduction by an electron arising from the (4Fe-4S)1+ center. The reduction of Mo(V)-nitrate to Mo(IV)-nitrate then yields a Mo center with the two electrons required for nitrate reduction to nitrite. The release of the nitrite product will leave an unbonded O ligand on the Mo ion (the oxotransferase reaction), which will yield a Mo(VI)=O species. This species may then protonate to give the stable Mo(VI)―OH2 resolved in the crystal structure.
The E. coli NrfA protein is a 50-kDa pentaheme c-type cytochrome. The analysis of the organization of all E. coli and Salmonella nrf gene clusters also reveals genes encoding a periplasmic pentaheme cytochrome (nrfB), a putative periplasmic ferredoxin with four (4Fe-4S) iron-sulfur clusters (nrfC), and a putative integral membrane quinol dehydrogenase (nrfD). In contrast to the highly variable structures of gene clusters encoding Nap systems (78, 93), the structures of the nrf gene clusters have been relatively conserved throughout evolution. Nevertheless, two distinct and well-characterized types of Nrf complexes can be recognized, those encoded by the seven-gene nrfABCDEFG operon and those encoded by the four-gene nrfHAIJ operon (112). The former arrangement is typical of the gammaproteobacteria, whereas the latter is found in the delta- and epsilonproteobacteria. Significantly, nrf genes have not been reported to be present in the alpha- and betaproteobacteria, which include essentially aerobic bacteria and facultatively anaerobic bacteria that are denitrifiers. Bacteria in which an nrfA gene has been identified include obligate anaerobes such as Desulfovibrio desulfuricans, Sulfospirillum deleyianum, Geobacter metallireducens, and Wolinella succinogenes; the fermicute Desulfitobacterium hafniense; and also the planctomycete Pirellula sp.
NrfA catalyzes the six-electron reduction of nitrite to ammonium, but sulfite and putative reaction intermediates nitric oxide (NO˙) and hydroxylamine (NH2OH) are alternative substrates, with sulfide and ammonia as products, respectively. NrfA crystallizes as a homodimer (Fig. 6), with each monomer containing five hemes. All hemes have bis-His ligands, except the active-site heme, heme 1, which is unusual as it is a high-spin species coordinated with a lysine on the proximal side and either water or nitrite on the distal side (Fig. 6). The active-site heme is thus attached to the novel CXXCK motif, while the other four hemes are attached to the conventional CXXCH motif (4, 39). There are four further highly conserved residues around the active site, including a glutamine (Q263) positioned 7 Å from the heme iron; the side chain of this glutamine, unusually, contributes to the coordination sphere of a conserved essential calcium ion. While aspartate, glutamate, and asparagine contribute around 70% of the calcium ion ligands in calcium binding proteins, glutamine is responsible for only around 3%. Among these cases, the NrfA enzymes represent the only family in which the glutamine-calcium ligand is close enough to the active site to be involved in enzyme activity. The mutation of this glutamine to glutamate results in an increase in the Km of 10-fold, while the Vmax is unaltered. The comparison of high-resolution crystal structures of the wild-type and Q263E-mutated enzymes reveals an increase in the Fe―H2O bond distance of the distal water ligand upon mutation and further changes to the structure and mobility of active-site water molecules. These results suggest that an important function of the unusual Q263-calcium ion pair of NrfA is to modulate substrate affinity through its role in supporting a network of hydrogen-bonded water molecules stabilizing the active-site heme distal ligand, the site of nitrite reduction (24).
The NrfA crystal structures from E. coli, Wolinella succinogenes, Sulfurospirillum deleyianum, and Desulfovibrio desulfuricans have been resolved, and comparisons have been made (23). These comparisons reveal differences in inlet and outlet channels of the substrate and the product, respectively, that are thought to impact the substrate specificities. There are variations in the dimer-monomer dissociation constant (Kd) as well, with 4 μM for E. coli NrfA (25) and 1 to 3 nM for Wolinella succinogenes NrfA (112). These numbers are quite different, especially when brought into the context of the NrfA concentration in the periplasm, thought to be ~3 μM (89). The similarities are also of importance, though, and include the conservation of the sequence, especially around the hemes. For example, heme 1 has the characteristic lysine ligand in all of the structures. The active site (Fig. 6), with its catalytically important residues, is also highly conserved.
Fe-Fe distances throughout the NrfA monomer are less than 13 Å, small enough to allow the transfer of electrons. Structural analysis reveals that the only plausible site for NrfA-NrfB interaction in the NrfA dimer, which also allows electron transfer, is at the solvent-exposed edge of NrfA heme 2 (4). A logical, natural flow of electrons would take place along a decreasing redox gradient, but heme 2 has the highest midpoint potential (−37 mV); it is thus forced to transfer electrons to a lower-potential (−107-mV) heme. Electron transfer steps along increasing redox gradients are of course slower, but given that the distance between the two redox centers is small enough, transfer should still be faster than catalytic turnover and not be a rate-limiting step.
It has been postulated that there can be intermolecular electron transfer in the NrfA homodimer. The interface heme 5 (Fig. 6) is only 12 Å away from its counterpart in the other monomer, potentially allowing the transfer of electrons between monomers (4). A recently determined structure of the Desulfovibrio vulgaris NrfAH reveals a most interesting Nrf complex that supports this idea. In Desulfovibrio vulgaris, NrfH, a tetraheme membrane protein, is the direct electron donor to NrfA. The resolved structure shows two NrfA dimers bound to one NrfH dimer, an α4β2 arrangement (102) with both NrfAs in close contact with the NrfH. Heme 2 is the most likely candidate for electron entry into NrfA. Another striking feature is that heme 4 of NrfH is coordinated to a His of NrfH and a Lys of NrfA, explaining the stability of the complex, which can be dissociated only under denaturing conditions (102). Thus, it is highly likely that this system allows electron transfer between NrfA subunits.
The NrfA-NrfB Kd for the E. coli enzyme has been estimated to be around 10 nM (25), low enough to speculate that NrfA and NrfB exist as a heterodimer in vivo. Indeed, reduced NrfB is rapidly oxidized in the presence of nitrite and 5 nM NrfA, a concentration at which most of NrfA is monomeric (Kd = 4 μM). This outcome suggests that even though electrons may be able to flow through the NrfA homodimer interface, this route may not be the primary electron path for nitrite reduction in vivo. Instead, electrons may flow primarily through a decaheme NrfA-NrfB heterodimer. Support of this hypothesis is provided by the finding that nitrite reduction is catalyzed at potentials reducing heme 1 and 3 (3) but at which heme 4 and 5 would not be reduced.
Recently, the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO than the parent strain led to the proposal that NrfA may also participate in NO detoxification. To assess whether NO is a true substrate of NrfA, protein film voltammetry has been used to quantify the NO reductase activity (127). This approach avoids the nonspecific reduction of NO that is a problem encountered with many chemical reductants. The Km is of the order of 300 μM, and the turnover number is ca. 840 s−1. These parameters compare to a Km of ~35 μM and a turnover number of about 650 s−1 for nitrite. Thus, while the catalytic efficiency (Km/kcat) is higher for nitrite than NO, NrfA is nevertheless a highly competent NO reductase and, indeed, has a much higher turnover number for NO than the dedicated respiratory NO reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO detoxification (31, 46, 47, 48, 72). NrfA NO reduction is initiated at potentials similar to those initiating NrfA nitrite and hydroxylamine reductase activities. In addition, all three activities are strongly inhibited by cyanide. Together, these results suggest a common active site for the reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme 1, rather than nonspecific NO reduction at one of the four bishistidine-coordinated hemes also present in each NrfA subunit.
A mechanism for nitrite reduction by Wolinella succinogenes NrfA that involves a bound NO derivative as an intermediate has been proposed (40). In such a mechanism, nitrite would bind to ferrous heme 1, displacing coordinated water. This step would initiate the reaction by heterolytic cleavage of the first N―O bond, with help from proton donors Tyr216 and His264, and back bonding to yield bound nitrosonium (NO+). The reaction would then proceed through transient nitric oxide (NO˙) and hydrogen nitroxide (HNO) intermediates. The transfer of two electrons and a protonation step would generate hydroxylamine (NH2OH). Hydroxylamine has also been shown to serve as an exogenous substrate for E. coli NrfA (3), supporting the evidence for this mechanism. The hydroxylamine would then be readily reduced to ammonium (NH4+) by another two-electron cascade and a proton transfer. The concerted two-electron transfers involved could be facilitated by the heme pairing between hemes 1 and 3 at the active site of the enzyme (3).
Periplasmic nitrate reductases are, like NarGHI, linked to quinol oxidation in respiratory electron transport chains but do not conserve the free energy in the QH2-nitrate couple. However, nitrate reduction via Nap can be coupled to energy conservation if the primary quinone reductase, for example, the NADH dehydrogenase Nuo or formate dehydrogenase FdhN, generates a proton electrochemical gradient. In many Nap systems, quinol oxidation is catalyzed by NapC, which has a single N-terminal transmembrane helix that anchors a globular domain with four c-type hemes onto the periplasmic surface of the cytoplasmic membrane. All four hemes have relatively low midpoint redox potentials, are low-spin hexacoordinates, and are apparently bishistidinyl axial ligated (19, 103). Likely candidates for the distal histidines were suggested from sequence alignment studies and verified by mutagenesis studies (19). The results of these studies also suggested that the soluble domain of NapC is made up of two diheme pairs, which was confirmed when the structure of a homologue (NrfH) emerged (25). NapC belongs to a large family of bacterial tetraheme and pentaheme cytochromes that have been proposed to participate in electron transfer between the quinol/quinone pool and periplasmic redox enzymes such as the trimethylamine N-oxide reductase, dimethyl sulfoxide reductase, fumarate reductase, nitrite reductases, and hydroxylamine oxidoreductase (HAO).
The E. coli nap gene cluster includes genes encoding both the tetraheme quinol dehydrogenase NapC and, surprisingly, a second quinol dehydrogenase complex, NapGH, that like NrfCD is predicted to be dependent on iron-sulfur clusters rather than hemes (5, 54). It has been established that electron transfer from the ubiquinol pool to nitrate is strongly dependent on NapG and NapH, which are involved in ubiquinol oxidation (11, 12). This function is consistent with the roles of NapGH in quinol oxidation in Wolinella succinogenes and Campylobacter jejuni, bacteria that lack a napC gene in the nap gene cluster (109). However, in E. coli NapGH activity appears to be dependent on the presence of NapC, and results of two-hybrid studies suggest that a complex of NapC and NapH is formed in vivo (12). This idea raises the possibility of a dual-specificity menaquinol (NapC)- and ubiquinol (NapG)-oxidizing complex as illustrated in Fig. 7. The topological model for the organization of the NapGH protein is depicted in Fig. 7 and is supported by the results of subcellular fractionation studies (83). On the basis of these localization studies, the putative 4Fe-4S clusters of NapG and NapH are located on opposite sides of the membrane (Fig. 7). Electron transport from quinol to NapA would minimally require only redox centers on the periplasmic side of the membrane if the site of quinol oxidation were also on this side. Intriguingly, there are conserved CXXXCP motifs predicted to be present in, or on, the cytoplasmic side of the membrane; the roles of these motifs are unclear, but they may be involved in the binding of a redox center or disulfide bond formation or be redox active. The basic NapH type of organization can be predicted from in silico analyses of genes found in a number of other electron transfer gene clusters that are widespread in bacteria, such as those of the nitrous oxide reductase (Nos) and the high-affinity cytochrome cbb3 oxidase (5). Similar to NapH, the transmembrane iron-sulfur flavoprotein NosR has a cytoplasmic iron-sulfur domain and two CXXXCP motifs. NosR is involved in the maturation of the nitrous oxide reductase NosZ (135). The mutation of the CXXXCP motifs in NosR showed that both motifs are functionally important, though not to equivalent degrees, in nitrous oxide respiration but not in the activation of the periplasmic nitrous oxide reductase. However, the functional roles of both the motifs and the cytoplasmic domain are still poorly understood. Redox centers in newly synthesized proteins may require reductive or oxidative activation steps. NapA is a twin-arginine transport (Tat) protein substrate that is translocated across the membrane as a folded protein with the iron-sulfur center and Mo-bis-MGD incorporated. It is possible that a reductive process is required in the assembly or activation of these cofactors in the maturing enzyme. Such reductive activation requires a source of electrons, which may be provided by the cytoplasmic iron-sulfur domain of NapH. The arrangement of redox centers may allow the NapGH complex to be multifunctional in respiratory electron transport to mature periplasmic NapA and in the reductive activation of maturing cytoplasmic NapA.
Despite the importance of periplasmic nitrite reduction to ammonium in enteric bacteria and the developing structure-informed biochemical understanding of the enzyme that catalyzes this process, the nature of electron delivery from the quinol pool has never been addressed structurally or spectroscopically. However, the key to coupling the NrfAB complex to energy conservation lies in the communication with the menaquinol pool and, thereby, electrogenic quinone reductases, such as formate dehydrogenase. Results from genetic studies suggest that quinol oxidation is catalyzed by NrfCD (60), but no biochemical information on what would be a structurally novel quinol dehydrogenase complex that relies on iron-sulfur centers is available for any organism. However, NrfCD-type quinol dehydrogenases are widespread in bacterial anaerobic electron transport systems. For example, genes for proteins homologous to NrfCD are found in the gene clusters encoding enzymes involved in polysulfide reduction (Psr) (38, 74) (e.g., in Wolinella succinogenes and Erwinia caratova) and tetrathionate reduction (Ttr) (58) (e.g., in Salmonella serovar Typhimurium).
The structure of the redox partner to NrfA, NrfB, has also been determined (22, 25). The overall structure of the NrfB polypeptide is completely unique among those of multiheme c-type cytochromes. The approximate overall dimensions are 40 by 30 by 20 Å3, with a chain of five hemes distributed through the structure (Fig. 8). A heme group is covalently attached approximately every 20 to 25 amino acids via thiol ether linkages to the cysteines of five classical CXXCH attachment sites so that all five of the minimum interporphyrin ring distances between neighboring hemes lie within 6 Å. The hemes are arranged in a series of alternative parallel and perpendicular pairs such that there are two parallel pairs (hemes 1 and 2 and hemes 3 and 4) and two perpendicular pairs (hemes 2 and 3 and hemes 4 and 5). The C-terminal heme 5 and N-terminal heme 1 are the most solvent exposed, suggesting that they are routes of electron entry into and exit from the NrfB heme wire. The area around heme 5 is predominantly conserved, suggestive of a potential redox partner binding site. This region includes an exposed conserved tryptophan residue, Trp102. The next most conserved region is that around the surface of heme 1, suggesting a potential second binding site. This arrangement supports the hypothesis that hemes 1 and 5 are the likely sites of electron input into or output from NrfB.
Recently, the structure of a (NrfA)4-(NrfH)2 complex from Desulfovibrio vulgaris was published (102). In contrast to NrfAB, NrfH and NrfA form a very tight hard-wired interaction; indeed, NrfH has never been purified in a native form independent from NrfA. The crystal structure provides some insight into this tight interaction since two of the NrfA proteins provide Lys ligands to one of the hemes on each NrfH protein in the (NrfA)4-(NrfH)2 complex. It has not been possible to purify an NrfAB complex or to cocrystallize such a complex, but findings from solution-state protein-protein interaction studies strongly suggest that the NrfA and NrfB form a 2:2 complex that is structurally distinct from the (NrfA)4-(NrfH)2 complex (25). However, NrfB and NrfH are structurally distinct in many ways, so there is no reason to expect the NrfAB and NrfAH stoichiometries to be the same. The level of sequence identity between NrfB and NrfH molecules is very low (~15%), and this identity is largely attributed to the alignment of four CXXCH motifs. NrfH has a transmembrane helix located towards the N terminus, whereas NrfB is a soluble periplasmic protein in which the N-terminal signal peptide has been cleaved.
The polypeptide chains of NrfB and the soluble domain of NrfH cannot be superimposed. Despite this, four of the NrfB hemes (hemes 1 to 4) adopt heme-heme packing motifs similar to those of the four NrfH hemes. However, two of these four superimposable hemes have different heme ligations. In NrfB, all four heme irons are low-spin hexacoordinates with bishistinyl axial ligation. However, in the (NrfA)4-(NrfH)2 complex, one of the NrfH hemes is a pentacoordinate (and therefore likely to be high spin) with a Met distal ligand and an aspartate occupying the proximal position but not being within bonding distance. NrfH heme 4 is ligated by a His ligand provided by NrfH and a Lys ligand from NrfA. The major difference between NrfB and NrfH is the presence in NrfB of heme 5, which is absent in NrfH. This heme is the most likely electron output site for NrfB, and its presence makes the construction of an NrfAB complex model by using an NrfAH structural template impossible. However, it is notable that hemes 1 to 5 of NrfA can be superimposed onto hemes 4 to 8 of the octaheme HAO subunit and the remaining hemes 1 to 3 of HAO can be superimposed onto hemes 3 to 5 of NrfB (25). Thus, it has been suggested that the octaheme HAO polypeptide provides a template model for the NrfAB arrangements. Hemes 1 and 2 of NrfB have no corresponding hemes in the HAO structure, and these two additional hemes would thus provide the link to the quinol-oxidizing NrfCD complex, with electrons originating from the quinol pool entering NrfB heme 1 via the NrfC iron-sulfur cluster electron transfer chain (Fig. 1).
Strong clues to the physiological role of any biochemical process can often be deduced by studying how the transcription of the structural genes is regulated. For example, the key role of Nap in aerobic denitrification by Paracoccus pantotrophus is consistent with both a redox-balancing role for this enzyme and the observation that nap gene expression is optimal during aerobic growth on a highly reduced carbon source such as butyrate (41, 108). In E. coli, the expression of both nap and nrf genes is repressed during growth on excess nitrate, raising the question of why the expression of genes encoding a nitrate reductase should be repressed by the pathway substrate. The explanation is simple, at least for E. coli (there are few comparable data for other bacteria). When nitrate is sufficiently abundant, enteric bacteria exploit the energy-efficient, highly active, but low-affinity NarG enzyme to reduce nitrate in the cytoplasm. When nitrate is scarce, Nap provides a high-affinity but low-activity pathway that does not require nitrate transport for nitrate to serve as an effective electron sink. These reciprocal roles for Nap and NarG were demonstrated previously in chemostat competition experiments with strains expressing either only Nap or only NarG (92). Furthermore, the reciprocal regulatory circuits described below involving dual two-component regulatory systems, NarX- NarL and NarQ-NarP, consolidate this kinetic advantage by enhancing the expression of nap genes during nitrate-limited growth (129). Nap can also fulfill a redox-balancing role in E. coli, one of the few bacteria for which multiple physiological roles for Nap have been documented experimentally (12).
In E. coli K-12, the synthesis of the NADH-dependent nitrite reductase is regulated almost, but not quite, coordinately with that of nitrate reductase A. Both are repressed during aerobic growth, induced during anaerobic growth by the oxygen-sensing transcription factor FNR, and further induced by the two-component regulatory system NarX-NarL. NarX is a membrane-spanning environmental sensor that detects and responds primarily to moderately high concentrations of nitrate: nitrate stimulates its autokinase activity, resulting in the transfer of the phosphate group to the response regulator NarL. Phospho-NarL activates the expression of both the narGHJI and nirBDC operons (NirC is a nitrite transport protein). Nitrite promotes the dephosphorylation of NarL (97). The significant difference between the two operons is that the narG promoter is essentially insensitive to a second two-component system, NarQ-NarP (97), but the nirB promoter is strongly activated by phosphorylated NarP in response to nitrite. It is significant that the fdnGHI operon is regulated in parallel with the expression of nitrate reductase A (119). Conversely, the expression of the nap and nrf genes for the periplasmic pathway is repressed by nitrate-activated NarL but induced by NarP in response to low concentrations of nitrate (33, 130).
How nitrite activates the expression of the nir and nrf operons has been the source of some debate. High (millimolar) concentrations of nitrate fully activate the expression of the cytoplasmic NarG-NirB pathway for nitrite reduction to ammonia, but as noted above, millimolar concentrations of nitrite accumulate transiently under these conditions. Phosphorylated NarL activates the expression of the nir operon but represses nrf transcription (13, 14, 15, 16, 97, 124, 125). The transcription of the nrf operon is activated in response to low concentrations of nitrate, such as those commonly found in the human gastrointestinal tract (97, 130). Nevertheless, nitrite still activates nrf operon expression. This activation was recently shown to be mediated not only by NarXL and NarQP (which are 10- to 1,000-fold less sensitive to nitrite than to nitrate [76, 129, 130]), but also by nitric oxide generated as a by-product of nitrite reduction. Nitric oxide nitrosylates the iron-sulfur center of NsrR, which is a member of the Rrf2 family of transcription factors that represses the transcription of about nine operons in E. coli (43). Nitrosylation results in the inactivation of the repressor activity of NsrR. This regulation is physiologically significant in light of the high-level nitric oxide reductase activity of NrfA (89).
The realization that both E. coli and S. enterica synthesize three nitrate reductases, two nitrate transporters, two nitrite reductases, and three formate dehydrogenases, as well as at least three systems for reducing nitric oxide generated as a toxic by-product of nitrate and nitrite reduction, has led to statements that these parallel pathways are mutually redundant. Such a conclusion is incorrect, however, because each redox process is regulated by different factors, resulting in the optimal expression of each process under the environmental conditions in which it confers a selective advantage. The primary role of NarG is to generate proton motive force when nitrate is abundant, for example, as occurs in carbon-limited soil, sediments, or wastewater treatment plants. Under these conditions, the nitrite reductase NirB protects the cytoplasm from nitrite toxicity, simultaneously generating ammonia to neutralize the negative charge accumulated by nitrate import and facilitating the indirect generation of ATP by substrate-level phosphorylation. In their alternative habitat in the bodies of warm-blooded animals, these bacteria encounter much lower concentrations of nitrate. A major unresolved question is whether the environmental sensor proteins NarX and NarQ respond to different concentrations of nitrate. The following points are relevant. The results of early studies established that either NarX or NarQ is sufficient for batch cultures to respond to millimolar concentrations of nitrate (21, 96). The transfer of phosphate from either NarX or NarQ to NarL was confirmed by in vitro experiments (107), but as only the cytoplasmic domains of NarX and NarQ were used for these experiments, their autokinase activities were insensitive to the presence or absence of nitrate. In the first of two studies, half-maximal nitrate-stimulated phosphorylation of NarX was estimated to require about 1 mM nitrate (132), though the subsequent study under different conditions concluded that 5 μM is sufficient to elicit a response to nitrate and that half-maximal NarX phosphorylation requires 35 μM nitrate (76). With the important caveats that no comparable data are available for NarQ and that the experiments cited above were completed in vitro, evidence from chemostat experiments in two laboratories suggests that NarQ may be exquisitely more sensitive to nitrate than NarX. First, strains expressing both NarX and NarQ are able to respond to nitrate, as shown by the nitrate-induced expression of napF-lacZ and nrfA-lacZ chromosomal fusions, even during nitrate-limited growth under conditions in which the concentration of nitrate in the fermentor was below the detection limit of 50 nM (129, 130). As this nitrate concentration is three orders of magnitude lower than that required for the half-maximal phosphorylation of NarX in vitro, we infer that this response is more likely to have been due to NarQ than to NarX. Furthermore, NarL-dependent activation of the transcription of the narG and nirB promoters, both of which are induced by phosphorylated NarL, requires much higher concentrations of nitrate than the activation of the nrfA and napF promoters, both of which are repressed by NarL when nitrate is abundant (129, 130). When these data are combined with the selective advantage conferred by the periplasmic nitrate reductase during nitrate-dependent growth, in contrast to the selective advantage conferred by the expression of nitrate reductase A when nitrate is more abundant (92), the fascinating question arises whether the NarQ-NarP system may be retained to enable E. coli to benefit from very low concentrations of nitrate, such as those found in the gastrointestinal tracts of animals, whereas the NarXL system and genes activate by phosphorylated NarL fulfill important roles in environments where nitrate is more abundant. Consistent with this proposal is the retention of the NarQ-NarP, Nap, and Nrf systems in the small-genome obligate human pathogen Haemophilus influenzae and other pathogens such as the gonococcus, both of which lack NarX-NarL, nitrate reductase A, and NirBD. We suggest that the periplasmic pathway for nitrate and nitrite reduction is active in the gastrointestinal tract, where the concentration of nitrate is insufficient to activate the NarX-NarL two-component system but sufficient to activate the alternative NarQ-NarP system and where the repressor activity of phosphorylated NarP also provides a delicate switch between nitrate-limited anaerobic respiration and fermentation (29; see also reference 51). Especially significant in the context of nitrate and nitrite reduction is that two of the four E. coli hydrogenases are differentially regulated by NarP in response to the availability of low concentrations of nitrate. The synthesis of hydrogenase A is significantly repressed by both nitrate-activated NarL and NarP, consistent with hydrogenase A fulfilling a hydrogen uptake role for dicarboxylate reduction during fermentative growth only when nitrate is unavailable (29). In contrast, hydrogenase B, the alternative uptake hydrogenase, is strongly repressed by phospho-NarL but insensitive to NarP (29). Hydrogenase B is therefore a prime candidate for transferring electrons from hydrogen released during fermentative growth via the menaquinone pool to the periplasmic nitrate and nitrite reductases, facilitating the use of hydrogen as a physiological source of electrons for nitrate reduction to ammonia (27).
Table 1 lists the many factors that regulate the expression of genes for nitrate and nitrite reduction: nucleoid-associated proteins provide fine-tuning mechanisms for all of these components. Indeed, yet more transcription factors, such as Crp and FhlDC, have also been proposed to play regulatory roles, but such isolated reports remain to be confirmed. (See the EcoCyc [Encyclopedia of Escherichia coli K-12 Genes and Metabolism] website [http://ecocyc.org] for further details.)
Table 1Transcription factors and environmental signals that regulate expression of genes for nitrate reduction to ammonia. |
NapA is located in the periplasmic compartment and is predicted to be exported by the Tat system, which is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In E. coli, many Tat substrates bind redox-active cofactors in the cytoplasm before transport. The coordination of cofactor insertion with protein export involves a Tat proofreading process in which chaperones bind twin-arginine signal peptides, thus preventing premature export. The initially described Tat proofreading chaperones belong to the TorD family and are required for the assembly of S- and N-oxide reductases of E. coli (105). In the case of NapA, ensuring the correct sequence of events in the insertion of the molybdenum cofactor and the iron-sulfur cluster and in the export of the active folded protein is critical, and the Nap systems appear to have two proteins involved in this process, NapD and NapF (83). E. coli napD mutants are deficient in active periplasmic nitrate reductase (94). NapD is a cofactorless ~10-kDa cytoplasmic protein, and two-hybrid analysis has demonstrated that it can interact with immature NapA in the cell cytoplasm (83). Recent studies have resolved the nuclear magnetic resonance structure of NapD, which adopts a β-α-β-β-α-β ferredoxin-type fold (77). The purified protein can bind tightly and specifically to the NapA twin-arginine signal peptide, and in intact cells, this tight binding serves to suppress signal peptide translocation activity such that transport on the Tat pathway is retarded.
NapF is predicted to be a cytoplasmic 16-kDa protein that has a number of Cys residues that may bind Fe-S clusters (5). For E. coli, N-terminal sequence analysis, cell fractionation coupled with immunoblotting, and the construction of LacZ and PhoA fusion proteins have been used to establish that NapF is indeed located in the cytoplasm (83). Two-hybrid protein-protein interaction studies have demonstrated that NapF interacts in the cytoplasm with NapA but that it does not self-associate or interact with other electron transport components of the Nap system, NapC, NapG, or NapH, or with the NapD chaperone described above. Purified NapF exhibits spectral properties characteristic of an iron-sulfur protein and is able to pull down NapA from soluble extracts of E. coli. NapF proteins have a highly conserved N-terminal double-arginine motif and a conserved double-proline motif, but neither motif appears to be essential for in vivo function (83). Although the role of NapF in E. coli NapA maturation is presently less clear than that of NapD, the combined available data are consistent with the proposal that NapF plays a role in the posttranslational modification of NapA prior to the export of folded NapA. The evidence from Rhodobacter sphaeroides is that NapF assists in the insertion of the 4Fe-4S cluster into NapA, perhaps transiently binding Fe-S during this process (85). In E. coli, the isc and suf operons are required for the biogenesis of different iron-sulfur proteins. The Isc system is the housekeeping Fe-S cluster assembly system, whereas the Suf system is important for Fe-S biogenesis under stress conditions. In the Suf system, there are two key proteins, SufA and SufS. SufA plays a role as a scaffold protein for the assembly of iron-sulfur clusters and delivery to target proteins, and SufS is a cysteine desulfurase that mobilizes the sulfur atom from cysteine and provides it to the cluster. It would then appear that NapF may augment the Isc and Suf systems by also serving as a scaffold for Fe-S clusters, specifically for a NapA-specific function in the latter stage of Fe-S incorporation, perhaps acting, as suggested for NapD, to prevent premature export through Tat until insertion is complete.
Although NarG is not a periplasmic protein, the formation of the active membrane-bound NarGHI complex with all of the cofactors correctly inserted is still a challenging assembly problem, particularly in terms of ensuring the correct sequence of events in the assembly process and insertion into the membrane. In this respect, NarJ appears to be a key protein. NarJ is encoded in the narGHJI operon. In a narJ mutant, the NarG and NarH subunits are associated in unstable and inactive NarGH complexes in which the NarG molybdenum cofactor and iron-sulfur cluster are absent. The NarGH complex can be activated in vitro if purified NarJ is added to the cell supernatant (9). However, once the apoenzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro, illustrating the importance of the correct sequencing of the assembly process; i.e., the NarJ-assisted molybdenum cofactor insertion step must precede membrane anchoring of the apoenzyme (75). It has been shown that NarJ interacts at two distinct binding sites of the NarG apoenzyme, one interfering with its membrane anchoring and the other involved in molybdenum cofactor insertion (128). It is possible that one of these interaction sites is a remnant of a Tat signal peptide of the type with which NapD interacts during NapA assembly and export.
The NrfA nitrite reductase also requires private assembly factors. Unlike NapA, this enzyme is exported as an unfolded protein via the Sec system, rather than as a folded protein via the Tat system. In the periplasm, the five hemes of NrfA must be covalently attached to the polypeptide chain at the CXXCH attachment sites, and this attachment is catalyzed by the cytochrome c maturation system (Ccm) that is required for the assembly of other c-type cytochromes and that is widespread elsewhere in the bacterial world. However, although NrfA has five covalently attached hemes, it has only four CXXCH motifs. The remaining heme, the active-site heme, is attached to a CXXCK motif. This one amino acid change requires the provision of system-specific heme attachment proteins, NrfEFG, that may form a so-called heme lyase complex. Thus, mutants with changes in these nrfEFG genes cannot assemble an active NrfA because they cannot attach a heme to the CXXCK motif (39, 60). A recently determined crystal structure of NrfG suggests that the contact between the heme lyase complex (NrfEFG) and NrfA is accomplished via a tetratricopeptide repeat domain in NrfG which serves as a binding site for the C-terminal CXXCK motif of NrfA. The portion of NrfA that binds to the tetratricopeptide repeat domain of NrfG has a unique secondary motif, a helix followed by an approximately 6-residue C-terminal loop that adopts a hook conformation.
There are many examples of bacteria that are able to both assimilate nitrate and either denitrify or catalyze the rapid anaerobic reduction of nitrate to ammonia. This pattern reemphasizes the totally independent physiological roles of these processes in assimilatory and respiratory metabolism. In contrast, there are no well-established examples of bacteria that both denitrify and dissimilate nitrate to ammonia: the very few occasional statements to the contrary await confirmation. Generalizations can also be made—at least based on the current, inadequately small database—concerning the distribution of genes for the various nitrate and nitrite reductases. In different bacteria, nitrate reductase A (NarGHI) can apparently function in partnership with any of the cytoplasmic or periplasmic nitrite reductases involved in denitrification or nitrite reduction to ammonia.
In contrast, no bacterium has yet been shown to couple a periplasmic nitrate reductase solely to the cytoplasmic nitrite reductase NirB. The cytoplasmic pathway for nitrate reduction to ammonia is restricted almost exclusively to a few groups of facultative anaerobic bacteria that encounter high concentrations of environmental nitrate. Although the enteric bacteria have a complete fermentative metabolism when no inorganic terminal electron acceptors are available, they also have a fully competent respiratory metabolism in which oxygen, nitrate, nitrite, and other oxidants can be used to conserve energy. Klebsiella pneumoniae typically lives outside the bodies of warm-blooded animals, for example, in soil, where nitrate is likely to be abundant. It expresses nitrate reductase A and Nir but lacks nap and nrf genes. Does this reflect a response to evolutionary pressures on organisms living in environments in which nitrate is more abundant than in the human body? Consistent with this suggestion is the exploitation of nitrate reductase A and Nir in German salami sausage production when Staphylococcus carnosus is inoculated into meat mixed with a high concentration of nitrate. The NarG-NirB cytoplasmic pathway also occurs in Bacillus subtilis, which lacks a fermentative metabolism. Conversely, pathogenic bacteria with small genomes that survive in oxygen-limited environments where both nitrate and oxygen are scarce have retained Nap and Nrf but lack nitrate reductase A and Nir. This finding suggests that Nap and Nrf may provide a selective advantage to bacteria other than E. coli, where it is essential in the competition for trace concentrations of any terminal electron acceptor that may become available. It is significant that denitrifying bacteria are rarely isolated from normal human flora.
Although neither E. coli nor S. enterica carries genes for a complete denitrification pathway, the first defining intermediate of denitrification, nitric oxide, is generated enzymatically by enteric bacteria and is also formed chemically from nitrite at acidic pHs (30, 90, 114). Nitric oxide is generated as a by-product during the anaerobic reduction of nitrate or nitrite. In contrast to bacteria in laboratory experiments in which artificial environments are created, in their natural habitats, enteric bacteria are concomitantly exposed to nitrate, nitrite, NO, hydroxylamine, oxygen, and reactive oxygen and nitrogen species generated either as products of their own metabolism or as part of host defense mechanisms. Chemical and enzyme-catalyzed interactions between these species generate a plethora of secondary compounds that enhance or moderate their toxicities. Bacterial survival within warm-blooded animal hosts therefore depends upon the ability of bacteria to mitigate the consequences of the generation of by-products during nitrate reduction to ammonia. This section will provide an overview of the responses to nitric oxide, nitrosating agents generated from nitric oxide, and hydroxylamine. Upon exposure to air, NO becomes highly mutagenic in a reaction that requires both oxygen and the nfi-encoded endonuclease V, a DNA repair enzyme that recognizes hypoxanthine and xanthine (131). How enteric bacteria cope with DNA damage caused by transnitrosation or peroxynitrite generation from NO and superoxide (73) is outside the scope of this chapter.
Spiro (117) has emphasized the need to distinguish between transcription factors that respond directly to NO and those that are chemically damaged by NO, leading to indirect transcription responses. During aerobic growth, the primary mechanism for removing NO from the environment is by oxidation to nitrate, catalyzed by the dioxygenase reaction of the bacterial flavohemoglobin Hmp (48, 57). The expression of the hmpA gene is regulated primarily by NO-activated derepression of NsrR (43, 69, 70, 95). The expression of hmpA is also significantly repressed by FNR and MetR, both of which are inactivated by chemical reaction with nitric oxide (10, 43, 69, 80, 91).
The flavohemoglobin Hmp is also active during anaerobic growth, when it catalyzes the reduction of NO to nitrous oxide. However, the catalytic efficiency of Hmp under anoxic conditions is significantly less than those of two other proteins, NorV and the nitrite reductase NrfA. The norVW operon encodes a nitric oxide reductase, NorV, and a NorV reductase, NorW. This operon is regulated by NorR, which was the first transcription factor in enteric bacteria to be shown to respond specifically to NO (47, 61). NorR is essentially a three-domain transcription activator that interacts with the σ54 form of RNA polymerase to activate the expression of the norVW operon (34, 121, 122). No other transcription units are significantly regulated by NorR (95). The addition of NO is essential to activate the ATPase activity of the central AAA+ domain of NorR, but the removal of the N-terminal GAF domain results in the constitutive transcription of norVW. On the basis of EPR spectra, the direct binding of NO to the GAF domain appears to result in the formation of a mononitrosyl [Fe(NO)]7 (spin = 3/2) complex. The function of the N-terminal GAF domain is therefore to inhibit the ATPase activity of the central AAA+ domain in the absence of NO. Hence, NorR is a highly specific and sensitive NO sensor with a Kd of 50 nM (34).
Nitric oxide is reduced directly to ammonia by the cytochrome c nitrite reductase, and the synthesis of NrfA is partially repressed by the NsrR protein, a repressor of the Rrf2 family of transcription factors (43). Unlike NorR, NsrR regulates at least nine operons, NsrR repression being lifted by the inactivation of the NsrR iron-sulfur center resulting from the formation of a dinitrosyl derivative upon exposure to nitric oxide. There has been some debate concerning whether Hmp or NrfA plays the more significant role in protection against NO damage during anaerobic growth (72, 89). Most likely, the answer depends upon the growth conditions. The expression of hmpA during anaerobic growth requires the inactivation of the repressor activity of FNR, which will occur only when the bacteria are exposed to high concentrations of NO (31). In contrast, very low concentrations of NO are sufficient to activate NorR or to inactivate NsrR, so conditions prevalent in the gastrointestinal tracts of humans favor the expression of the nrfA and norVW operons rather than that of hmpA. We propose that Hmp provides a last line of defense during anaerobic growth when the concentration of NO is too high for alternative defense mechanisms to function. During nitrate reduction to ammonia, enteric bacteria are vulnerable to reactive oxygen species and reactive nitrogen species generated as part of host defense mechanisms, especially if damage is so severe that the transcription activator FNR is nonfunctional. Thus, even during anaerobic growth when NrfA is unavailable to remove NO as fast as it is encountered in the human body, relief of FNR repression of hmp expression provides an alternative protection mechanism as a backup to the NorR-activated norV-norW system.
Although the direct binding of NO to NorR and NsrR and its reduction by Hmp, NorV, and NrfA are the major direct effects of nitric oxide on E. coli and Salmonella, these responses are augmented by a plethora of secondary consequences of exposure to NO. Both proteins and small molecules become nitrosated, and some of these products, for example, S-nitrosoglutathione, are themselves excellent transnitrosylating reagents (44). S-nitrosoglutathione, but not NO, nitrosates homocysteine, the cofactor for MetR-mediated repression of genes required for methionine and S-adenosylmethionine biosynthesis (44, 80). Other transcription factors are inactivated by the nitrosylation of their metal or sulfhydryl active sites. In some cases, NO can directly inactivate the transcription factor, for example, by inactivating the iron-sulfur centers of FNR, SoxR, Fur, and probably also NsrR or by transnitrosylating key cysteine residues, for example, those in OxyR. Nitric oxide also inactivates key enzymes, for example, the citric acid cycle enzymes aconitase and fumarase, both of which contain iron-sulfur centers. It has now been shown conclusively that the product of the ytfE gene, YtfE (also known as DnrN) (101), repairs NO-induced damage to iron-sulfur centers of both enzymes and transcription factors (71). Other proteins that may fulfill similar roles include products of genes of unknown function that are part of the NsrR regulon, some of which were initially identified from microarray analyses of the NarXL and NarP regulons of E. coli (29). They include YeaR-YoaG, YibIH, and YgbA, as well as the enigmatic prismane, or hybrid cluster protein (HCP) (43). The synthesis of HCP is optimal during anaerobic growth in the presence of nitrite, and it has been shown to catalyze hydroxylamine reduction, albeit under nonphysiological conditions (133). As the Km for hydroxylamine is orders of magnitude greater than the concentration required to inhibit growth completely and since both the nitrite reductases Nir and Nrf have hydroxylamine reductase activities similar to that of HCP, functions of HCP other than the recently proposed peroxidase activity should be reconsidered (1).
Nitrate reduction to ammonia is now recognized to be a widely distributed process and to be ecologically significant. NarGH has recently been shown to be essential for the successful colonization of the gut by E. coli (66). The complex bacterial communities found in wastewater treatment plants include many bacteria that either denitrify nitrate or reduce nitrate anaerobically to ammonia. Are there also environments in which bacteria have evolved both pathways in the same organism, and if so, what are the environmental determinants of this selection?
The NapGH and NrfCD proteins and their homologues are widespread in bacteria. It is emerging that their activities in anaerobic and microoxic respiratory systems are very important for the survival of pathogens. Structure-function research on bacterial integral membrane quinol-dependent respiratory proteins has attracted a great deal of high-profile exposure through the determination of structures for the quinone-dependent formate dehydrogenase and succinate dehydrogenases and the quinol-dependent fumarate reductase, nitrate reductase A, and cytochrome bo oxidase of E. coli. Such structures reveal the diversity of quinol/quinone-dependent respiratory electron transfer proteins, but none of these systems have any relationship at the primary structure level with the E. coli NapGH and NirCD systems described here. Critically, these quinol dehydrogenases are not homologous with the key quinol/quinone-dependent respiratory enzymes of mammalian mitochondria such as NADH dehydrogenase, succinate dehydrogenase, and the cytochrome bc1 complex. Thus, in the long term, structure-function studies of NapGH and NrfCD may lead to the development of antimicrobial compounds that bind specifically to the quinol binding sites of these enzymes and do not target the host mitochondrial respiratory system.
References
1. Almeida, C. C., C. V. Romão, P. F. Lindley, M. Teixeira, and L. M. Saraiva. 2006. The role of the hybrid-cluster protein in oxidative stress. J. Biol. Chem. 281:32445–32450.[PubMed] [CrossRef]
2. Anderson, L., D. J. Richardson, and J. N. Butt. 2001. Protein film electrochemistry of the membrane-bound nitrate reductase. Biochemistry 40:11294–11307.[PubMed] [CrossRef]
3. Angove, H., J. A. Cole, D. J. Richardson, and J. N. Butt. 2002. Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome c nitrite reductase. J. Biol. Chem. 277:23374–23381.[PubMed] [CrossRef]
4. Bamford, V., H. Angove, H. Seward, J. N. Butt, J. A. Cole, A. J. Thomson, A. H. Hemmings, and D. J. Richardson. 2002. The structure and spectroscopy of the cytochrome c nitrite reductase of Escherichia coli. Biochemistry 41:2921–2931.[PubMed] [CrossRef]
5. Berks, B. C., S. J. Ferguson, J. W. B. Moir, and D. J. Richardson. 1995. Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim. Biophys. Acta 1232:97–173.[PubMed] [CrossRef]
6. Bertero, M. G., R. A. Rothery, M. Palak, C. Hou, D. Lim, F. Blasco, J. H. Weiner, and N. C. Strynadka. 2003. Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nat. Struct. Biol. 10:681–687.[PubMed] [CrossRef]
7. Bertero, M. G., R. A. Rothery, N. Boroumand, M. Palak, F. Blasco, N. Ginet, J. H. Weiner, and N. C. Strynadka. 2005. Structural and biochemical characterization of a quinol binding site of Escherichia coli nitrate reductase A. J. Biol. Chem. 280:14836–14843. [PubMed] [CrossRef]
8. Björklöf, K., V. Zickermann, and M. Finel. 2000. Purification of the 45 kDa, membrane bound NADH dehydrogenase of Escherichia coli (NDH-2) and analysis of its interaction with ubiquinone analogues. FEBS Lett. 467:105–110.[PubMed] [CrossRef]
9. Blasco, F., J. P. Dos Santos, A. Magalon, C. Frixon, B. Guigliarelli, C. L. Santini, and G. Giordano. 1998. NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli. Mol. Microbiol. 28:435–47.[PubMed] [CrossRef]
10. Bodenmiller, D. M., and S. Spiro. 2006. The yjbE (nsrR) gene of Escherichia coli encodes a nitric oxide-sensitive transcriptional regulator. J. Bacteriol. 188:874–881.[PubMed] [CrossRef]
11. Brondijk, T. H. C., D. Fiegen, D. J. Richardson, and J. A. Cole. 2002. Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation. Mol. Microbiol. 44:245–255.[PubMed] [CrossRef]
12. Brondijk, T. H. C., A. Nilavongse, N. Filenko, D. J. Richardson, and J. A. Cole. 2004. The NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology, and physiological roles in quinol oxidation and redox balancing. Biochem. J. 379:47–55.[PubMed] [CrossRef]
13. Browning, D. F., J. A. Cole, and S. J. W. Busby. 2000. Suppression of FNR-dependent transcription activation at the Escherichia coli nir promoter by Fis, IHF and H-NS: modulation of transcription initiation by a complex nucleo-protein assembly. Mol. Microbiol. 37:1258–1269.[PubMed] [CrossRef]
14. Browning, D. F., C. M. Beatty, A. J. Wolfe, J. A. Cole, and S. J. W. Busby. 2002. Independent regulation of the divergent Escherichia coli nrfA and acsP1 promoters by a nucleo-protein assembly at a shared regulatory region. Mol. Microbiol. 43:687–701.[PubMed] [CrossRef]
15. Browning, D. F., J. A. Cole, and S. J. W. Busby. 2004. Transcription activation by remodelling of a nucleoprotein assembly: the role of NarL at the FNR-dependent Escherichia coli nir promoter. Mol. Microbiol. 53:203–215.[PubMed] [CrossRef]
16. Browning, D. F., D. C. Grainger, C. M. Beatty, A. J. Wolfe, J. A. Cole, and S. J. W. Busby. 2006. The Escherichia coli K-12 NarL and NarP proteins insulate the nrf promoter from the effects of integration host factor. J. Bacteriol. 188:7449–7456.[PubMed] [CrossRef]
17. Butler, C. S., J. M. Charnock, B. Bennett, H. J. Sears, A. J. Reilly, S. J. Ferguson, C. D. Garner, D. J. Lowe, A. J. Thomson, B. C. Berks, and D. J. Richardson. 1999. Models for molybdenum co-ordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy. Biochemistry 38:9000–9012.[PubMed] [CrossRef]
18. Campbell, J. W., R. M. Morgan-Kiss, and J. E. Cronan, Jr. 2003. New Escherichia coli metabolic competency: growth on fatty acids by a novel anaerobic β-oxidation pathway. Mol. Microbiol. 47:793–805.[PubMed] [CrossRef]
19. Cartron, M. L., D. Roldán, S. J. Ferguson, B. C. Berks, and D. J. Richardson. 2002. Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases. Biochem. J. 368:425–432.[PubMed] [CrossRef]
20. Chang, L., I.-C. Wei, J. P. Audia, R. A. Morton, and H. E. Schellhorn. 1999. Expression of the Escherichia coli NRZ nitrate reductase is highly growth phase dependent and is controlled by RpoS, the alternative vegetative sigma factor. Mol. Microbiol. 34:756–766. [CrossRef]
21. Chiang, R. C., R. Cavicchioli, and R. P. Gunsalus. 1992. Identification and characterisation of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli. Mol. Microbiol. 6:1913–1923.[PubMed] [CrossRef]
22. Clarke, T., V. Dennison, H. Seward, B. Burlat, J. A. Cole, A. Hemmings, and D. J. Richardson. 2004. Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex. J. Biol. Chem. 279:41333–41339.[PubMed] [CrossRef]
23. Clarke, T. A., A. M. Hemmings, B. Burlat, J. N. Butt, J. A. Cole, and D. J. Richardson. 2006. Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans. Biochem. Soc. Trans. 34:143–145.[PubMed] [CrossRef]
24. Clarke, T. A., G. Kemp, J. van Wonderen, R. Doyle, J. N. Butt, M. R. Cheesman, D. J. Richardson, and A. M. Hemmings. 2007. The role of a conserved glutamine residue in tuning the catalytic activity of Escherichia coli cytochrome c nitrite reductase. Biochemistry 47:3789–3799. [CrossRef]
25. Clarke, T. C., J. A. Cole, D. J. Richardson, and A. M. Hemmings. 2007. The structure of the pentaheme c-type cytochrome NrfB and characterisation of its solution-state interaction with the pentaheme nitrite reductase, NrfA. Biochem. J. 406:19–30.[PubMed] [CrossRef]
26. Clegg, S., Y. Feng, L. Griffiths, and J. A. Cole. 2002. The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters. Mol. Microbiol. 44:143–155.[PubMed] [CrossRef]
27. Clegg, S., W. Jia, and J. A. Cole. 2006. Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth. Microbiology 152:2091–2100.[PubMed] [CrossRef]
28. Cole, J. A., and C. M. Brown. 1980. Nitrite reduction to ammonia by fermentative bacteria: a short circuit in the biological nitrogen cycle. FEMS Microbiol. Lett. 7:65–72. [CrossRef]
29. Constantinidou, C. C., J. L. Hobman, M. D. Patel, C. W. Penn, J. A. Cole, and T. W. Overton. 2006. A reassessment of the fumarate and nitrate reduction regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL and NarQP as Escherichia coli adapts from aerobic to anaerobic growth. J. Biol. Chem. 281:4802–4808.[PubMed] [CrossRef]
30. Corker, H., and R. K. Poole. 2003. Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase, the NO-sensing regulator, Fnr, and the flavohemoglobin Hmp. J. Biol. Chem. 278:31584–31592.[PubMed] [CrossRef]
31. Cruz-Ramos, H., J. Crack, G. Wu, M. N. Hughes, C. Scott, A. J. Thompson, J. Green, and R. K. Poole. 2002. NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohemeoglobin, Hmp. EMBO J. 21:3235–3244.[PubMed] [CrossRef]
32. Darwin, A. J., and V. Stewart. 1995. Nitrate and nitrite regulation of the Fnr-dependent aeg-46.5 promoter of Escherichia coli K-12 is mediated by competition between homologous response regulators (NarL and NarP) for a common DNA-binding site. J. Mol. Biol. 251:15–29.[PubMed] [CrossRef]
33. Darwin, A. J., K. L. Tyson, S. J. W. Busby, and V. Stewart. 1997. Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement. Mol. Microbiol. 25:583–595.[PubMed] [CrossRef]
34. D’Autréaux, B. D., N. P. Tucker, R. Dixon, and S. Spiro. 2005. A non-heme iron centre in the transcription factor NorR senses nitric oxide. Nature 437:769–772.[PubMed] [CrossRef]
35. de Graef, M. R., S. Alexeeva, J. L. Snoep, and M. J. Teixeira de Mattos. 1999. The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J. Bacteriol. 181:2351–2357.[PubMed]
36. Dementin, S., P. Arnoux, B. Frangioni, S. Grosse, C. Leger, B. Burlat, B. Guigliarelli, M. Sabaty, and D. Pignol. 2007. Access to the active site of periplasmic nitrate reductase: insights from site-directed mutagenesis and zinc inhibition studies. Biochemistry 46:9713–9721.[PubMed] [CrossRef]
37. DeMoss, J. A., and P.-Y. Hsu. 1991. NarK enhances nitrate uptake and nitrite excretion in Escherichia coli. J. Bacteriol. 173:3303–3310.[PubMed]
38. Dietrich, W., and O. Klimmek. 2002. The function of methyl-menaquinone-6 and polysulfide reductase membrane anchor (PsrC) in polysulfide respiration of Wolinella succinogenes. Eur. J. Biochem. 269:1086–1095.[PubMed] [CrossRef]
39. Eaves, D. J., J. Grove, W. Staudenmann, P. James, R. K. Poole, S. A. White, L. Griffiths, and J. Cole. 1998. Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase of Escherichia coli. Mol. Microbiol. 28:205–216.[PubMed] [CrossRef]
40. Einsle, O., P. Stach, A. Messersmidt, J. Simon, A Kröger, R. Huber, and P. M. H. Kroneck. 2000. Cytochrome c nitrite reductase from Wolinella sucinogenes: structure at 1.6 Å resolution, inhibitor binding, and heme packing motifs. J. Biol. Chem. 275:39608–39616.[PubMed] [CrossRef]
41. Ellington, M. J. K., K. K. Bakoo, G. Sawers, D. J. Richardson, and S. J. Ferguson. 2002. Hierarchy of carbon source selection in Paracoccus pantotrophus: strict correlation between reduction state of the carbon substrate and aerobic expression of the nap operon. J. Bacteriol. 184:4767–4774.[PubMed] [CrossRef]
42. Elliott, S. J., K. R. Hoke, K. Heffron, M. Palak, R. A. Rothery, J. H. Weiner, and F. A. Armstrong. 2004. Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site. Biochemistry 43:799–807.[PubMed] [CrossRef]
43. Filenko, N., S. Spiro, D. Browning, D. Squire, T. Overton, J. Cole, and C. Constantinidou. 2007. The NsrR regulon of Escherichia coli K-12 includes genes encoding the hybrid cluster protein and the periplasmic, respiratory nitrite reductase. J. Bacteriol. 189:4410–4417. [CrossRef]
44. Flatley, J., J. Barrett, S. T. Pullan, M. N. Hughes, J. Green, and R. K. Poole. 2005. Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis. J. Biol. Chem. 280:10065–10072.[PubMed] [CrossRef]
45. Forsythe, S. J., J. M. Dolby, A. D. B. Webster, and J. A. Cole. 1988. Nitrate and nitrite-reducing bacteria in the achlorhydric stomach. J. Med. Microbiol. 25:253–259.[PubMed] [CrossRef]
46. Gardner, A. M., R. A. Helmick, and P. R. Gardner. 2002. Flavorubredoxin, an inducible catalyst for nitric oxide reduction and detoxification in Escherichia coli. J. Biol. Chem. 277:8172–8177.[PubMed] [CrossRef]
47. Gardner, A. M., C. R. Gessner, and P. R. Gardner. 2003. Regulation of the nitric oxide reduction operon (norRVW) in Escherichia coli. Role of NorR and sigma54 in the nitric oxide stress response. J. Biol. Chem. 278:10081–10086.[PubMed] [CrossRef]
48. Gardner, P. R., A. M. Gardner, L. A. Martin, and A. L. Saltzman. 1998. Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc. Natl. Acad. Sci. USA 95:10378–10383.[PubMed] [CrossRef]
49. Giel, J. L., D. Rodionov, M. Liu, F. R. Blattner, and P. J. Kiley. 2006. IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol. Microbiol. 60:1058–1075.[PubMed] [CrossRef]
50. Gilberthorpe, N. J., and R. K. Poole. 2008. Nitric oxide homeostasis in Salmonella Typhimurium: roles of respiratory nitrate reductase and flavohemoglobin. J. Biol. Chem. 283:11146–11154. [PubMed] [CrossRef]
51. Goh, E. B., P. J. Bledsoe, L. L. Chen, P. Gyaneshwar, V. Stewart, and M. M. Igo. 2005. Hierarchical control of anaerobic gene expression in Escherichia coli K-12: the nitrate-responsive NarX-NarL regulatory system represses synthesis of the fumarate-responsive DcuS-DcuR regulatory system. J. Bacteriol. 187:4890–4899. [PubMed] [CrossRef]
52. Gott, P., and W. Boos. 1988. The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions. Mol. Microbiol. 2:655–663.[PubMed] [CrossRef]
53. Green, J., and J. R. Guest. 1994. Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors. Mol. Microbiol. 12:433–444.[PubMed] [CrossRef]
54. Grove, J., S Tanapongpipat, G. Thomas, L. Griffiths, H. Crooke, and J. Cole. 1996. Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. Mol. Microbiol. 19:476–481. [CrossRef]
55. Gwyer, J. D., D. J. Richardson, and J. N. Butt. 2005. Diode or tunnel-diode characteristics? Resolving the catalytic consequences of proton coupled electron transfer in a multi-centered oxidoreductase. J. Am. Chem. Soc. 127:14964–14965.[PubMed] [CrossRef]
56. Harborne, N. R., L. Griffiths, S. J. W. Busby, and J. A. Cole. 1992. Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon. Mol. Microbiol. 6:2805–2813.[PubMed] [CrossRef]
57. Hausladen, A., A. Gow, and J. S. Stamler. 1998. Nitrosative stress: metabolic pathway involving the flavohemoglobin. Proc. Natl. Acad. Sci. USA 95:14100–14105.[PubMed] [CrossRef]
58. Hinsley, A. P., and B. C. Berks. 2002. Specificity of respiratory pathways involved in the reduction of sulfur compounds by Salmonella enterica. Microbiology 148:3631–3640.[PubMed]
59. Huang, Y., M. J. Lemieux, Y. Song, Y., M. Auer, and D. N. Wang. 2003. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301:616–620.[PubMed] [CrossRef]
60. Hussain, H., J. Grove, L. Griffiths, S. Busby, and J. Cole. 1994. A seven gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria. Mol. Microbiol. 12:153–163.[PubMed] [CrossRef]
61. Hutchins, M. I., N. Mandhana, and S. Spiro. 2002. The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species. J. Bacteriol. 184:4640–4643. [PubMed] [CrossRef]
62. Iuchi, S., and E. C. C. Lin. 1988. arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc. Natl. Acad. Sci. USA 85:1888–1892.[PubMed] [CrossRef]
63. Jackson, R., A. Cornish-Bowden, and J. A. Cole. 1981. Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12. Biochem. J. 193:861–867.[PubMed]
64. Jayaraman, P.-S., K. L. Gaston, J. A. Cole, and S. J. W. Busby. 1988. The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite. Mol. Microbiol. 2:527–530.[PubMed] [CrossRef]
65. Jepson, B. J. N., S. Mohan, T. A. Clarke, A. J. Gates, J. A. Cole, C. S. Butler, J. N. Butt, A. M. Hemmings, and D. J. Richardson. 2007. Spectrophotometric and structural analysis of the periplasmic nitrate reductase from Escherichia coli. J. Biol. Chem. 282:6425–6437.[PubMed] [CrossRef]
66. Jones, S. A., F. Z. Chowdhury, A. J. Fabich, A. Anderson, D. M. Schreiner, A. L. House, S. M. Autieri, M. P. Leatham, J. J. Lins, M. Jorgensen, P. S. Cohen, and T. Conway. 2007. Respiration of Escherichia coli in the mouse intestine. Infect. Immun. 75:4891–4899. [PubMed] [CrossRef]
67. Jormakka, M., S. Tornroth, B. Bytne, and S. Iwata. 2002. Molecular basis of proton motive force generation: structure of formate dehydrogenase-N. Science 295:1863–1868.[PubMed] [CrossRef]
68. Jormakka, M., D. J. Richardson, B. Byrne, and S. Iwata. 2004. The architecture of NarGH reveals a structural classification of MGD enzymes. Structure 12:95–104.[PubMed] [CrossRef]
69. Justino, M. C., C. C. Almeida, V. L. Goncalves, M. Teixeira, and L. M. Saraiva. 2005. Escherichia coli YtfE is a di-iron protein with an important function in assembly of iron-sulphur clusters. FEMS Microbiol. Lett. 257:278–284. [CrossRef]
70. Justino, M. C., J. B. Vicente, M. Teixeira, and L. M. Saraiva. 2006. New genes implicated in the protection of anaerobically grown Escherichia coli against nitric oxide. J. Biol. Chem. 280:2636–2643. [CrossRef]
71. Justino, M. C., C. C. Almeida, M. Teixeira, and L. M. Saraiva. 2007. Escherichia coli di-iron YtfE protein is necessary for the repair of stress-damaged iron-sulfur clusters. J. Biol. Chem. 282:10352–10359.[PubMed] [CrossRef]
72. Kim, S.O., Y. Orii, D. Lloyd, M. N. Hughes, and R. K. Poole. 1999. Anoxic function for the Escherichia coli flavohemeoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide. FEBS Lett. 445:389–394.[PubMed] [CrossRef]
73. Koppernol, W. H., J. J. Moreno, W. A. Pryor, H. Ischiropoulos, and J. S. Beckman. 1982. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol. 5:834–842. [CrossRef]
74. Krafft, T., R. Gross, and A. Kröger. 1995. The function of Wolinella succinogenes psr genes in electron transport with polysulphide as the terminal electron acceptor. Eur. J. Biochem. 230:601–606.[PubMed] [CrossRef]
75. Lanciano, P., A. Vergnes, S. Grimaldi, B. Guigliarelli, and A. Megalon. 2007. Biogenesis of a respiratory complex is orchestrated by a single accessory protein. J. Biol. Chem. 282:17468–17474. [PubMed] [CrossRef]
76. Lee, A. I., A. Delgado, and R. P. Gunsalus. 1999. Signal-dependent phosphorylation of the membrane-bound NarX two-component sensor-transmitter protein of Escherichia coli: nitrate elicits a superior anion ligand response compared to nitrite. J. Bacteriol. 181:5309–5316.[PubMed]
77. Maillard, J., C. Spronk, G. Buchanan, V. Lyall, D. J. Richardson, T. Palmer, G. W. Vuister, and F. Sargent. 2007. Structural diversity in twin-arginine signal peptide-binding proteins. Proc. Natl. Acad. Sci. USA 104:15641–15646.[PubMed] [CrossRef]
78. Marietou, A., D. Richardson, J. Cole, and S. Mohan. 2005. Nitrate reduction by Desulfovibrio desulfuricans: a periplasmic nitrate reductase that lacks NapB, but includes a unique tetraheme c-type cytochrome, NapM. FEMS Microbiol. Lett. 248:217–225.[PubMed] [CrossRef]
79. McNicholas, P. M., and R. P. Gunsalus. 2002. The molybdate-responsive Escherichia coli ModE transcriptional regulator coordinates periplasmic nitrate reductase (napFDAGHBC) operon expression with nitrate and molybdate availability. J. Bacteriol. 184:3253–3259.[PubMed] [CrossRef]
80. Membrillo-Hernández, J., M. D. Coopamah, A. Channa, M. N. Hughes, and R. K. Poole. 1998. A novel mechanism for upregulation of the Escherichia coli K-12 hmp (flavohaemoglobin) gene by the ‘NO releaser,’ S-nitrosoglutathione: nitrosation of homocysteine and modulation of MetR binding to the glyA-hmp intergenic region. Mol. Microbiol. 29:1101–1112.[PubMed] [CrossRef]
81. Mills, P., G. Rowley, S. Spiro, J. Hinton, and D. J. Richardson. 2008. A combination of cytochrome c nitrite reductase (NrfA) and flavorubredoxin (NorV) protects Salmonella enterica serovar Typhimurium against killing by NO in anoxic environments. Microbiology 154:1218–1228.[PubMed] [CrossRef]
82. Moir, J. W., and N. J. Wood. 2001. Nitrate and nitrite transport in bacteria. Cell. Mol. Life Sci. 58:215–224.[PubMed] [CrossRef]
83. Nilavongse, A., T. H. C. Brondijk, T. W. Overton, D. J. Richardson, E. Leach, and J. A. Cole. 2006. The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA. Microbiology 152:3227–3237.[PubMed] [CrossRef]
84. Noji, S., T. Nohno, T. Saito, and S. Taniguchi. 1989. The narK gene product participates in nitrate transport induced in Escherichia coli nitrate-respiring cells. FEBS Lett. 252:139–143.[PubMed] [CrossRef]
85. Olmo-Mira, F., D. J. Richardson, F. Castillo, C. Moreno-Vivian, and D. Roldan. 2004. NapF is a cytoplasmic iron-sulfur protein required for Fe-S cluster assembly in the periplasmic nitrate. J. Biol. Chem. 279:49727–49735.[PubMed] [CrossRef]
86. Park, S.-J., C.-P. Tseng, and R. P. Gunsalus. 1995. Regulation of succinate dehydrogenase (sucCDAB) operon expression in Escherichia coli response to carbon supply and anaerobiosis: role of ArcA and FNR. Mol. Microbiol. 15:473–482.[PubMed] [CrossRef]
87. Peakman, T., J. Crouzet, J. F. Mayaux, S. Busby, S. B. Mohan, J. Wootton, R. Nicolson, and J. A. Cole. 1990. Nucleotide sequence organisation and structural analysis of the products of the nirB to cysG region of the Escherichia coli chromosome. Eur. J. Biochem. 191:315–323.[PubMed] [CrossRef]
88. Pommier, J., M. A. Mandrand, S. E. Holt, D. H. Boxer, and G. Giordano. 1992. A second phenazine methosulphate-linked formate dehydrogenase isoenzyme in Escherichia coli. Biochim. Biophys. Acta 1107:305–313. [CrossRef]
89. Poock, S. R., E. R. Leach, J. W. B. Moir, J. A. Cole, and D. J. Richardson. 2002. Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli. J. Biol. Chem. 277:23664–23669.[PubMed] [CrossRef]
90. Poole, R. K., and M. N. Hughes. 2000. New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress. Mol. Microbiol. 36:775–783.[PubMed] [CrossRef]
91. Poole, R. K., M. F. Anjum, J. Membrillo-Hernández, S. O. Kim, M. N. Hughes, and V. Stewart. 1996. Nitric oxide, nitrite, and FNR regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12. J. Bacteriol. 178:5487–5492.[PubMed]
92. Potter, L., P. Millington, G. Thomas, and J. Cole. 1999. Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth? Biochem. J. 344:77–84.[PubMed] [CrossRef]
93. Potter, L., H. Angove, D. J. Richardson, and J. A. Cole. 2001. Nitrate reduction in the periplasm of Gram negative bacteria. Adv. Microb. Physiol. 45:52–102.
94. Potter, L. C., and J. A. Cole. 1999. Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12. Biochem. J. 344:69–76. [PubMed] [CrossRef]
95. Pullan, S. T., M. D. Gidley, R. A. Jones, J. Barrett, T. M. Stevanin, R. C. Read, J. Green, and R. K. Poole. 2007. Nitric oxide in chemostat-cultured Escherichia coli is sensed by Fnr and other global regulators: unaltered methionine biosynthesis indicates lack of S nitrosation. J. Bacteriol. 189:1845–1855.[PubMed] [CrossRef]
96. Rabin, R. S., and V. Stewart. 1992. Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K12. Proc. Natl. Acad. Sci. USA 89:8419–8423.[PubMed] [CrossRef]
97. Rabin, R. S., and V. Stewart. 1993. Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12. J. Bacteriol. 175:3259–3268.[PubMed]
98. Rabin, R. S., L. A. Collins, and V. Stewart. 1992. In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12. Proc. Natl. Acad. Sci. USA 89:8701–8705.[PubMed] [CrossRef]
99. Richardson, D. J. 2007. Structural and functional flexibility of bacterial respiromes, p. 97–128. In W. El-Sharoud (ed.), Bacterial Physiology: a Molecular Approach. Springer, New York, NY.
100. Richardson, D. J., and G. Sawers. 2002. PMF through the redox loop. Science 295:1842–1843.[PubMed] [CrossRef]
101. Rodionov, D. A., I. L. Dubchak, A. P. Arkin, E. J. Alm, and M. S. Gelfand. 2005. Dissimilatory metabolism of nitrogen oxides in Bacteria: comparative reconstruction of transcriptional networks. PLoS Comput. Biol. 1:e55.
102. Rodriques, M. L., T. F. Oliveira, I. A. C. Pereira, and M. Archer. 2006. X-ray structure of the membrane-bound cytochrome c quinol dehydrogenase NrfH reveals novel haem coordination. EMBO J. 25:5951–5960.[PubMed] [CrossRef]
103. Roldán, M. D., H. J. Sears, M. R. Cheesman, S. J. Ferguson, A. J. Thomson, B. C. Berks, and D. J. Richardson. 1998. Spectroscopic characterization of a novel multiheme c-type cytochrome widely implicated in bacterial electron transport. J. Biol. Chem. 273:28785–28790.[PubMed] [CrossRef]
104. Rowe, J. J., T. Ubbink-Kok, D. Molenaar, W. N. Konings, and A. J. M. Driessen. 1994. NarK is a nitrite extrusion system involved in anaerobic nitrate respiration by Escherichia coli. Mol. Microbiol. 12:579–586.[PubMed] [CrossRef]
105. Sargent, F. 2007. Constructing the wonders of the bacterial world: biosynthesis of complex enzymes. Microbiology 153:633–651.[PubMed] [CrossRef]
106. Sazanov, L. A., and P. Hinchliffe. 2006. Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 311:1430–1436. [PubMed] [CrossRef]
107. Schröder, I., C. D. Wolin, R. Cavicchioli, and R. P. Gunsalus. 1994. Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli. J. Bacteriol. 176:4985–4902.[PubMed]
108. Sears, H. J., G. Sawers, B. C. Berks, S. J. Ferguson, and D. J. Richardson. 2000. Control of periplasmic nitrate reductase gene expression (napEDABC) from Paracoccus pantotrophus in response to oxygen and carbon substrates. Microbiology 146:2977–2985.[PubMed]
109. Sellars, N. J., S. J. Hall, and D. J. Kelly. 2002. Growth of Campylobacter jejuni supported by respiration of fumarate, nitrate, nitrite, trimethylamine-N-oxide, or dimethyl sulfoxide requires oxygen. J. Bacteriol. 184:4187–4196. [CrossRef]
110. Siegel, L. M., M. J. Murphy, and H. Kamin. 1973. Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of enterobacteria. I. The Escherichia coli hemoflavoprotein: molecular parameters and prosthetic groups. J. Biol. Chem. 248:251–264.[PubMed]
111. Simon, J. 2002. Enzymology and bioenergetics of respiratory nitrite ammonification. FEMS Microbiol. Rev. 26:285–309.[PubMed] [CrossRef]
112. Simon, J., R. Pisa, T. Stein, R. Eichler, O. Klimmek, and R. Gross. 2001. The tetraheme cytochrome c NrfH is required to anchor the cytochrome c nitrite reductase (NrfA) in the membrane of Wolinella succinogenes. FEBS J. 268:5776–5782.
113. Smith, M. S. 1983. Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity. Appl. Environ. Microbiol. 45:1545–1547.[PubMed]
114. Sobko, T., L. Huang, T. Midtvedt, E. Norin, L. E. Gustafsson, M. Norman, E. A. Jansson, and J. O. Lundberg. 2006. Generation of NO by probiotic bacteria in the gastrointestinal tract. Free Radic. Biol. Med. 41:985–991.[PubMed] [CrossRef]
115. Sorensen, J. 1978. Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Microbiol. 35:301–305.[PubMed]
116. Spector, M. P., F. Garcia del Portillo, S. M. D. Bearson, A. Mahmud, M. Magut, B. B. Finlay, G. Dougan, J. W. Foster, and M. J. Pallen. 1999. The rpoS-dependent starvation-stress response locus sti encodes a nitrate reductase (narZYWV) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium. Mol. Microbiol. 45:3035–3045.
117. Spiro, S. 2007. Regulators of bacterial responses to nitric oxide. FEMS Microbiol. Rev. 31:193–211.[PubMed] [CrossRef]
118. Stewart, V. 1982. Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12. J. Bacteriol. 151:1320–1325.[PubMed]
119. Stewart, V. 1993. Nitrate regulation of anaerobic gene expression in Escherichia coli. Mol. Microbiol. 9:425–434.[PubMed] [CrossRef]
120. Stewart, V., Y. Lu, and A. J. Darwin. 2002. Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12. J. Bacteriol. 184:1314–1323.[PubMed] [CrossRef]
121. Studholm, D. J., and R. Dixon. 2003. Domain architectures of σ54-dependent transcriptional activators. J. Bacteriol. 185:1757–1767.[PubMed] [CrossRef]
122. Tucker, N. P., B. D’Autreaux, D. J. Studholm, S. Spiro, and R. Dixon. 2004. DNA binding activity of the Escherichia coli nitric oxide sensor NorR suggests a conserved target sequence in proteobacteria. J. Bacteriol. 186:6656–6660.[PubMed] [CrossRef]
123. Tyson, K., S. Busby, and J. Cole. 1997. Catabolite regulation of two Escherichia coli operons encoding nitrite reductase: role of the Cra protein. Arch. Microbiol. 168:240–244.[PubMed] [CrossRef]
124. Tyson, K. L., A. I. Bell, J. A. Cole, and S. Busby. 1993. Definition of the nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL. Mol. Microbiol. 7:151–157.[PubMed] [CrossRef]
125. Tyson, K. L., J. A. Cole, and S. J. W. Busby. 1994. Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase. Mol. Microbiol. 13:1045–1055.[PubMed] [CrossRef]
126. Ueda, K., A. Yamashita, J. Ishikawa, M. Shimada, T. O. Watsuji, K. Morimura, H. Ikeda, M. Hattori and T. Beppu. 2004. Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalisms. Nucleic Acids Res. 32:4937–4944.[PubMed] [CrossRef]
127. van Wonderen, J., B. Burlat, D. J. Richardson, M. R. Cheeseman, and J. N. Butt. 2008. The nitric oxide reductase activity of cytochrome c nitrite reductase from Escherichia coli. J. Biol. Chem. 283:9587–9594.[PubMed] [CrossRef]
128. Vergnes, A., J. Pommier, R. Toci, F. Blasco, G. Giordano, and A. Megalon. 2006. NarJ chaperone binds on two distinct sites of the aponitrate reductase of Escherichia coli to coordinate molybdenum cofactor insertion and assembly. J. Biol. Chem. 281:2170–2176.[PubMed] [CrossRef]
129. Wang, H., C. P. Tseng, and R. P. Gunsalus. 1999. The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite. J. Bacteriol. 181:5303–5308. [PubMed]
130. Wang, H., and R. P. Gunsalus. 2000. The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. J. Bacteriol. 182:5813–5822.[PubMed] [CrossRef]
131. Weiss, B. 2006. Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli. J. Bacteriol. 188:829–833.[PubMed] [CrossRef]
132. Williams, S. B., and V. Stewart. 1997. Discrimination between structurally related ligands nitrate and nitrite controls autokinase activity of the NarX transmembrane signal transducer of Escherichia coli K-12. Mol. Microbiol. 26:911–925.[PubMed] [CrossRef]
133. Wolfe, M. T., J. Heo, J. S. Garavelli, and P. W. Ludden. 2002. Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli. J. Bacteriol. 184:5898–5902.[PubMed] [CrossRef]
134. Wood, N. J., T. Alizadeh, D. J. Richardson, S. J. Ferguson, and J. W. Moir. 2002. Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus. Mol. Microbiol. 44:157–170.[PubMed] [CrossRef]
135. Wunsch, P., and W. G. Zumft. 2005. Functional domains of NosR, a novel transmembrane iron-sulfur flavoprotein necessary for nitrous oxide respiration. J. Bacteriol. 187:1992–2001.[PubMed] [CrossRef]
136. Zhang, X., and J. A. DeMoss. 1996. Structure modification induced in the narG promoter by binding of integration host factor and NARL-P. J. Bacteriol. 176:3971–3973.
137. Zopfi, J., T. Kjaer, L. P. Nielsen, and B. B. Jorgensen. 2001. Ecology of Thioploca spp.: nitrate and sulfur storage in relation to chemical microgradients and influence of Thioploca spp. on the sedimentary nitrogen cycle. Appl. Environ. Microbiol. 67:5530–5537.[PubMed] [CrossRef]
Module3.2.5
