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Bacterial respiration: a flexible process for a changing environment

School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK¹

Tel: +44 1603 593250. Fax: +44 1603 592250. e-mail: d.richardson{at}uea.ac.uk

Keywords: bacterial respiration, nitrate reductase, nitric oxide reductase, Fe(III) respiration, nitrogen cycle

1999 Fleming Lecture (Delivered at the 144th meeting of the Society for General Microbiology, 8 September 1999)

	Overview

TOP Overview Bacterial Fe(III) respiration Nitrate respiration and the... Denitrification, nitric oxide... Concluding remarks REFERENCES

 
The respiration of oxygen is fundamental to the life of higher animals and plants. The basic respiratory process in the mitochondria of these organisms involves the donation of electrons by low-redox-potential electron donors such as NADH. This is followed by electron transfer through a range of redox cofactors, bound to integral membrane or membrane-associated protein complexes. The process terminates in the reduction of the high-redox-potential electron acceptor, oxygen (Fig. 1). The free energy released during this electron-transfer process is used to drive the translocation of protons across the mitochondrial membrane to generate a trans-membrane proton electrochemical gradient or protonmotive force (p) that can drive the synthesis of ATP (Fig. 1). The respiratory flexibility of the mammalian mitochondrion is rather poor. There is some flexibility at the level of electron input (Fig. 1), but none at the level of electron output where cytochrome aa₃ oxidase provides the only means of oxygen reduction. In the case of plant mitochondria, a slightly greater degree of respiratory flexibility is encountered with a number of alternative NADH dehydrogenases and two oxidases being apparent. This respiratory flexibility affords plant mitochondria with the capacity to contribute to processes other than the generation of ATP. For example, electron transfer from the alternative NADH dehydrogenase to the alternative oxidase is not coupled to the generation of p and instead serves to release energy as heat, which can volatilize insect attractants to aid pollination. In the American skunk cabbage this same mechanism for heat production serves to permit growth at subzero temperatures (Nicholls & Ferguson, 1992 ). There is also some respiratory flexibility in the mitochondria of yeast, filamentous fungi and ancient protozoa, but it is amongst the Bacteria and Archaea that respiratory flexibility can be found at its most extreme. In these organisms, a diverse range of electron acceptors can be utilized including elemental sulphur and sulphur oxyanions (Hamilton, 1998 ), organic sulphoxides and sulphonates (Lie et al., 1999 ; McAlpine et al., 1998 ), nitrogen oxy-anions and nitrogen oxides (Berks et al., 1995 ), organic N-oxides (Czjzek et al., 1998 ), halogenated organics (Dolfing, 1990 ; Louie & Mohn, 1999 ; van de Pas et al., 1999 ), metalloid oxy-anions such as selenate and arsenate (Krafft & Macy, 1998 ; Macy et al., 1996 , 1993 ; Schroder et al., 1997 ), transition metals such as Fe(III) and Mn(IV) (Lovley, 1991 ), and radionuclides such as U(VI) (Lovley & Phillips, 1992 ) and Tc(VII) (Lloyd et al., 1999 ). This respiratory diversity can be found amongst pyschrophiles, mesophiles and hyperthermophiles and contributes to the ability of prokaryotes to colonize many of Earth’s most hostile micro-oxic and anoxic environments.

View larger version (19K): [in this window] [in a new window]   Fig. 1. A summary of the topology and bioenergetics of a basic aerobic respiratory electron transport system of a mammalian mitochondrion. UQ, ubiquinone; UQH2, ubiquinol; Cyt, cytochrome.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Respiratory flexibility in Paracoccus denitrificans (a) and Escherichia coli (b). MQ, menaquinone; UQ, ubiquinone; DMQ, demethylmenoquinone.

Amongst the Archaea, methanogens can utilize CO₂, acetate or methanol as electron acceptors, with H₂ as the electron donor, in the strictly anaerobic process ofmethanogenesis, which can be considered as a respiratory process as it is coupled to the generation of a H⁺ or Na⁺ electrochemical gradient. The complexity of methanogenesis is clearly apparent from analysis of the genomes of the strictly anaerobic hyperthermophile Methanococcus jannaschii (Bult et al., 1996 ) and moderate thermophile Methanobacterium thermoautotrophicum (Smith et al., 1997) and was reviewed recently by Thauer (1998) . The genome of the anaerobic hyperthermophilic sulphate respirer Archeoglobus fulgidus has also been sequenced and reveals a genetic potential for respiratory flexibility not yet recognized by biochemical studies and which might include the ability to utilize Fe(III), nitrate, DMSO, polysulphide, fumarate, heterodisulphides and oxygen as electron acceptors (Klenk et al., 1997 ). Another sulphate-reducing hyperthermophile, Pyrobacterium aerophilum, has been demonstrated to utilize nitrate or oxygen (at low partial pressures) as growth-supporting respiratory substrates (Volkl et al., 1993 ). Amongst the aerobic Archaea (e.g. Sulfolobus acidocaldarius), the study of oxygen respiration has been the focus of some attention, leading to the identification of haem–copper oxidases (HCOs) that are related to the HCOs of Bacteria. Indeed, analysis of protein sequences in current databases of Bacteria and Archaea reveal that a number of respiratory proteins are homologous in both domains of life. In addition to HCOs these include nitrate reductase, DMSO reductase, adenylylsulphate reductase, sulphite reductase and polysulphide reductase, and cytochrome bd oxidase. Thus, it seems likely that these respiratory pathways arose early in evolution and that the last common ancestor of living organisms was not a simple organism in its energetic metabolism and that its respiratory flexibility enabled it to proliferate under a range of environmental conditions (Castresana & Moreira, 1999 ).

One of the respiratory processes that probably evolved before the last common ancestor was denitrification, in which nitrate is reduced via nitrite, nitric oxide and nitrous oxide to dinitrogen (Berks et al., 1995 ). One of the enzymes involved in this process, nitrate reductase, binds a complex organic cofactor [the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor], which lies at the heart of a number of different respiratory enzymes (Kisker et al., 1997 ). Another two, nitric oxide reductase and nitrous oxide reductase, have together evolved into the cytochrome aa₃ oxidase that catalyses much of the oxygen respiration on earth today (Saraste, 1994 ; Saraste & Castresana, 1994 ; van der Oost et al., 1994 ; Watmough et al., 1999 ). The absence of photosynthetic reaction centres in Archaea, and the presence of oxidases of similar primary structure in both Bacteria and Archaea, has led to the ‘respiration-first’ hypothesis. It is argued that oxygen levels began to increase on early Earth before the onset of oxygenic photosynthesis (Castresana & Saraste, 1995 ; Schafer et al., 1996 ). Thus the original oxidases were high-affinity enzymes that evolved from the nitric oxide reductase. Some of these may still exist today in the guise of the high-affinity bacterial cytochrome cbb₃ oxidases (Preisig et al., 1993 ; Saraste & Castresana, 1994 ). The original role of these early HCOs and the unrelated high-affinity cytochrome bd oxidase may have been to protect oxygen-labile enzymes from destruction by oxygen. Such roles can be seen, for example in the respiratory protection of nitrogenase by cytochrome bd in Azotobacter vinelandii (Poole & Hill, 1997 ) and by cytochrome cbb₃ in Bradyrhizobium japonicum (Preisig et al., 1993 ). The lower-affinity cytochrome aa₃ oxidases may then have evolved subsequently in response to increasing oxygen levels in many environments.

A review detailing progress of study on all of the prokaryotic respiratory systems is beyond the scope of any article and in this paper only some of the key respiratory reactions currently under study in the author’s laboratory will be addressed, namely Fe(III) respiration, nitrate respiration and nitric oxide reductase. In doing so, attention will be paid to the flexible use of particular proteins or cofactors in a range of other respiratory reactions and also to flexible use of the respiratory process itself in functions other than the generation of p.

	Bacterial Fe(III) respiration

TOP Overview Bacterial Fe(III) respiration Nitrate respiration and the... Denitrification, nitric oxide... Concluding remarks REFERENCES

 
Fe(III) reduction: an ancient respiratory process
It is generally agreed that the first respiratory processes to evolve in the hyperthermal reducing environment of early Earth, over 3·5 billion years ago, would have utilized either Fe(III) or S(0) as electron acceptors. It has recently been argued that Fe(III) respiration preceded sulphur respiration (Vargas et al., 1998 ) since Fe(III), derived from photochemical oxidation of Fe(II) from Archean seas and hydrothermal-vent fluids, was abundant on early earth and Fe(III) respiration has been demonstrated in a number of deep-branching hyperthermophilic Archaea and Eubacteria, many of which do not respire S(0) (Vargas et al., 1998 ). Intriguingly, it has been pointed out that extracellular magnetite accumulations characteristic of Fe(III) respiring prokaryotes are associated with some of the putative fossilized microbes found in ancient Martian meteorites (McKay et al., 1996 ; Vargas et al., 1998 ).

An early importance for Fe(III) respiration in microbial life is also attractive because it would not have required the evolution of complex cofactors and redox proteins. The respiratory reduction of Fe(III) and other transition metals is largely dependent on a sufficient thermodynamic driving force and an appropriate long-range electron-transfer system. This need not require specific enzymes, indeed it is unlikely that there are specific iron reductases operating in any of the hyperthermophiles recently reported as Fe(III)-respiring bacteria (Vargas et al., 1998 ). A simple early respiratory process for Fe(III) is likely to have utilized hydrogen as an electron donor with electrons being extracted via an extra-membranous hydrogenase. The scalar protons produced from hydrogen oxidation would contribute to the generation of a proton gradient across the membrane. In this context, it is also notable that the hydrogenase (Hyc) of the formate hydrogen lyase system of Escherichia coli has been reported to provide the organism with the capacity for technetium reduction (Lloyd et al., 1997 ). Hydrogen-driven Fe(III) respiration in hyperthermophiles may account for observations of magnetite accumulation at depths of 6·7 km in the Earth’s core (Gold, 1992 ; Lovley, 1997 ).

Fe(III) respiration in mesophilic Gram-negative bacteria
Although Fe(III) respiration may have originally evolved in hydrothermal environments on early Earth and can be identified in a number of hyperthermophilic Archaea, the process is also known to be widespread amongst a number of branches of the Bacteria (Lonergan et al., 1996 ) and contributes significantly to the biogeochemical development of a number of mesophilic anoxic environments (Lovley, 1991 ). Indeed, the activity of Fe(III)-respiring bacteria can have an impact on the populations of both sulphate- and nitrate-reducing bacteria in these environments, and hence impact on the nitrogen and sulphur cycles in general (Lovley, 1991 ). The activity of Fe(III)-respiring bacteria can be detrimental, for example in the corrosion of deep-sea oil pipes, but it can also potentially be harnessed for the bioremediation of polluted anoxic subsurface zones (Lovley et al., 1994 ). The major problem of utilizing Fe(III) as a respiratory substrate is the insolubility of the cation at circum-neutral pH. The soluble Fe^III(H₂O)₆ species only exists at pH <2 and the use of Fe(III) as an electron acceptor by sulphur-oxidizing acidophiles (e.g. Thiobacillus ferrooxidans) has been reported (Sugio et al., 1992 ). However, as the pH increases, deprotonation and dehydration events lead to the formation of complex oxo/hydroxo bridged precipitates (e.g. rust). An Fe(III)-respiring Gram-negative bacterium then faces the problem of moving electrons generated from carbon metabolism in the cytoplasm across two cell membranes and the intervening periplasm to the site of reduction of the insoluble extracellular species.

In the case of the members of the Fe(III)-respiring genus Shewanella, it is emerging that this problem may be solved by using a number of tetra-haem and deca-haem c-type cytochromes to form a multi-haem electron ‘wire’ between the inner and the outer membranes (Beliaev & Saffarini, 1998 ; Field et al., 2000 ; Myers & Myers, 1997a ,b , 1998 ) (Fig. 3a). Accordingly, when cells of Shewanella frigidimarina are grown under anaerobic conditions with Fe(III) present as the electron acceptor, the expression of cytochromes greatly increases compared to cells grown anaerobically with fumarate present as the electron acceptor (Dobbin et al., 1999 ), and addition of Fe(III) complexes to anaerobically incubated cells results in the ‘drain’ of electrons from the total cytochrome pool (Dobbin et al., 1995 , 1996a ). Some of the multi-haem cytochromes purified from Shewanella frigidimarina grown with Fe(III) include a 20 kDa tetra-haem membrane-anchored quinol dehydrogenase (CymA), a 35 kDa periplasmic deca-haem cytochrome (Pcc35), a 60 kDa iron-induced flavocytochrome c₃ (Ifc₃) and an outer-membrane 80 kDa deca-haem lipoprotein (OmcA) (Dobbin et al., 1999 ; Field et al., 2000 ; P. S. Dobbin, S. J. Field, M. Ellington & D. J. Richardson, unpublished) (Fig. 3a). It is becoming apparent that the genes for many of these cytochromes are present in multiple copies on the Shewanella putrefaciens genome, for example one recently characterized gene cluster (GenBank accession no. AF083240) contains three outer-membrane deca-haem lipoproteins and two periplasmic deca-haem proteins (Beliaev & Saffarini, 1998 ). This increased gene dosage presumably enables the bacteria to drive the high levels of cytochrome expression required to facilitate rapid reduction of the surrounding insoluble Fe(III) species.

View larger version (67K): [in this window] [in a new window]   Fig. 3. Metal ion reduction by Gram-negative bacteria. (a) A hypothetical scheme showing the involvement of inner-membrane, periplasmic and outer-membrane multi-haem cytochromes in Fe(III) respiration by Shewanella frigidimarina. (b) A hypothetical electron-transfer system for soluble metal ion reduction and sulphite reduction in sulphate-reducing bacteria involving multi-haem cytochromes of the cytochrome c3 family. HmcA, B, C, E and F are the products of orfs 1, 2, 3, 5 and 6 of the hmc operon. The model is based on the data of Rossi et al. (1993) and the arguments of Berks et al. (1995) . Haem groups are represented by black ovals. OM, outer membrane; IM, inner membrane.

In the model for Fe(III) respiration shown in Fig. 3a, the reduction of Fe(III) is probably via non-specific long-range electron transfer. The multi-haem cytochromes themselves are not specific Fe(III) reductases. Indeed the soluble periplasmic multi-haem cytochromes have Fe(III) reductase activity but are not physiological reductases for the insoluble species because of their cellular location (Dobbin et al., 1999 ). However, adding Fe(III) chelators such as nitriloacetic acid can accelerate the rate of Fe(III) reduction, which may be because the soluble species can enter the periplasm where they can be reduced by the large pool of low-potential periplasmic cytochromes (Dobbin et al., 1995 , 1996a , b , 1999 ). Furthermore, the non-specific nature of the multi-haem Fe(III) reductase system makes it suitable for reduction of a range of extracellular substrates, such as Mn(IV) and insoluble sulphur species (Beliaev & Saffarini, 1998 ; Lovley, 1991 ).

Diverse respiratory functions of periplasmic multi-haem c-type cytochromes
Consideration of the multi-haem cytochromes involved in Fe(III) respiration in conjunction with recent structural studies on multi-haem cytochromes is beginning to give insight into the evolution of these proteins. Sequence analysis of the deca-haem cytochromes involved in Fe(III) respiration suggests that they are composed of two penta-haem units which show some similarity to the 16 kDa penta-haem NrfB protein of some enteric bacteria (Beliaev & Saffarini, 1998 ; Hussein et al., 1994 ). NrfB is involved in electron transfer to the 50 kDa NrfA protein which is a nitrite reductase and which also has a penta-haem core (Darwin et al., 1993 ; Einsle et al., 1999 ) (Fig. 4). Recent structural analysis increases the members of this multi-haem protein family further to include some tetra-haem and octa-haem proteins. Fig. 4 shows the haem arrangement of four such proteins. One of these is the Fe(III)-induced periplasmic flavocytochrome c₃, which is an isozyme of a soluble fumarate reductase (Fcc₃) also expressed by the Shewanella species and for which structures are also known (Bamford et al., 1999 ; Taylor et al., 1999 ; Leys et al., 1999 ). The tetra-haem arrangement of the haems in this protein includes an intriguing haem–haem pair in which the haem-irons are only 9  apart and the closest haem edges are only 4  apart. Inspection of the octa-haem arrangement in the hydroxylamine oxidoreductase (HAO) (Igarashi et al., 1997 ) reveals that the four Ifc₃/Fcc₃ haems can be superimposed onto four of the HAO haems. The four Ifc₃ haems also have a similar arrangement to four of the haems from the penta-haem NrfA-type nitrite reductase (Einsle et al., 1999 ) and all five haems from this enzyme overlay on five of the HAO haems. Finally two of the haems from the Ifc₃ overlay onto two of the four haems of cytochrome c-554 (Iverson et al., 1998 ), from which all four haems will superimpose on the HAO (Fig. 4). Despite this conservation of haem organization, little similarity can be seen between these four proteins at the primary-structure level. Nevertheless, this analysis suggests that all four multi-haem cytochromes share a common evolutionary origin but have diverged for use in four distinct periplasmic electron-transfer processes: Fe(III) reduction, fumarate reduction (Fig. 3a), hydroxylamine oxidation (Fig. 5) and nitrite reduction to ammonia (Fig. 5). The conservation of the haem arrangements must reflect the importance of haem–haem angles and distances in these periplasmic electron-transfer processes. Given the non-enzymic nature of long-range electron transfer, it seems likely that these multi-haem cytochromes evolved for functions such as protein–protein electron transfer and non-specific reduction of respiratory substrates, and later evolved other catalytic activities through the evolution of the polypeptide chain yielding specialized active sites around a catalytic haem.

View larger version (25K): [in this window] [in a new window]   Fig. 4. A comparison of the organization of the haem groups in the multi-haem cytochromes of the HAO/Nrf family, cytochrome c3 and the photosynthetic reaction centre tetra-haem cytochrome c. The sources of the proteins are: Ifc3, Shewanella frigidimarina; HAO, Nitrosomonas europaea; Nrf, Sulfurospirillum deleyianum; Cyt c-554, Nitrosomonas europaea; Cyt c3, Desulfovibrio gigas (Simoes et al., 1998 ); and photosynthetic reaction centre tetra-haem cyt c, Rhodopseudomonas viridis

View larger version (58K): [in this window] [in a new window]   Fig. 5. Model schemes of the electron-transport systems in which the multi-haem cytochromes of the HAO/Nrf family participate. (a) The scheme for HAO represents a possible electron-transport system for ammonia oxidation in Nitrosomonas europaea. (b).The scheme for Nrf represents a possible electron-transport system for periplasmic nitrate reduction to ammonium in Escherichia coli. Haem groups are represented by black ovals. P, periplasm; C, cytoplasm.

The inter-membrane electron-transfer system that operates during Fe(III) respiration in Shewanella species requires a means of extracting electrons from membrane-entrapped quinol and directing them into the periplasmic compartment. This is true for a number of bacterial respiratory systems that involve periplasmic oxido-reductases and one solution to the problem seems to have been the evolution of the NapC family of membrane-anchored tetra/penta-haem cytochromes (Roldan et al., 1998 ). Members of this family have been implicated in mediating electron transfer from quinols to a range of oxido-reductases that include the nitrate reductase (NapC) (Roldan et al., 1998 ), the periplasmic DMSO reductase (DorC) (Shaw et al., 1999 ), the TMAO reductase (TorC) and some cytochrome cd₁ nitrite reductases (NirT) (Jungst et al., 1991 ). In the case of CycB (Bergmann et al., 1994 ), they may also serve to transfer electrons into the ubiquinone (UQ) pool during nitrification by Nitrosomonas europaea (Fig. 5a). The CymA protein involved in Fe(III) respiration in Shewanella putrefaciens is also a member of this family and additionally serves to transfer electrons to the periplasmic fumarate reductase and the periplasmic nitrate reductase of this organism (Myers & Myers, 1997a ). This demonstrates a promiscuous role for this multi-haem quinol dehydrogenase that contributes to the respiratory flexibility of the organism. The biology of one of its redox partners, the periplasmic nitrate reductase, will be discussed below.

	Nitrate respiration and the bis-MGD enzymes

TOP Overview Bacterial Fe(III) respiration Nitrate respiration and the... Denitrification, nitric oxide... Concluding remarks REFERENCES

 
Nitrate is an important component of the biological nitrogen cycle (Fig. 6). It serves as the substrate for the denitrification process in which nitrate is reduced via nitrite, nitric oxide and nitrous oxide to dinitrogen. Each reaction is catalysed by an enzyme that is coupled to energy-conserving electron-transport pathways. The process as a whole is important in agriculture where it results in the loss of nitrate fertilizers from fields and in waste-treatment processes where nitrate must be removed from waste waters before release into the environment. Nitrate is also an end product of the nitrification process whereby ammonia is oxidized, via hydroxylamine and nitrite, to nitrate by the combined action of species such as Nitrosomonas europaea ( oxidizer) and Nitrobacter vulgaris ( oxidizer). Nitrate can also be reduced via nitrite to ammonium by some enteric and sulphate-reducing bacteria in a respiratory process (Berks et al., 1995a ; Moura et al., 1997 ). The same series of reactions, though catalysed by distinct enzymes, can also occur as part of nitrogen assimilation into cellular biomass in ammonium-limited environments.

View larger version (28K): [in this window] [in a new window]   Fig. 6. The biological nitrogen cycle. Figures in parentheses denote the oxidation state of the nitrogen. ANAMMOX, anaerobic ammonium oxidation.

View larger version (41K): [in this window] [in a new window]   Fig. 7. The catalytic centre of the nitrate reductases. (a) The bis-MGD cofactor. (b) A possible catalytic cycle for NAP of Paracoccus pantotrophus. (c) An alignment of the segment 3 region of some members of the bis-MGD family. PpNapA, Paracoccus pantotrophus NAP; EcNapA, Escherichia coli NAP; HiNapA, Haemophilus influenzae NAP; ReNapA, Ralstonia eutropha NAP; SynNarB, Synechocystis NAS; BsNarB, Bacillus subtilis NAS; KpNasA, Klebsiella pneumoniae NAS; SpNasA, Shewanella putrefaciens NAS; EcFdhF, Escherichia coli formate dehydrogenase F, MfFdh, Methanobacterium formicicum formate dehydrogenase; MtFdhA, Methanobacterium thermoautotrophicum formate dehydrogenase; EcFdoG, Escherichia coli formate dehydrogenase O; RsDmsA, Rhodobacter sphaeroides DMSO reductase; RcDorA, Rhodobacter capsulatus DMSO reductase; EcBisA, Escherichia coli biotinsulphoxide reductase; EcTorA, Escherichia coli TMAO reductase; HiDmsA, Haemophilus influenzae DMSO reductase; EcNarG, Escherichia coli membrane-bound nitrate reductase A; PpNarG, Paracoccus pantotrophus NAR; PfNarG, Pseudomonas fluorescens NAR; ScNarG, Staphylococcus carnosus NAR; BsNarG, Bacillus subtilis NAR A; TtNarG, Thermus thermophillus NAR. The asterix indicates proven or speculative Mo-coordinating residues.

Almost all the characterization of bis-MGD enzymes has been carried out in Bacteria. However, nitrate-reducing Archaea are known (Volkl et al., 1993 ) and putative bis-MGD-binding enzymes can be identified from primary-structure analysis of ORFs in the genome sequence of Archaeoglobus fulgidus which suggest the presence of nitrate, DMSO and polysulphide reductases. It should be noted that no biochemical data are available on these enzymes and the possibility that they bind tungsten rather than molybdenum at the active site cannot be excluded. Indeed, the inclusion of tungstate in the growth medium of Pyrobacterium aerophilum stimulates anoxic growth with nitrate (Volkl et al., 1993 ) and active W-substituted TMAO reductase from Escherichia coli has been reported (Buc et al., 1999 ). However, the presence of putative bis-M(W)GD enzymes in Archaea suggests an early evolution for these respiratory reactions, before the last universal ancestor.

The structures of some of the catalytic subunits of members of the bis-MGD family have emerged in recent years from the formate dehydrogenase (Boyington et al., 1997 ), DMSO reductase (McAlpine et al., 1998 ; Schindelin et al., 1996 ), TMAO reductase (Czjzek et al., 1999) and a periplasmic nitrate reductase from the suphate-reducing bacterium Desulfovibrio desulfuromonas (Dias et al., 1999 ). All the subunits show a high degree of similarity in the structural organization with the bis-MGD cofactor lying at the bottom of a deep substrate cleft. However, there is some debate over the number of oxo groups bound to the Mo in these enzymes. Crystal structures and spectroscopic studies indicate one oxo group in the Mo(VI) form of Rhodobacter sphaeroides DMSO reductase (Schindelin et al., 1996 ) and two in the Mo(VI) form of Rhodobacter capsulatus DMSO reductase (McAlpine et al., 1998 ). Similarly with nitrate reductases, EXAFS (Extended X-ray Absorption Fine Structure) studies on Paracoccus denitrificans periplasmic nitrate reductase indicate a di-oxo Mo(VI) state (Fig. 7b) (Butler et al., 1999 ) and crystal-structure studies on Desulfovibrio desulfuricans periplasmic nitrate reductase suggest a mono-oxo Mo(VI) state (Dias et al., 1999 ). Studies with model oxo-molybdenum complexes have demonstrated that some can exhibit a broad specificity of oxo-transferase chemistry. Thus, for example, some can exhibit both nitrate reductase and DMSO reductase activity (Craig & Holm, 1989 ). This contrasts to the bis-MGD enzymes where a nitrate reductase does not exhibit DMSO reductase activity and a DMSO reductase does not exhibit nitrate reductase activity. Clearly the evolution of the polypeptide around the bis-MGD cofactor has played a major role in conferring catalytic selectivity and this is reflected in differences in the amino acids that line the substrate cleft and that lie in the catalytic pocket. In the case of DMSO reductase, mutation of the Mo-coordinating serine residues has altered catalytic specificity (Hilton et al., 1999 ).

The multiple nitrate reductases of Paracoccus species
Perhaps not surprisingly, given the multiple roles for nitrate in bacteria, it has emerged that many bacteria can express multiple biochemically distinct nitrate reductases. For example, Paracoccus denitrificans and Paracoccus pantotrophus have three nitrate reductases (Sears et al., 1997 ). One of these, NAS (cytoplasmic assimilatory nitrate reductase), is located in the cytoplasmic compartment, is ammonium repressible and participates in nitrogen assimilation. The other two, however, are both linked to respiratory electron-transport systems, each ultimately taking electrons from the quinol pool (Fig. 8b). One of the enzymes (NAR, membrane-bound nitrate reductase) is a three-subunit complex anchored to the cytoplasmic face of the membrane with its active site located in the cytoplasmic compartment. The other (NAP, periplasmic nitrate reductase) is a two-subunit enzyme located in the periplasmic compartment that is coupled to quinol oxidation via a membrane-anchored tetra-haem cytochrome of the NapC quinol dehydrogenase family, discussed earlier in the context of Fe(III) respiration (Berks et al., 1995a , c ; Roldan et al., 1998 ; Richardson & Watmough, 1999 ).

View larger version (27K): [in this window] [in a new window]   Fig. 8. (a) The topological organization of some bis-MGD enzymes. FdhN, Escherichia coli formate dehydrogenase N; Dor, Rhodobacter capsulatus periplasmic DMSO reductase; Ttr, Salmonella typhimurium tetrathionate reductase. (b) The nitrate reductases of Paracoccus species. NarGHI, the membrane-bound nitrate reductase; NapABC, the periplasmic nitrate reductase. P, periplasm; C, cytoplasm.

Different physiological roles for the membrane-bound and periplasmic nitrate reductases in Paracoccus species
An early question that emerged, on discovering two respiratory nitrate reductases in a single organism, was what are their physiological roles? Studies on enzyme expression revealed that NAR was predominantly expressed under anaerobic denitrifying growth conditions, whilst NAP was predominantly expressed under aerobic growth conditions (Bell et al., 1990 ). Consideration of the bioenergetic properties of each system offers a physiological rationale for this. In the case of NAR, quinol is oxidized at the periplasmic face of the cytoplasmic membrane by the NarI subunit (Fig. 8b). Protons are ejected into the periplasm whilst the electrons flow back across the membrane via the two stacked NarI haems (Rothery et al., 1999 ). They then pass, via multiple NarH iron–sulphur centres (Magalon et al., 1998 ), to the cytoplasmic NarG bis-MGD cofactor where nitrate is reduced to nitrite with the associated consumption of two protons (Fig. 7b). This electron-transfer process represents a classic Mitchellian electrogenic redox loop and ensures that the free energy in the QH₂/ redox couple is conserved as p (Berks et al., 1995b ). The role of an integral di-b-haem cytochrome in these sort of redox loops is widespread in bacterial electron-transfer systems (Berks et al., 1995b ) (see, for example, the formate dehydrogenase depicted in Fig. 5b) and these simple loops may have evolved before the more complex proton-motive Q cycle of the di-haem cytochrome bc₁ complex (Nicholls & Ferguson, 1992 ).

In NAP, quinol is also oxidized at the periplasmic face of the cytoplasmic membrane by NapC, but the electrons also flow into the periplasm where they ultimately reduce nitrate to nitrite. Thus, by contrast to the membrane-bound nitrate reductase, the free energy in the QH₂/ redox couple is not conserved as p and is therefore dissipated (Fig. 8b). In physiological terms, it makes bioenergetic sense to express the energy-coupled NAR system under anaerobic conditions when the organism is dependent on nitrate reduction for energy conservation. The expression of an energy-dissipating system under aerobic conditions immediately raises the possibility of a role for NAP in redox balancing. During chemoheterotrophic growth on reduced carbon sources, the carbon substrate must be oxidized to the level at which it can be assimilated. If this oxidation results in the release of more reductant than is needed for the generation of the ATP required for the metabolism of the carbon then a means of disposing of the excess reductant must be available. If it is not, the growth rate will be slowed to that allowed by the reoxidation of NADH in cell-maintenance reactions. One means of disposing of the excess reductant is via the respiratory electron-transport pathways. However, efficiently coupled pathways (e.g. the cytochrome bc₁ complex/cytochrome-oxidase-dependent pathways) will not turn over at high rates in the presence of a large steady-state p. Consequently, the maximum rate of growth-substrate utilization is only possible if electrons are disposed of by relatively uncoupled pathways, such as that provided by NAP.

The need to dissipate reductant is likely to be most acute during the metabolism of a reduced carbon substrate under conditions that are both oxygen and energy sufficient (Sears et al., 1997 ). Laboratory cultures of denitrifying bacteria are routinely grown on relatively oxidized carbon substrates such as malate or succinate. However, higher levels of intracellular reductant may be generated through the oxidation of more reduced substrates, such as fatty acids, to carbon intermediates suitable for biosynthesis and assimilation. Accordingly, expression of the periplasmic nitrate reductase is 10–40-fold higher following growth of Paracoccus species aerobically with butyrate or caproate compared to malate or succinate (Richardson & Ferguson, 1992 ; Sears et al., 1993 ). Futhermore, analysis of steady-state cultures of Paracoccus denitrificans in butyrate-limited and malate-limited chemostat cultures revealed that aerobic nitrate respiration was only significant in the butyrate-limited cultures. The malate-limited cultures were carbon limited and energy limited, whilst the butyrate-limited cultures were carbon limited but energy sufficient with the excess reductant dissipated through the NAP system (Sears et al., 1997 ).

A model for the integrated aerobic respiratory and NAP electron-transport systems of Paracoccus species is shown in Fig. 9a. Unregulated electron flux through the NAP system would be detrimental to the cell as it would result in an unnecessary wastage of redox energy. A major factor that is likely to influence the destination of electrons entering the respiratory chain from low-potential electron donors is the redox state of the QH₂/Q pool, since this is a redox component that is common to both the oxygen- and nitrate-respiratory pathways. Cellular overreduction will ultimately result in overreduction of the QH₂/Q pool as a consequence of the high NADH/NAD⁺ ratio driving turn over of the Q-dependent NADH dehydrogenase, even in the presence of a high p. The resulting increase in the QH₂/Q ratio could ultimately limit turn over of the cytochrome bc₁ complex, since this requires Q as well as QH₂ as substrate (Fig. 9b). The redox components that comprise the NAP electron-transport system have rather low redox potentials and the system depends only on QH₂ (Fig. 9b). Thus electron flow through the NAP system may be slow when the QH₂/Q ratio is low (high E_h) and cytochrome bc₁-dependent electron transport is favoured. At high QH₂/Q ratios, turn over of the cytochrome bc₁ complex becomes limited by Q availability and there is a stronger thermodynamic driving force for electron transport through the NAP system. Reoxidation of QH₂ via NAP will serve to repoise the Q pool, lowering the QH₂/Q ratio so that electron flux switches back to the more highly coupled cytochrome bc₁-dependent oxidase system.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Aerobic nitrate respiration in Paracoccus pantrophus. (a) Scheme for electron transport to oxygen and nitrate during aerobic metabolism of a reduced carbon substrate. The boxed chemical reactions show oxidation of butyryl CoA. ETF, electron-transfer flavoprotein. (b) The midpoint redox potentials of some of the electron-transfer components detailed in (a). White boxes denote the periplasmic nitrate reductase system. Black boxes denote the cytochrome bc1 complex-dependent oxidase system. For details of the NAP subunits, see Fig. 8b.

View larger version (29K): [in this window] [in a new window]   Fig. 10. Generalized electron-transport systems of Rhodobacter species. (a) A scheme for cyclic photosynthetic electron transport. (b) Routes for electron input and output into the Q pool. It should be noted that some Rhodobacter species and strains within a species are deficient in some of the oxido-reductases indicated. For example, Rhodobacter capsulatus does not have a cytochrome aa3 oxidase, but Rhodobacter sphaeroides does, and not all strains of Rhodobacter capsulatus have a nitrate reductase. If a strain has a nitrate reductase it is usually periplasmic (NAP) but some have the membrane-bound (NAR) type; some strains of Rhodobacter sphaeroides may have both NAP and NAR.

NAP and photo-nitrate respiration
Members of the Rhodospirallaceae family of -proteobacteria, which includes Rhodobacter species, possess a cyclic photosynthetic electron-transport system. This cyclic system relies on a single reaction centre, the cytochrome bc₁ complex, the QH₂/Q pool and one or more c-type cytochromes that mediate electron transfer between the two integral membrane complexes (Fig. 10). Under illuminated conditions the photosynthetic reaction centre will only turn over if there is a supply of oxidized Q as electron acceptor, whilst the protonmotive Q cycle of the cytochrome bc₁ complex requires the provision of both Q and QH₂ (Fig. 10a). Consequently, the cyclic electron-transport system is critically dependent on the QH₂/Q ratio in the Q pool. However, the cyclic electron-transport system is not a closed system, there are a number of routes for electron input during photoheterotrophic metabolism (Fig. 10). Thus the UQ/UQH₂ (E⁰'=+80 mV) pool can be coupled to low-potential electron donors such as NADH (E⁰' NADH/NAD⁺=-330 mV). This could lead to extensive reduction of the UQH₂/UQ pool, restricting the rate of cyclic electron transport. Many strains of Rhodobacter capsulatus and Rhodobacter sphaeroides have the ability to express a NAP and it has become clear that nitrate reduction can serve to repoise the cyclic electron-transport system when it has become perturbed by overreduction (Ferguson et al., 1987 ; Jones et al., 1990 ). This may be particularly important during photoheterotrophic growth of Rhodobacter capsulatus on reduced carbon substrates, such as butyrate (Richardson et al., 1988 ). Many species of the family Rhodospirallaceae are only able to photometabolize butyrate anaerobically in the presence of carbon dioxide. Excess reductant generated through the oxidative photometabolism of butyrate can be consumed by the reductive fixation of CO₂, resulting in extensive deposition of poly-3-hydroxybutyrate. However, in the absence of CO₂, photoheterotrophic growth of Rhodobacter capsulatus on butyrate can be facilitated through the reduction of nitrate (Richardson et al., 1988 ). Under energy rich conditions, in the presence of both CO₂ and nitrate, reductive CO₂ fixation is the selected mechanism by which NAD⁺ is regenerated, presumably because it conserves carbon. However, CO₂ fixation also consumes ATP and the choice of mechanism may be different under energy-limited (i.e. light limited) conditions. In order for nitrate reduction to effectively consume excess reductant, it may be advantageous if the transfer of electrons from ubiquinol to the reductases can continue in the presence of a substantial light-dependent p. Since oxidation of ubiquinol by nitrate, via NAP, is not thought to be coupled to the generation of p it will not be subject to thermodynamic back-pressure mediated by light-dependent p.

During steady-state cyclic electron-transfer on oxidized carbon substrates, thermodynamic back-pressure of the light-dependent p on the protonmotive NADH dehydrogenase is a major factor that prevents overreduction of the cyclic electron-transport chain. Thus at high light intensities nitrate respiration by bacteria utilizing malate (and therefore predominantly NADH) as the electron donor is sensitive to inhibition by the light-dependent p (which may be around 200 mV). However, intermittent periods of darkness or prolonged periods of low light intensity could result in the redox poise of the photosynthetic electron-transport system becoming disturbed even during photometabolism of such a relatively oxidized carbon substrate. Under these conditions, the light-dependent protonmotive force could collapse to as low as +50 mV, leading to electron flux into the Q pool from the NADH pool. The effect of prolonged periods of anaerobic dark incubation, in malate medium, on the redox poise of the electron-transfer system of intact cells of Rhodobacter capsulatus has been examined and has indicated that the Q/QH₂ pool was extensively reduced, restricting turn over of the reaction centre (Jones et al., 1990 ). This problem was relieved if nitrate was present during the dark incubation period since NAP, by drawing electrons from the UQ/UQH₂ pool, was serving to maintain the optimal redox poise of the cyclic electron-transfer pathway during the period of darkness (Jones et al., 1990 ). The light intensity on sediment surfaces is probably rarely as high as routinely used for growth of photosynthetic organisms in laboratory cultures. At low light intensities (and therefore low-light-dependent p), the reduction of nitrate, during growth of Rhodobacter capsulatus on oxidized carbon substrates such as malate and succinate, is more extensive than at high light intensities. This may be related to the electron-transfer system being more susceptible to overreduction under these conditions (Richardson et al., 1988 ).

An extreme example of the use of nitrate reduction as a means of regulating the redox poise of the cyclic photosynthetic electron-transport chain of purple non-sulphur photosynthetic bacteria can be found in Roseobacter denitrificans which can not grow photosynthetically under anaerobic conditions unless auxilliary oxidants, such as nitrate, are present. The reaction-centre complex of Roseobacter denitrificans is very similar to that of the photosynthetically competent Rhodobacter species, with the exception that the redox potential of the primary acceptor quinone (Q_A) is higher (E⁰'=+35 mV compared to -20 mV in Rhodobacter sphaeroides). Thus the QH₂/Q pool has to be maintained in a very oxidized state (i.e. at a high redox potential) in order for the reaction centre to turn over and this can be facilitated by the reduction of nitrate (Takamiya, 1988 ).

Nitrate reductase is not the only accessory oxidant available to photosynthetic bacteria during photoheterotrophic metabolism. The respiratory flexibility of these bacteria is such that DMSO, TMAO, , N₂O, (Ferguson et al., 1987 ), NO (Bell et al., 1992 ) and Fe(III) (Dobbin et al., 1996a ) can all be used as respiratory substrates and many of these have been demonstrated to serve redox-poising roles (Jones et al., 1990 ; McEwan et al., 1985 ; Richardson et al., 1988 ; Takamiya et al., 1988 ). Futhermore, in strains of Rhodobacter sphaeroides in which the CO₂-fixation pathway has been inactivated, spontaneous mutants can arise which have deregulated nitrogenase so that reductive fixation of nitrogen can serve to facilitate cellular redox balance (Qian & Tabita, 1998 ).

NAP and nitrate scavenging
Escherichia coli can, in principle, express either a NAR or a NAP type of nitrate reductase under anaerobic growth conditions. The q⁺/e^- (ratio of the charges translocated across the membrane per electron transferred through the respiratory system) for nitrate reduction by NAR would be 6 or 4 with NADH or formate as electron donor, but only 4 (NADH) and 2 (formate) when NAP is used to reduce nitrate. Given that NAR is more highly coupled than NAP, the question then arises as to when the NAP system is expressed and physiologically important. This has been addressed in recent competition experiments in continuous cultures where a strain expressing only NAR has been placed in competition with a strain expressing only NAP. Under nitrate-limited conditions the strain expressing NAR is out-competed, but the situation is reversed under carbon-limited conditions where the strain expressing NAP is out-competed (Potter et al., 1999 ). This may reflect a low K_s (higher affinity) for intact cells for nitrate when the NAP system is expressed. Thus NAP may be important in scavenging nitrate from nitrate-limited environments and consequently under these conditions coupling efficiency is sacrificed in favour of substrate affinity. Expression studies of Nap in Escherichia coli support this view point since the nap operon is induced at low nitrate concentrations but repressed at higher nitrate concentrations which induce the narG operon (Wang et al., 1999 ). In this context, it becomes significant that many of the pathogenic bacteria that may have to scavenge nitrate from the low levels present in many bodily fluids have the genetic information for NAP. Indeed, in some of these (e.g. Haemophilus influenzae) NAP is the only nitrate reductase present. It should be noted that consideration of the nap gene clusters of enteric bacteria reveals some heterogeneity in their composition. For example, in addition to the napAB genes that encode the NAP enzyme, the nap gene cluster of Escherichia coli contains additional genes (napFGH) that are predicted to encode membrane-associated iron–sulphur clusters (NapFGH; Fig. 5b). These are not essential for electron transport to NAP (Potter & Cole, 1999 ) and are absent in the nap cluster of Paracoccus denitrificans which instead has a gene encoding a small single-helix transmembrane protein, NapE. It is however notable that there are genes encoding structural homologues of NapFGH elsewhere on the Paracoccus denitrificans chromosome (see Berks et al., 1995a for a detailed discussion of the NapFGH protein fa milies).

The periplasmic reduction of nitrate to ammonium via the NAP and Nrf systems can also support anaerobic growth in some sulphate-reducing bacteria, for example Desulfovibrio desulfuricans and Sulfurospirillum deleyianum, studies on which have recently provided X-ray crystal structures of both enzymes (Dias et al., 1999 ; Einsle et al., 1999 ). These species thus have a rather flexible respiratory metabolism, enabling them to grow as either sulphate reducers or nitrate reducers (Moura et al., 1997 ) and they can express multi-haem cytochromes of both the HAO/Nrf family (Fig. 5) and the cytochrome c₃ family to facilitate this (Fig. 3b).

NAP and anaerobic denitrification
In many of the best characterized denitrification systems NAR catalyses the first stage of anaerobic nitrate reduction to nitrite (e.g. Pseudomonas stutzeri and Paracoccus species). In Paracoccus pantotrophus a mutation in the structural genes of the nar operon led to the generation of a mutant strain that could de-repress NAP under anaerobic conditions. Consequently it could still grow under anaerobic denitrifying conditions using NAP, rather than NAR, in the first step (Bell et al., 1993). The strain did, however, have a lower specific growth rate and growth yield. Recently, it has become apparent that some Rhizobium species (e.g. G179) can express a NAP and that disruption of the nap genes is lethal for growth under denitrifying conditions (Bedzyk et al., 1999 ). Also, in Rhodobacter sphaeroides f. sp. denitrificans, which can express both NAR and NAP, mutation of NAP is lethal for anaerobic denitrification (Liu et al., 1999 ). Thus, in these organisms one of the physiological roles of NAP is in anaerobic denitrification. When considered in isolation, the energy coupling of NAR and NAP appear markedly different: q⁺/2e^-=6 (NAR) and 4 (NAP) with NADH as electron donor and 2 (NAR) and 0 (NAP) with succinate as electron donor. However, when considered in the context of the entire denitrification pathway the q⁺/2e^- ratio is 24 (NAR) or 22 (NAP) with NADH and 8 (NAR) or 6 (NAP) with succinate. Thus the energetic loss of using NAP rather than NAR is only 8% when NADH is the electron donor to the respiratory system.

	Denitrification, nitric oxide reductase and the evolution of oxygen respiration

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In many bacteria, the nitrite generated from nitrate respiration can be further reduced in the reactions of denitrification (Berks et al., 1995a ; Zumft, 1997 ) (Fig. 6). In Paracoccus species this proceeds via a periplasmic nitrite reductase containing c and d₁ haems, an integral membrane NO reductase complex and a periplasmic copper-