Download Look on the positive side! The orientation, identi¢cation and

MINIREVIEW
Look on the positive side! The orientation, identi¢cation and
bioenergetics of ‘Archaeal’membrane-bound nitrate reductases
Rosa Maria Martinez-Espinosa1, Elizabeth J. Dridge2, Maria J. Bonete1, Julea N. Butt3, Clive S. Butler2,
Frank Sargent3 & David J. Richardson3
1
División de Bioquı́mica y Biologı́a Molecular, Departamento de Agroquı́mica y Bioquı́mica, Facultad de Ciencias, Universidad de Alicante, Alicante,
Spain; 2School of Biosciences, University of Exeter, Exeter, UK; and 3School of Biological Sciences, University of East Anglia, Norwich, UK
Correspondence: David J Richardson,
Centre for Metalloprotein Spectroscopy and
Biology, School of Biological Sciences,
University of East Anglia, Norwich NR4 7TJ,
UK. Tel.: 144 1603 593250; fax: 144 1603
592250; e-mail: [email protected]
Received 25 April 2007; revised 13 July 2007;
accepted 13 July 2007.
First published online 20 September 2007.
DOI:10.1111/j.1574-6968.2007.00887.x
Editor: Rustam Aminov
Keywords
nitrate reductase; Archaea; nitrogen cycle;
Rieske protein; molybdoenzyme; Q-cycle.
Abstract
Many species of Bacteria and Archaea respire nitrate using a molybdenumdependent membrane-bound respiratory system called Nar. Classically, the
‘Bacterial’ Nar system is oriented such that nitrate reduction takes place on the
inside of this membrane. However, the active site subunit of the ‘Archaeal’ Nar
systems has a twin arginine (‘RR’) motif, which is a suggestion of translocation to
the outside of the cytoplasmic membrane. These ‘Archaeal’ type of nitrate
reductases are part of a group of molybdoenzymes with an ‘RR’ motif that are
predicted to have an aspartate ligand to the molybdenum ion. This group includes
selenate reductases and possible sequence signatures are described that serve to
distinguish the Nar nitrate reductases from the selenate reductases. The ‘RR’
sequences of nitrate reductases of Archaea and some that have recently emerged in
Bacteria are also considered and it is concluded that there is good evidence for
there being both Archaeal and Bacterial examples of Nar-type nitrate reductases
with an active site on the outside of the cytoplasmic membrane. Finally, the
bioenergetic consequences of nitrate reduction on the outside of the cytoplasmic
membrane have been explored.
Introduction
Coupling the reduction of nitrate to energy-conserving
electron transport pathways is a widespread means of
sustaining growth and cell maintenance in anoxic environments (Richardson, 2000). In the process of nitrate respiration, the membrane-bound nitrate reductases of the
g proteobacterium Escherichia coli and the a proteobacterial
Paracoccus species have for many years been paradigm
enzymes for bioenergetic and biochemical elucidation of
the process. These so-called Nar enzymes comprise three
subunits: NarG, the ‘a-subunit’ of about 140 kDa, which
contains the molybdenum bis molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor at its catalytic site and an
[4Fe–4S] cluster; NarH, the b-subunit of about 60 kDa,
which contains one [3Fe–4S] and three [4Fe–4S] clusters;
and NarI, the integral membrane g-subunit of about 25 kDa
with five transmembrane helices that bind two haems b: one
low potential (bL) located at the periplasmic side, and one
high potential (bH) located at the cytoplasmic side (Bertero
et al., 2003) (Fig. 1). NarG and NarH are located in the
FEMS Microbiol Lett 276 (2007) 129–139
cytoplasm and associate with NarI at the membrane potential-negative cytoplasmic face (Dc ) of the cytoplasmic
membrane. Most published bioinformatic analyses of membrane-bound nitrate reductase sequences currently in the
databases suggest that this arrangement is conserved among
Gram-negative bacteria and indeed, for many years, it was
assumed that this orientation of membrane-bound nitrate
reductases would be conserved among prokaryotes in general. However, recent evidence suggests that this is not the
case. Where respiratory nitrate reduction has been identified
in Archaea, it is predicted to take place in a catalytic subunit
that has a signal sequence that is characteristic of twinarginine signal peptides, which serve to export folded redox
proteins across the cytoplasmic membrane (Cabello et al.,
2004). Examples of Archaea that probably have this type of
nitrate reductase at the time of writing include Haloferax
mediterranei (Lledo et al., 2004), Haloarcula marismortui
(Yoshimatsu et al., 2002), Pyrobaculum aerophilum (Afshar
et al., 2001) and Archaeoglobus fulgidus (Richardson et al.,
2001; Dridge et al., 2006). In this minireview, the ‘RR’
sequences of nitrate reductases of Archaea and some that
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c
130
R.M. Martinez-Espinosa et al.
NapB
NapA
cytcH
NO3− + 2H+
Mo
Periplasm
cytcL
2H+
NO2− + H2O
[4Fe– 4S]
pNarG
pNarH
[3Fe–4S]
NO3− + 2H+
Mo
[4Fe–4S] [4Fe–4S]
∆Ψ+
[4Fe–4S]
2H +
NarI
2H +
cytc
NapC
cytc cytc
−
NO2 + H2O
2e−
cytc
∆Ψ+
[4Fe–4S]
QH2
?
QH2
cytbL
2e−
Q
QH2
2
Q
Q
[3Fe – 4S]
∆Ψ−
cytbH
∆Ψ−
[4Fe – 4S] [4Fe – 4S]
NO3− + 2H+
Mo
[4Fe – 4S]
nNarH
−
NO2 + H2O
NO2− + H2O
[4Fe – 4S]
Cytoplasm
NO3− + 2H+
nNarG
Nar Group of Nitrate Reductases
[4Fe-4S]
Mo
XH2
X
Nas
Nap Group of Nitrate Reductases
Fig. 1. The different classes of prokaryotic Mo-bis-MGD nitrate reductases. Note that: (i) the Nap system illustrated is the rather simple system from
Paracoccus pantotrophus (Berks et al., 1995), some Nap systems do not use a NapB as electron donor and that NapC may be substituted by a different
quinol dehydrogenase (Jepson et al., 2006; Marietou et al., 2005) and (ii) the Nas enzymes characteristically have one [4Fe–4S] cluster, but some are
predicted to bind an additional [2Fe–2S] cluster (Richardson et al., 2001).
have recently emerged in Bacteria will be considered and it
will be assessed whether they can be used to argue for a
Dc1 location of the active site subunit by also considering
the genetic context of the narG gene and available biochemical evidence on the limited number of systems that have
been characterized.
The ‘Archaeal’ type of nitrate reductase is part of
a group of Mo-bis-MGD enzymes with an ‘RR’ motif that
are predicted to have an aspartate ligand in the molybdenum
ion co-ordination sphere provided by the polypeptide chain.
This group includes selenate reductase and there is
a high level of sequence identity between enzymes designated selenate reductases in databases and enzymes of
the Archaeal nitrate reductase group. Possible sequence
signatures are sought that can aid in distinguishing
a nitrate reductase from a selenate reductase with the
aim of removing confusion over the annotation of these
closely related enzymes when sequences emerge in those
databases.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
There is a high percentage of sequence similarity between
the putative Tat-dependent Archaeal nitrate reductase NarG
subunits and the cytoplasmically active NarG subunits of
Gram-negative bacteria and thus they are often given the
same gene nomenclature ‘narG’ in the literature. However,
this can serve to obscure important bioenergetic differences
in the two types of nitrate reductase system. Thus, the
bioenergetic consequences of nitrate reduction on the
Dc1 of the cytoplasmic membrane and the possibility that
a protonmotive Q-cycle might operate in at least one group
of Archaeal Nar enzymes that would be bioenergetically
equivalent to the Q-loop mechanism in operation in the
paradigm bacterial Nar enzymes are explored.
Cellular location of the active site
‘Archaeal’ NarG
Archaeal membrane-bound nitrate reductase systems have
attracted some interest because of the extreme conditions
FEMS Microbiol Lett 276 (2007) 129–139
131
Archaeal nitrate respiration
under which they can operate, for example the hyperthermophilic systems of Pyrobaculum aerophilum (Afshar et al.,
2001) or halophilic systems of Haloferax mediterranei (Lledo
et al., 2004) and Haloarcula marismortui (Yoshimatsu et al.,
2000). Despite this, there are still only a few Archaeal
respiratory nitrate reductase sequences available. In all cases,
though, analysis of the N-terminal region of the nitrate
reductases reveals the conservation of a twin arginine (‘RR’)
motif that has similarities to a Tat signal peptide n-region
consensus sequence: S/T-RR-X-FLK (Berks et al., 2000;
Sargent, 2007). The Tat translocase system is a protein
transport apparatus present in Bacteria and Archaea
that can transport folded proteins across the
cytoplasmic membrane. In the cases of the Archaeal Nar
systems, the ‘RR’ motifs are followed by characteristic h- and
c-regions and are strongly predicted by the authors’
analysis, using the modelling programme TatP (Bendtsen
et al., 2005), to indeed be signal peptides for protein export
(Fig. 2). Is there biochemical evidence to support this
assertion?
Where data are available, cell fractionation studies with
nitrate-reducing Archaea suggest that the NarG protein is
strongly associated with the membrane fraction and requires
detergent solublization to release it (Yoshimatsu et al., 2000,
2002; Afshar et al., 2001; Lledo et al., 2004). This raises the
question of whether the subunit is located on the inside or
outside of the cytoplasmic membrane. In nitrate-reducing
Gram-negative bacteria, such as Paracoccus pantotrophus
and E. coli, the widely accepted diagnostic method for
assessing the cellular location of the active site of a nitrate
reductase in intact cells has been to assay for activity with
the nonphysiological electron donors methylviologen (MV)
and benzylviologen (BV) (Bell et al., 1990) The membranepermeant benzylviologen is a more effective electron donor
to enzymes with their active site located on the inside of the
cytoplasmic membrane than the relatively more membraneimpermeant methylviologen, despite the latter redox dye
being more reducing. Thus, for example, in intact cells of
E. coli (Yoshimatsu et al., 2000) expressing nar, an MV/BV
activity ratio of 0.12 was measured, but a much higher ratio
of 1.2 was measured for the halophilic Haloarcula marismortui by Yoshimatsu et al. (2000) and this was confirmed in
studies for the present minireview where a ratio of MV/BVdependent Nar c. 3.0 has been measured in whole cells of
halophilic Haloferax mediterranei Nar. These results are
diagnostic for electron donation to the active site of an
enzyme that is on the outside, rather than inside, of the
cytoplasmic membrane (Table 1). Such experiments have
not yet been reported for the other putative Archaeal Nars
with Tat sequences thus far identified. However, the data
show that where experimental evidence is available, there is
compelling evidence that the active site of these Archaeal
Nar systems is indeed on the outside of the cytoplasmic
membrane. This gives confidence that this will be true of
STRRXFLK
Archaea
Haloferax mediterranei_pNarG
Haloarcula marismortui_pNarG
Pyrobaculum aerophilum_pNarG
Aeropyrum pernix_pNarG
Archaeoglobus fulgidus_pNarG
Bacteria
Candidatus Kuenia stuttgartensis_pNarG
Carboxydothermus hydrogenoformans_pNarG
Moorella thermoacetica_pNarG
D-group
Thauera selenatis_SerA
Dechloromonas agitata_PcrA
Ideonella dechloratans_ClrA
Rhodovulum sulfidophilum_DdhA
Azoarcus sp. EbN1_ebdA
Desulfococcus oleovorans_ebdA
nNar
Bacillus subtilis_nNarG
Staphylococcus carnosus_nNarG
Escherichia coli_nNarG
Paracoccus denitrificans_nNarG
SGVSRRTFLEGIGVASLLGIGTSAASDDSLF-QMGGLKPVD
AGISRRDFVRGLGAASLLGATGLSFADD----GMDGLEAVD
LKTTRRRMLAGVATISAAAWVMALAQNLQYL-QPLAQFVNT
VKLSRRGFLKVLAAAGLLSSLGPLASALSSN-RYLTTIETP
MKVSRRDFIKLS-AATAFASG-LGLG-YFQKSRGVQQN--E
MKLTRRAFLQVAGATGATLTLAKNAMAFRLLKPAVVVDNPL
VKLTRRQFLKGT-AAAGVLLG-AGGGRVLVKKALADNR--A
FKLSRRQFLKAS-AATAVLAGTAGATRYFIPKAGAENTSPL
DGNGRRRFLQFSMAALASAAAPSSVWAF-S-KIQPIEDPLARLSRRDFLKASAATLLGN-SL-TFKTLAATMDLSGAFEYS
EHNGRRRFLQFSAAALASAAASPSLWAF-S-KIQPIEDPL--TTRRTLMQGASLVGAGLFAAGRGWAL-N-RLEPIGDTLA
QDQDRRDFLKRSGAAVLSLSLSSLATGV-VPGFLKDAQAGT
--ISRRTFLKGTSATVALLSLNSLGFLG-GNTIANATEKIF
SPLFRRLNYFS---PIEHHSNKHSQTTREDRDWENVYRNRW
--MNFFK---PTEKFNGNWSVLTDKSREWEKMYRERWSHDK
SKFLDRFRYFK--QKGETFADGHGQLLNTNRDWEDGYRQRW
SHLLDRLNFLKP-TRKDVFSEGHGQTTTENRDWEDTYRSRW
Fig. 2. N-terminal Tat-like signal peptides of pNar and related enzymes. The ‘RR’ sequences or remnant ‘RR’ are underlined. The Tat consensus sequence is
indicated at the top of the alignment.
FEMS Microbiol Lett 276 (2007) 129–139
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Published by Blackwell Publishing Ltd. All rights reserved
c
132
R.M. Martinez-Espinosa et al.
Table 1. Ratios of the methylviologen-dependent and benzylviologendependent nitrate and selenate reductase activities in intact cells
Species/enzyme
MV: BV
Ratio
Predicted
location
Escherichia coli Nar
0.12
Dc
Haloarcula marismortui 1.2
Nar
Haloferax mediterranei 3.0
Nar
Enterobacter cloacae 0.5
Nar
Enterobacter cloacae 2.0
Ser
Thauera selantis Nar
0.17
Thauera selantis
10
Ser
References
Dc1
Yoshimatsu et al.
(2000)
Yoshimatsu et al.
(2000)
This work
Dc
Watts et al. (2003)
Dc1
Watts et al. (2003)
Dc
Dc1
This work
This work
Dc1
those that have not been experimentally tested but have
predicted Tat signal sequences. However, in the absence of
whole-cell MV/BV Nar activities, can other bioinformatic
analysis be brought in to support the assertion that the ‘RR’
sequence directs a Nar for export?
In Nar systems in which nitrate is reduced on the
cytoplasmic (Dc ) face of the membrane, the quinol is
oxidized at the membrane potential-positive face (Dc1)
(Fig. 1). The consequence of this is that electrons have to be
moved some 40 Å across the membrane from the site of
quinol oxidation to the iron–sulphur clusters of the NarH
subunit. This electron transfer is catalysed by NarI, which
binds two b-haems that are stacked in a five helix bundle. By
contrast, in a Nar system in which the active site subunit is at
the Dc1 side of the membrane, the electrons from quinol
oxidation at this face do not need to pass back across the
membrane. Thus, a di-haem NarI type of subunit is not
needed. As the narI gene characteristically clusters with the
other nar genes, then its absence in a nar cluster in
conjuction with the NarG gene having an ‘RR’ motif
will add weight to the assignment of such a subunit to the
Dc1 of the membrane.
An example of the worth of bringing the ‘RR’ and NarI
analyses together comes when the Bacillus subtilis Nar
system is considered (Fig. 2). In this case, the NarG subunit
has an ‘RR’ motif, albeit a very poor one. However, analysis
of the genetic context of the narG gene reveals that it is
located in a classical narGHJI cluster with the presence of the
narI gene (Hoffmann et al., 1995), suggesting that the NarG
protein is located at the Dc face of the membrane. In fact,
the remnants of a Tat signal sequence towards the
N-terminus can be seen in many bacterial NarG proteins
where the presence of a single R in the position of the ‘RR’
motif is quite common (Sargent, 2007). But can any
Bacterial Nar systems be identified where the combined
application of ‘RR’ and ‘NarI’ bioinformatics analysis
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c
strongly suggests an Archaeal-like Dc1 orientation? The
evolutionary question of ‘what came first, pNar or nNar?’ is
not one that can be answered with any degree of certainty.
However, there seems to be no biochemical reason why each
should not be present in both Archaea and Bacteria.
Evidence for pNar enzymes in Archaea has not been found
(note, though, that the number of genome sequences is
relatively small). However, searching Bacterial genome databases for pNar enzymes using the defining characters
discussed above produced three hits that could be pNarG
enzymes. This first is in Carboxydothermus hydrogenoformans Z-2901. This thermophilic (optimum growth
temperature = 78 1C), Gram-positive Firmicutes is a bacterial hydrogenogen that can grow anaerobically utilizing
carbon monoxide as the sole carbon source and water as an
electron acceptor, producing carbon dioxide and hydrogen
as waste products. The organism has been shown to grow
slowly under heterotrophic conditions with lactate as an
electron donor and nitrate as an electron acceptor (Wu et al.,
2005). The second hit is in Moorella thermoacetica ATCC
39073. This strict anaerobe is also a Gram-positive Firmicutes and a moderately thermophilic acetogen (optimum
growth temperature = 58 1C). A gene locus (ABC20208) is
strongly predicted by the present analysis to be a pNarG and,
accordingly, the organism has been reported to utilize nitrate
as an electron acceptor under some conditions (Seifritz et al.,
2002). The third example of a bacterial pNar can be found in
Candidatus Kuenia stuttgartensis (Strous et al., 2006). This
strictly anaerobic ammonia-oxidizing planktomycete has
internal membranes, in addition to the cytoplasmic membrane, that compartmentalize anammoxosomes. It is known
to be able to reduce nitrate and is strongly predicted to have a
pNar, but it is not possible at present to predict which
membrane pNar is transported across.
It seems certain that as more genome sequences emerge,
other examples will arise of Archaeal-type Nar systems being
present in Bacteria. Thus, rather than a kingdom-based
subclassification of Nar systems, it is now proposed to adopt
a location-based classification of nNar for a system in
which the NarG subunit is located on the membrane
potential-negative (Dc ) side and pNarG for a system in
which the NarG subunit is located on the membrane
potential-positive (Dc1) side (Fig. 1). It is noted that in
all pNar systems, the NarH proteins encoded in the pnarG
gene clusters do not have Tat signal peptides. This is also
true of the iron–sulphur subunits of structurally defined
nitrate-inducible formate dehydrogenase of E. coli where it
is proposed that the iron–sulphur-containing b-subunit
is exported as a passenger with the Tat-dependent
Mo-bis-MGD-containing a subunit (Sargent, 2007). The
same mechanism is thus likely to apply to the Nar systems
so that a pNarH that will be associated will be coexported
by Tat with a pNarG.
FEMS Microbiol Lett 276 (2007) 129–139
133
Archaeal nitrate respiration
Is there a sequence signature that allows a
putative pNar sequence to be assigned as
a nitrate reductase?
The recent X-ray crystal structures of the nNarG subunit of
E. coli have revealed that the Mo ion at the active site is coordinated by an aspartate residue, Asp222 (Bertero et al.,
2003; Jormakka et al., 2004). This residue is conserved in all
the pNarG subunits and so it is likely that it is also a Mo
ligand in these enzymes (Fig. 3). However, some of the
pNarG proteins [c. 900 amino acids (aa)] are considerably
smaller than the nNar proteins (c. 1200 aa). In fact, they
are very similar in size to the c. 900 aa catalytic subunits
of selenate reductase (SerA) that are also Mo-bis-MGD
enzymes. These molybdoenzymes also have Tat-like signal
peptides (Fig. 2) and are located in the periplasm, on
the membrane potential-positive side of the cytoplasmic
membrane (Krafft et al., 2000). In addition, the Mo–Asp
ligand is also conserved (Fig. 3); in fact, SerA is part of a
group of enzymes (the D group of Mo-bis-MGD enzymes;
Jormakka et al., 2004) in which this aspartate ligand is
conserved (Fig. 3). This group also includes dimethylsulphide dehydrogenase (McDevitt et al., 2002), ethylbenzene
dehydrogenase (Kniemeyer & Heider, 2001), chlorate reductase (Thorell et al., 2003) and perchlorate reductase
(Bender et al., 2005) and all have Tat signal peptides
(Fig. 2). This raises the question of when, from the sort of
bioinformatic analyses described in the previous section, can
one predict whether a putative pNarG is actually a nitrate
reductase and not a selenate reductase? Can one be confidently distinguished from the other clearly on the basis of
bioinformatics?
To illustrate the problem, initially, two enzymes will be
considered on which the authors work in their laboratories:
Haloferax mediterranei pNarG and Thauera selenatis SerA
(Fig. 4). Haloferax mediterranei pNarG shows 31% identity
and 63% similarity to T. selenatis SerA and the processed
proteins are both of a similar size (c. 920 aa). It also shows
(a) Iron–sulphur ‘Asparagine’ signature
Carboxydothermus hydrogenoformans_pNarG
Moorella thermoacetica_pNarG
Archaeoglobus fulgidus_pNarG
Thauera selenatis_SerA
Ideonella dechloratans_ClrA
Rhodovulum sulfidophilum_DdhA
Azoarcus sp. EbN1_EbdA
Candidatus Desulfococcus oleovorans_EbdA
Escherichia coli_nNarG
Paracoccus denitrificans_nNarG
Staphylococcus carnosus_nNarG
Bacillus subtilis_nNarG
Pyrobaculum aerophilum_pNarG
Aeropyrum pernix_pNarG
Haloferax mediterranei_pNarG
Haloarcula marismortui_pNarG
Dechloromonas agitata_PcrA
Candidatus Kuenia stuttgartensis_pNarG
*
*
*
*
TCSPNC
CTS--ACGIKAMVVDGQIKALFPTNDYPDP ----------EYNPRGCLRGISFIN
TCSP NCTL--ACGIRAMVVDGQIKALLPSNDYPEP ----------EYGPRGCLRGLSFIN
VCSP NCT--GACGFDALVYNGRIETLTQAADYPEP ----------EYNPRGCLRGQSMMN
THSNGCV --AGCAWRVFVKNGVPMREEQVSEYPQL -PGV----- PDMNPRGCQKGAVYCS
THSNGCV --AGCAWNVFVKNGIPMREEQISKYPQL -PGI----- PDMNPRGCQKGAVYCS
AHCI NCL--GNCAFDIYVKDGIVIREEQLAKYPQISPDI -----PDANPRGCQKGAIHST
SHLNICWPQGSCKFYVYVRNGIVWREEQAAQTPACNVDY ----- VDYNPLGCQKGSAFNN
THLVDCYP-GNCLWRVYSKDGVVFREEQAAKYPVIDPSG -----PDFNPRGCQKGASYSL
THGV NCT--GSCSWKIYVKNGLVTWETQQTDYPRTRPDL -----PNHEPRGCPRGASYSW
THGV NCT--GSCSWKIYVKSGIVTWETQQTDYPRTRPDL -----PNHEPRGCARGASYSW
THGV NCT--GSCSWKVFVKNGVITWENQQTDYPSCGPDM -----PEFEPRGCPRGASFSW
THGV NCT--GSCSWNIYVKNGIVTWEGQNLNYPSTGPDM -----PDFEPRGCPRGASFSW
THGV NCT--GSCSWNVYVKDGLIVWELQATDYPDISPDI -----PNYEPRGCPRGASFSW
THGV NCT--GSCSWMVYVKDGIVAYELQAGDYPDIGPSY -----PNYEPRGCPRGASTSW
THSV NCT--GSCSWNVYVKNGQVWREEQSGDYPRFDESL -----PDPNPRGCQKGACYTD
THSV NCT--GSCSWNVYVKDGQVWREEQAGDYPTFDESL -----PDPNPRGCQKGACYTD
AHLI NCT--GACPHFVYTKDGVVIREEQSKDIPPM -PNI-----PELNPRGCNKGECAHH
CCSPNDT—-HACRIRAFVRNNVMMRVEQNYDHQNYSDLYGNKATRNWNPRMCLKGYTFHR
(b) The substrate pocket signature
Carboxydothermus hydrogenoformans_pNarG
Moorella thermoacetica_pNarG
Archaeoglobus fulgidus_pNarG
Thauera selenatis_SerA
Ideonella dechloratans_ClrA
Rhodovulum sulfidophilum_DdhA
Azoarcus sp. EbN1_EbdA
Candidatus Desulfococcus oleovorans_EbdA
Escherichia coli_nNarG
Paracoccus denitrificans_nNarG
Staphylococcus carnosus_nNarG
Bacillus subtilis_nNarG
Pyrobaculum aerophilum_pNarG
Aeropyrum pernix_pNarG
Haloferax mediterranei_pNarG
Haloarcula marismortui_pNarG
Dechloromonas agitata_PcrA
Candidatus Kuenia stuttgartensis_pNarG
LAGWTLHHAYDLNGDLPMFWPQTFGV QTEEL
MAGWSLIHP YDQNGDLPMFWPQTFGV QTEEL
MFGWSALHG YTMNGDLPAFWSQTFGV QTEEF
LIGAIKPDVSSMTG DLYPGIQTVRMPARTVS
LLGAISPDATSMTG DLYTGIQTVRVPASTVS
LVGGVQLDIFTDVG DLNTGAHLAYGNALESF
LMDGVSPDINVDIG DTYMGAFHTFGKMHMGY
SLGATVLDLDSTIG DFNRGIYETFGKFMFMD
LIGGTCLSF YDWYCDLPPASPQTWGE QTDVP
LLGGTCMSF YDWYCDLPPASPQTWGE QTDVP
LMGGEMLSF YDWYADLPPASPQIWGE QTDVP
LIGGPMLSF YDWYADLPPASPQIWGD QTDVP
LIGGAMGSF YDWYADLPPASPQMWGE QTDVP
LIGGSMGSF YDWYADLPPASPQVWGE QTDVP
LLGGVSHSF YDWYSDLPPGQPITWGT QTDNA
LLGGVSHSF YDWYSDLPPGQPITWGT QTDNA
YIGAHTHTFFDWYS DHPTGQTQTCGVQGDSA
ALGGRNWSNYTWHGDQAPGHPFSHGL QTSDV
Fig. 3. Signature sequences of pNar enzymes. Residues discussed in the text are underlined in bold. The residues that bind the predicted 4Fe4S cluster
are indicated by an asterisk.
FEMS Microbiol Lett 276 (2007) 129–139
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
134
R.M. Martinez-Espinosa et al.
100
(a)
75
50
25
0
100
(b)
75
50
25
0
100
(c)
75
50
25
Chlorate
Nitrate
Selenate
0
Fig. 4. Methylviologen-dependent selenate, chlorate and nitrate reductase activities of (a) Haloferax meditteranei pNar, (b) Thauera selenatis
Ser and (c) Paracoccus denitrificans nNar. All activities were determined
at 25 mM substrate. The activities are presented as the percentage of the
maximum rate detected with chlorate. Methylviologen activities
were measured as described in Anderson et al. (2001). All data
have been newly collected for this work. Haloferax meditteranei pNar
100% activity = 250 nmol MV oxidized mg 1 min 1; Thauera selenatis;
Ser 100% activity = 500 nmol MV oxidized mg 1 min 1; Paracoccus denitrificans nNar 1000 nmol MV oxidized mg 1 min 1.
29% identity and 53% similarity to E. coli nNarG, but is
c. 300 aa smaller. On this basis alone, one could not conclude
that Haloferax mediterranei pNarG should be classified as a
nitrate reductase, rather than a selenate reductase. In
the light of this, for this minireview purified Haloferax
mediterranei pNarG has been assessed for nitrate and
selenate reductase activity and it has been established that
the enzyme is catalytically similar to nNarGs in that it is
highly reactive towards nitrate and chlorate as substrates, but
shows no reactivity towards selenate (Fig. 4). Likewise, the
activity of T. selenatis SerA has been analysed and it has been
established that it is highly active towards selenate, but
essentially inactive towards nitrate (Fig. 4). There have been
some suggestions in the literature that nNar reduces selenate,
for example NarG and NarZ have been reported to confer a
low selenate reductase activity to E. coli membranes (Avazeri
et al., 1997). However, these experiments were carried
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c
out at comparatively high selenate concentrations of 160 mM
and significant selenate reduction by Paracoccus (Fig. 4;
Watts et al., 2003) or Enterobacter cloacae pNar has not been
detected (Watts et al., 2005; Ridley et al., 2006). pNar, nNar
and Ser, though, do have the common ability to reduce
chlorate at high rates and this can serve as a reference point
for normalizing activities across different types of enzymes
(Fig. 4).
Thus, the biochemical analysis provides clear information
on a Nar or Ser enzyme’s catalytic character that the overall
sequence identities are ambiguous about. With this information, one can then look further into the pNarG, nNarG
and SerA sequences using the structurally defined E. coli
nNarG as a platform from which to identify pNars correctly
in emerging genome databases. Using molecular modelling
(Dridge et al., 2006) based on the three-dimensional X-ray
structure of nNarG from E. coli (Bertero et al., 2003;
Jormakka et al., 2004) and also examining the recent X-ray
structure of Aromatoleum aromaticum EbdA (Kloer et al.,
2006) and the amino acid sequences of other D-group
Mo-bis-MGD enzymes, a number of key signatures of a
pNarG begin to emerge. The first is Asn52, which is located
within a cysteine-rich motif H/CX3CX3 5CX35 46C
located close to the N-terminal of all the D-group molybdoenzymes. This Cys cluster is thought to be involved in the
co-ordination of an iron–sulphur cluster of the [4Fe-4S]
type (Fig. 3a). The involvement of this motif in the coordination of such a cluster has been shown in the crystal
structures of E. coli nNarG (Bertero et al., 2003; Jormakka
et al., 2004) and Aromatoleum aromaticum EbdA (Kloer
et al., 2006). Among all the nNarGs, the first clusterco-ordinating residue is His, but this is not the case in the
putative pNarG proteins where it can be a Cys residue
(Archaeoglobus fulgidus). Asn52 is located in the cluster
(Fig. 3a). It is positioned c. 3.9 Å away from the Mo atom
and serves a structural role, positioning the [4Fe–4S] cluster
and could form a hydrogen bond to a bound substrate.
This asparagine residue is also conserved in the pNarGs,
but is replaced with glycine in the active site of EbdA and
SerA (Fig. 3a), which could affect enzyme specificity.
The second signature relates to the substrate entry channel.
In the E. coli nNarG, the conformation of the putative
substrate entry channel is dictated by Thr54, Gln235 and
Thr236. Gln235 and Thr236 are conserved in the Haloferax
mediterranei pNarG sequence and, indeed, all the nNar
and putative pNar sequences. This could therefore be a
fingerprint for a Nar as they are not conserved in the
other D-group members (Fig. 3b), for example they
are replaced by Ala221 and Arg222 in T. selenatis SerA.
This structure-based bioinformatics analyses suggests
that there are signatures that may enable a pNar to be
distinguished from a SerA among the D-group branch of
molybdoenzymes.
FEMS Microbiol Lett 276 (2007) 129–139
135
Archaeal nitrate respiration
Maintaining the bioenergetic equivalence
of nitrate reduction on the membrane
potential-positive and -negative sides of
the membrane
During respiratory electron transfer, Nar enzymes receive
electrons from quinols located within the lipid phase of the
cytoplasmic membrane. As discussed earlier, in the case of
the nNar system the oxidation of quinol takes place at the
periplasmic side of NarI where the protons are released and
the two electrons are moved from the periplasmic-face haem
bL to cytoplasmic-face haem bH. This charge separation
makes the enzyme electrogenic in that it contributes to the
generation of a proton electrochemical gradient across the
membrane (two charge separations, q1, during transfer of
two electrons from quinol to nitrate; 2q1/2e ). Electrons
from haem bH of NarI are donated to the [3Fe–4S] cluster of
NarH. From there, they flow via the iron–sulphur clusters in
NarH to the one in NarG, which is the direct electron donor
to the Mo-bis-MGD cofactor-containing catalytic site in
NarG where nitrate is reduced to nitrite (Richardson
& Sawers, 2002). In many species of bacteria that have
nNarG, the nitrite produced is further reduced to nitric
oxide, nitrous oxide and dinitrogen in a series of enzymecatalysed reactions of denitrification. In each case, these
reactions take place in the periplasm or towards the periplasmic face of the membrane and can be coupled to energy
conservation through the protonmotive activity of the
cytochrome bc1 complex (Q-cytochrome c oxidoreductase)
(Berks et al., 1995). Like NarI, this complex binds two
haems: one at either side of the membrane. However, rather
than oxidizing one quinol per turnover (like NarI), it
oxidizes two quinols at the periplasmic face and moves two
electrons to the cytoplasmic face where it reduces one
quinone. Overall, this so-called Q-cycle effectively translocates two positive charges per quinol oxidized (2q1/2e )
and so it is bioenergetically equivalent to nNarI. This then
makes the reduction of nitrate, nitrite, nitric oxide and
nitrous oxide bioenergetically equivalent, despite the active
site of the nNar system being on the Dc
side of the
membrane and the active sites of the nitrite, nitric oxide and
nitrous oxide reductases being on the Dc1 side.
The importance of the cytochrome bc1 complex to this
bioenergetic equivalence can be illustrated when another
kind of respiratory nitrate reductase is considered – the
periplasmic nitrate reductase or NapA (Berks et al., 1995).
This enzyme, like nNarG, is widely spread among Gramnegative proteobacteria and indeed is found in many
bacteria, like Paracoccus denitrificans and E. coli, that can
also express an nNar system. However, NapA is not closely
related to the Nar enzymes. The molybdenum is co-ordinated by a Cys rather than Asp ligand (Jormakka et al.,
2004) and there is a rather poor identity overall (e.g. only
FEMS Microbiol Lett 276 (2007) 129–139
12% identity between E. coli nNarG and NapA). Nap does
reduce selenate although this enzyme is catalytically quite
distinct from Nar enzymes (Butler et al., 1999; Sabaty et al.,
2001). NapA is commonly coupled to quinol oxidation via a
tetrahaeme cytochrome called NapC (Roldan et al., 1998;
Cartron et al., 2002) that is not electrogenic and so there is
no net charge translocation associated with quinol oxidation
(Fig. 1); consequently, electron transport to nitrate via the
Nap system can only be energy conserving if the electron
input into the Q-pool is protonmotive. This can be the
case if electrons enter by a proton-translocating enzyme
(e.g. NADH dehydrogenase) or an electrogenic enzyme (e.g.
formate dehydrogenase or hydrogenase).
Turning back to the pNarG and nNarG enzymes, one can
now ask the question of whether being located at the Dc1
face of the membrane consigns pNarG to being a poorly
coupled enzyme, like NapA, or whether there is a mechanism by which pNarG can maintain bioenergetic equivalence
with its nNarG cousin? At present, it is not known how
electrons move from the Q-pool to pNarG. However, the
genetic context of the Haloferax mediterranei pNar and
Haloarcula marismortui pNar provides an interesting possibility. Analysis of the genes of the pnar cluster reveals that
one of the genes encodes a protein (NarC) that has sequence
similarity (4 50%) with the dihaem subunits of quinolcytochrome c reductases, such as the well-studied cytochrome bc1 complex of mitochondria and b6f complex of
plants and cyanobacteria (Lledo et al., 2004; Yoshimatsu
et al., 2006). This subunit is predicted to fold into nine
transmembrane helices and bind two b-haemes, stacked
across the membrane, with bis-histidinyl co-ordination
between helices II and IV. It should be noted that the
Cys residue of Chlamydomas reinhardtii that makes a
thioether bond to a third haem (Stroebel et al., 2003) is not
conserved in the Haloferax mediterranei pNar and Haloarcula marismortui pNar and so the cytochrome b6 is predicted
to be a dihaem, rather than a trihaem protein. Recent
experimental evidence that can be drawn on to support for
this view comes from the recent purification of NarC that
showed that it does indeed bind two b-haems (Yoshimatsu
et al., 2006). Adjacent to narC is a gene (narB) that is
predicted to encode a Rieske iron–sulphur protein also of
the type found in the protonmotive Q-cycling cytochrome
bc1 or b6f complexes (Lledo et al., 2004; Yoshimatsu et al.,
2006) (Fig. 5). From the present analysis, NarB is predicted
to bear an N-terminal signal anchor with the redox-active
C-terminal domain located at the p-side of the cytoplasmic
membrane. Integration and orientation of NarB in the
membrane is likely to be conducted by the Tat system
because the signal anchor contains all the features of a
Tat signal peptide and assembly of the Rieske protein
has recently been shown to be Tat-dependent in bacteria
(Bachmann et al., 2006; De Buck et al., 2007). In addition to
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c
136
R.M. Martinez-Espinosa et al.
pNarG
NO3− + 2H+
Mo
NO2− + H2O
[4Fe– 4S]
[3Fe–4S]
[4Fe–4S] [4Fe– 4S]
pNarH
[4Fe–4S]
4H+
NarB
[2Fe– 2S]
cytb
Orf7
∆Ψ+
∆Ψ+
2e−
2QH
cytb
2Q
Q
2
cytb
QH
∆Ψ−
∆Ψ−
Orf4
NarC
Orf1
2H+
NarJ
Antimycin A
Cytoplasm
orf1
narB
narC
orf4
narG
narH
orf7
Fig. 5. A Q-cycle coupling mechanism for the pNar enzyme of Haloferax mediterranei.
having the residues (two Cys and two His) that bind a 2Fe2S
cluster, we note that NarB also has two additional conserved
Cys residues. These characteristically form a disulphide
bond in Rieske proteins that modulates the redox properties
of the iron–sulphur cluster so that it operates at a high
potential (Em c. 250 mV) (Leggate & Hirst, 2005). This
suggests that NarB is a true Rieske protein, rather than the
Rieske-type protein found in the bacterial aromatic ring
dioxygenases and the assimilatory nitrate reductases that
lack the disulphide and operate at much lower potentials
(Em o 0 mV) (Butler & Mason, 1997). This then raises the
possibility that the Haloferax mediterranei and Haloarcula
marismortui pNar systems maintain bioenergetic parity with
the nNar systems by being coupled to a protonmotive
Q-cycle mechanism. Such a coupling mechanism has thus
far been unprecedented in any other respiratory nitrate
reductase system so far studied. As a Q-cycle activity is
sensitive to the classical inhibitor antimycin A that binds to
the cytochrome b subunit of plant and bacterial cytochrome
bc1/b6f complexes, but not the structurally distinct NarI
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c
cytochrome b subunit of the pNar systems the sensitivity of
nitrate reduction by the Haloferax mediterranei pNar system
to this inhibitor has been assessed . The results show a high
level of sensitivity (Fig. 6) to this inhibitor at concentrations
that inhibit nitrite, nitric oxide and nitrous oxide reduction
in Paracoccus denitrificans, all of which are dependent on the
cytochrome bc1 complex, but do not inhibit nitrate reduction by the cytochrome bc1 complex-independent nNarG
system (Berks et al., 1995).
In the cytochrome bc1 or b6f complexes, electrons flow
from cytochrome b to the Rieske iron–sulphur centre and
then to cytochrome c1 or cytochrome f. In the Haloferax
mediterranei and Haloarcula marismortui nar gene clusters,
there is no gene encoding a homologue of cytochromes
c1 or f. There is, though, a gene (Orf7) that we note encodes
another putative cytochrome subunit that has some homology with a group of soluble or membrane-anchored b-type
cytochromes encoded in the operons of many of the other
D-group molybdoenzyme systems, for example SerC in the
T. selenatis selenate reductase system (Krafft et al., 2000)
FEMS Microbiol Lett 276 (2007) 129–139
137
Archaeal nitrate respiration
not exclude the possibility that the pNar system is coupled
to a cytochrome b6–Rieske-type system encoded elsewhere
on the chromosome. Genome analysis reveals cytochrome
b6 homologues in Pyrobaculum aerophilum and Aeropyrum
pernix. This leaves open the possibility that some, but not all
pNars are coupled at the level of the Q-pool. If there is no
cytochrome b–Rieske system and so no protonmotive
Q-cycle, electron transfer will simply be bioenergetically
equivalent to the periplasmic nitrate reductase (Nap)
system, further highlighting the observation that one cannot
assume that all pNar systems are bioenergetically equivalent.
1.2
1.0
OD600 nm
0.8
0.6
0.4
0.2
Time (h) vs. OD Control
Time (h) vs. OD Antimycin
0.0
0
20
40
60
80
100
120
140
160
180
Time (h)
Concluding remarks
110
Time (h) vs. NO Control
[Nitrate] − mM
100
Time (h) vs. NO Antimycin
90
80
70
60
0
20
40
60
80
100
120
140
160
180
Time (h)
Fig. 6. Effect of Antimycin A on anaerobic growth and nitrate reduction
by Haloferax meditteranei. Cells were grown under anaerobic conditions
in maximal culture media in the presence of 100 mM KNO3 as described
in Lledo et al. (2004). Antimycin A was added to a final concentration of
100 mM at the point indicated.
and EbdC in the ethylbenzene dehydrogenase system
(Kniemeyer & Heider, 2001) (Fig. 5). This once again draws
similarities between pNar systems and other D-type Mo-bisMGD systems that are orientated towards the membrane
potential-positive side of the membrane. Importantly, the
EdbC structure has recently been resolved (Kloer et al.,
2006) and shows it to be a direct redox partner with the
EdbAB subunits that are equivalent to pNarGH. Significantly, the key lysine and methionine residues that provide a
novel haeme co-ordination in EbdC are conserved in
Haloferax mediterranei Orf7. This then strongly suggests
that Orf7 is a functional homologue of EbdC and the direct
electron donor to pNarGH, mediating electron transfer that
is coupled from the Q-pool via the cytochrome b/Rieske
protein complex.
If there is a Q-cycle mechanism for energy conservation
for the Haloferax mediterranei and Haloarcula marismortui
pNar systems, how widespread is it among other pNars?
Examination of the pnar gene clusters of Pyrobaculum
aerophilum, Archaeoglobus fulgidus and Aeropyrum pernix
reveals that they do not encode NarC homologues. This does
FEMS Microbiol Lett 276 (2007) 129–139
In this minireview, the evidence has been discussed for the
active subunits of ‘Archaeal’ or ‘pNar’ membrane-bound
nitrate reductases being secreted by Tat to the outside of the
cytoplasmic membrane, it has been proposed how to confidently identify a pNar from amino acid sequence analysis
and the bioenergetic consequences of pNar being active on
the membrane potential-positive side of a biological membrane have been explored. When prokaryotic nitrate reduction is considered as a whole, it is apparent that there are two
broad subclasses of Mo-bis-MGD nitrate reductases, the
‘Nar group’ and the ‘Nap group’, which are structurally
distinguished by having aspartate and cysteine Mo-bisMGD ligands, respectively (Jormakka et al., 2004). Like the
‘Nar’ group, the ‘Nap’ group also includes nitrate reductases
that are exported by the TAT system (the catabolic periplasmic NapA) and nitrate reductases that are active in the
cytoplasm (the anabolic Nas enzymes associated with nitrogen assimilation) (Richardson et al., 2001). As yet, no Nap
system has been reported to be coupled to a protonmotive
Q-cycling cytochrome b–Rieske complex. The suggestion
that at least some pNar systems may be coupled to
such a complex highlights emerging bioenergetic, as well as
structural, differences between these two groups of nitratereducing systems located on the membrane potentialpositive side of the membrane.
Acknowledgements
This work was funded in part by a research grants from the
BBSRC (BB/D00781X/1, BB/D018986) and from the MECSpain (BIO2005-08991-C02-01). F.S. is a Royal Society
University Research Fellow. The authors would like to thank
Dr Rick Lewis (University of Newcastle) for help with the
molecular modelling.
Authors’contribution
R.M.M.E. and E.J.D. are joint first authors.
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Published by Blackwell Publishing Ltd. All rights reserved
c
138
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