The membrane-bound nitrate reductase A

from Escherichia coli: NarGHI

Michela Bertero
Centre for Genomic Regulation, Barcelona, Spain

FUNCTIONAL CLASS
Enzyme; membrane-bound quinol:nitrate oxidoreductase A;
EC 1.7.99.4; a prokaryotic quinol:nitrate oxidoreductase
containing three subunits (NarG, NarH, and NarI) and eight
redox centers (one molybdenum cofactor, five iron–sulfur
clusters, and two heme groups); known as NarGHI.
Bacterial nitrate reduction can be catalyzed by three
classes of nitrate reductases: assimilatory nitrate reduc-
tases (NASs), periplasmic nitrate reductases (NAPs), and
membrane-bound respiratory reductases (NARs).1 These
enzymes differ in their cellular location, function, subunit
composition, and number and identity of their redox centers.
NAS is formed by one subunit, located in the cytoplasm and
is involved in nitrogen assimilation. NAP is a two-subunit
complex in the periplasm and presents functional flexibility,
involved in nitrate respiration or energy dissipation according
to the organism. NAR is a three-subunit enzyme located in
the membrane exposed to the cytoplasm and participates in
anaerobic nitrate respiration.
NarGHI belongs to the NAR class and comprises three
subunits with distinct functionalities: NarI (the membrane
anchor subunit), NarH (the electron transfer subunit) and
NarG (the catalytic subunit). NarGHI catalyzes electron
transfer from the quinol oxidation site, located in NarI in
the membrane, through the redox cofactors, embedded in
the enzyme, to the molybdo-bis(molybdopterin) guanine dinu-
cleotide (Mo-bisMGD) cofactor located in the cytoplasmic
NarG, where nitrate is reduced to nitrite. The overall reac-
tion is a two-electron and two-proton process. NarGHI
can accept electrons from both menaquinol (MQH2 ) and
ubiquinol (UQ2 ) as physiological reductants.2 In addition to
nitrate, other substrates can be reduced, such as Cl− , F− ,
NO2 − , ClO4 − , and BrO4 − .3
OCCURRENCE
The membrane-bound nitrate reductases have been
described among different bacterial species, both gram

(a) (b)

3D Structure Ribbon representation of NarGHI as a monomer (a) and as a dimer (b). NarG is in green, NarH in red, and NarI in
blue. NarGHI monomer is rotated approximately 90° around the vertical axis compared to the right NarGHI in the dimer. Cofactors are
represented as sticks. PDB code is 1Q16. All the 3D structures presented in this manuscript were created using PyMOL.19

Handbook of Metalloproteins, Online © 2006–2008 John Wiley & Sons, Ltd. 1

This article is © 2008 John Wiley & Sons, Ltd.
This article was published in the Handbook of Metalloproteins in 2008 by John Wiley & Sons, Ltd.
DOI: 10.1002/0470028637.met235
The membrane-bound nitrate reductase A from Escherichia coli: NarGHI
negative and gram positive. The enzymes have been
reported also in a few obligate aerobic bacteria, such as
Mycobacterium tuberculosis and Streptomyces coelicolor.1
Interestingly, operons encoding NAR enzymes have been
reported in hyperthermophilic4 and halophilic Archaea.5
In a few species, such as E. coli, a second membrane-
bound nitrate reductase has been discovered, known as
NarZYW.6 This is expressed at cryptic levels and may assist
the bacterium in the transition from aerobic to anaerobic
respiration.
BIOLOGICAL FUNCTION
Anaerobic conditions and the presence of nitrate are
signals for E. coli to activate the synthesis of a respiratory
chain, which is initiated by the membrane-bound formate
dehydrogenase, FdnGHI, and terminated by the membrane-
bound nitrate reductase, NarGHI. Electrons are transferred
from the formate oxidation site in FdnGHI to the
nitrate reduction site in NarGHI, via a plethora of
redox prosthetic groups and the lipid soluble quinol
pool (Figure 1). Electron transfer is coupled to proton
translocation across the membrane, generating a proton
motive force by a redox loop mechanism as proposed
by the chemiosmotic theory of Mitchell.7 In physiological
terms, the production of a proton motive force results in
conservation of the free energy derived from the catalytic
reactions.
AMINO ACID SEQUENCE INFORMATION
The three distinct subunits of NarGHI are encoded by the
narGHJI operon. NarJ is not part of the final enzyme but
is required as a specific chaperone for the correct assembly
of the enzyme.8,9
Nitrate reduction
NarGHI
Cytoplasm
Periplasm
FdnGHI
Formate oxidation
Figure 1 Schematic representation of the nitrate respiratory
chain. Black arrows indicate electron transfer and red arrow
proton translocation.
Handbook of Metalloproteins, Online © 2006–2008 John Wiley & Sons, Ltd.
This article is © 2008 John Wiley & Sons, Ltd.
This article was published in the Handbook of Metalloproteins in 2008 by John Wiley & Sons, Ltd.
DOI: 10.1002/0470028637.met235
• Escherichia coli: NarG subunit, 1246 AA (amino acid)
residues, P09152 (UniProtKB/Swiss-Prot entry); NarH,
512 AA, P11349; NarI, 225 AA, P11350.
PROTEIN PRODUCTION, PURIFICATION,
AND MOLECULAR CHARACTERIZATION
E. coli NarGHI can be purified from a recombinant
system.10 The enzyme is overexpressed in the engineered
E. coli LCB2048 strain that carries a deletion of two
endogenous operons, narGHJI and narZYWV (
narGHJI,

narZYWV).9 The pVA700 expression vector encodes the
entire narGHJI operon under the control of the hybrid
trp (tryptophan) – lac (lactose) tac promoter and upon
isopropyl-thio-galactoside (IPTG) induction. Bacterial cells
are lysed through a French press cell, the membranes are
purified by sucrose step-gradient centrifugation, and the
enzyme is extracted with the detergent polyoxyethylene
9-dodecyl ether (Thesit). NarGHI is purified by two consec-
utive anion exchange chromatographic steps at pH 6.0
and 8.0 (diethylaminoethyl-dextran DEAE-sepharose) and
a final gel filtration experiment (Superdex 200).
As judged by light scattering analyses, the purified
form of NarGHI is a dimer of heterotrimers with a
total molecular weight of ∼500 kDa, and it is likely
(see below) that this is the form of the enzyme that
occurs in situ in the inner membrane of E. coli.10 The
membrane anchor subunit NarI (26 kDa) contains two
b-type hemes, whose coordination and redox properties
were previously determined by the use of optical and
electron paramagnetic resonance (EPR) spectroscopy in
combination with site-directed mutagenesis and redox
potentiometry.11 The electron transfer subunit NarH
(58 kDa) contains four ferredoxin-type [4Fe–4S] and one
[3Fe–4S] clusters, already recognized in the 1970s12 and
further characterized by EPR spectroscopy.13 The catalytic
subunit NarG (139 kDa) hosts the complex molybdenum
cofactor, Mo-bisMGD, identified by sequence alignment
comparison and spectroscopy studies, as well as a novel
[4Fe–4S] cluster.13,14
ACTIVITY
Quinol:nitrate oxidoreductase activity is determined using
a spectrophotometric protocol. Measurements are based on
the absorption changes produced by oxidation of reduced
quinol and menaquinol analogs, such as tetramethyl-p-
benzoquinol (duroquinol) or 2-methyl-1,4-napthoquinol
(menadiol), and plumbagin.3,15 A stock ethanolic solution
of each quinone analog is reduced by metallic zinc in
acidified ethanol. The assay is initiated by addition of
nitrate to the degassed assay buffer containing NarGHI
(either enriched membranes or the purified enzyme)
and the electron donors. The use of quinol analogs as
2
 The membrane-bound nitrate reductase A from Escherichia coli: NarGHI
substrates tests the entire electron transfer relay of NarGHI.
Alternative assays use reduced viologens as electron donors.
Typically, a compound such as benzyl viologen is reduced
with excess dithionite and its enzyme-catalyzed nitrate-
dependent oxidation is followed spectrophotometrically.16
Because viologens donate electrons nonspecifically, they are
used to measure activities in variants of NarGHI in which
the electron transfer relay is ‘broken’.
X-RAY STRUCTURE OF THE COMPLEX
Crystallization
Crystallization of membrane proteins is extremely chal-
lenging and one of the critical parameters is the choice of the
appropriate detergent. Key to the successful crystallization
of NarGHI was the presence of 0.7 mM Thesit in the protein
solution.10 First crystals were obtained by the vapor diffu-
sion method with sitting drops equilibrated against a reser-
voir solution containing 100 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES), pH 7.0, 20% (w/v)
poly ethylene glycol (PEG 3000), 200 mM sodium acetate,
200 mM KCl, and 5 mM ethylene diamine tetraacetic acid
(EDTA). Crystals were orthorhombic and belonged to the
space group C2221 with unit cell dimension a = 154.175 Å,
b = 241.376 Å, and c = 139.494 Å. Only one complex was
present per asymmetric unit. Optimization of the cryopro-
tectant solution (100 mM HEPES, pH 7.0, 35% (w/v) PEG
3000, 300 mM sodium acetate, 200 mM KCl, 5 mM EDTA,
and 0.7% Thesit) allowed crystal diffraction to 1.9 Å reso-
lution. Selemethionine-substituted crystals were obtained
following the same protocol. Similar experimental condi-
tions were used to obtain several NarGHI crystal structures,
containing single point mutations or in complex with the
quinol inhibitor pentachlorphenol (PCP).
Overall architecture of NarGHI
The three subunits of NarGHI form a highly stable
heterotrimer with dimensions of 90 × 128 × 70 Å (3D
Structure). The cytoplasmic NarG and NarH interact
through extensive interfaces (∼11.452 Å2 ) and are anchored
to the membrane by NarI, predominantly through
hydrophobic surfaces. In particular, the positively charged
‘stop-transfer’ sequences of NarI17 interact with NarGH,
suggesting that the latter subunits are exposed toward the
cytoplasm, as supported previously by chemical labeling
studies.18
One of the most striking features of NarGHI structure is
revealed by the high-quality experimental electron density
map: eight redox centers aligned on a single chain that runs
like an electrical wire inside the enzyme for a total length of
∼98 Å (Figure 2). Following the chain from the periplasm
to the cytoplasm, we encounter heme bD (distal), heme
Handbook of Metalloproteins, Online © 2006–2008 John Wiley & Sons, Ltd.
This article is © 2008 John Wiley & Sons, Ltd.
This article was published in the Handbook of Metalloproteins in 2008 by John Wiley & Sons, Ltd.
DOI: 10.1002/0470028637.met235
Mo-bisMGD
7Å
11.2 Å
FS0
FS1
9.7 Å
FS2
9.6 Å
FS3
98 Å
9.4 Å
FS4
8.9 Å
bP
5.4 Å
bD
Figure 2 Redox centers within NarGHI (similar orientation
as in 3D Structure). Edge-to-edge distances are shown by dotted
lines.
bP (proximal), one [3Fe–4S] cluster (FS4), four [4Fe–4S]
clusters (FS3, FS2, FS1, and FS0), and the Mo-bisMGD
cofactor. Edge-to-edge distances between the redox centers
are well within the 14-Å limit observed for physiological
electron transfer.20
The heterotrimer NarGHI forms butterfly-shaped dimers
in the crystals with dimensions 143 × 128 × 90 Å and a
twofold symmetry axis approximately perpendicular to the
membrane. An extensive buried surface area (∼12 374 Å2 )
exists between the two NarGHI heterotrimers in the
complex, suggesting that the dimeric ‘butterfly’ is likely
to be the physiologically relevant form of the enzyme. This
is supported by the observation of the ‘swapping’ of a NarH
helix-turn domain into the symmetry-related NarGHI
and the identification of a phospholipid molecule at the
interface between the two NarIs of one dimer. However,
the distances between the redox centers belonging to the
two symmetry-related complexes are too long to allow
electron transfer, precluding kinetically competent electron
transfer between the individual heterotrimers.
In the following paragraphs, each subunit is described in
more detail following the same order in which the electrons
travel through the enzyme.
NarI
NarI is the enzyme membrane anchor where quinol binds
and is oxidized (the so-called Q site) initiating the journey
3
The membrane-bound nitrate reductase A from Escherichia coli: NarGHI

C -terminus

bP
H56
TM II
H205

TM I TM III

H66
bD
TM IV

N -terminus TM V

H187
(a) (b)

Figure 3 Ribbon representation of NarI (a) and close view to the hemes bD and bP (b). The helices have been removed and the
coordinating histidine residues are shown.

of the electrons to nitrate within the complex. NarI contains the protein surface (AA 46–174, 272–341, 357–509) and
five transmembrane helices (TM, I–V) that are tilted provide subunit–subunit interactions as well as shielding
approximately 30° to the membrane normal (Figure 3(a)). for the clusters.
Two short helices, one α and one 310 , connect TM IV
and V, and a C-terminal tail extends toward the cytoplasm
to interact with NarGH. TM I, the first N-terminal helix,
is involved in dimerization; TM II–V form a four-helix
bundle that contains two heme groups, bD and bP . Each
heme is oriented approximately parallel to the membrane FS1
A
normal and presents a Fe atom coordinated by two
histidines: His66 and His187 coordinate bD and His56
and His205 coordinate bP , confirming previous functional FS2
studies (Figure 3(b)).21

NarH

The electron transfer subunit NarH contains four

FS3
ferredoxin-type iron–sulfur clusters: three [4Fe–4S] referred B
FS4
to as FS1, FS2, and FS3 and one [3Fe–4S] as FS4.10 The
redox potentials of the iron–sulfur clusters were previously
determined by electrochemical methods: FS1 +130 mV,
FS2 −420 mV, FS3 −55 mV, and FS4 +180 mV.13 The
NarH core is constituted by two domains, A and B, each
holding two iron–sulfur centers and related to each other
by twofold rotational symmetry (Figure 4). The fold of
A and B is similar to the fold of bacterial 2 × [4Fe–4S] Figure 4 Ribbon representation of NarH. Dark red indicates
ferredoxins with two clusters sandwiched between two the ‘core’ and light red the nonconserved motives. The positions
helices and one β-sheet.22 Extra ordered motifs decorate of the FS are indicated by arrows.

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DOI: 10.1002/0470028637.met235
 The membrane-bound nitrate reductase A from Escherichia coli: NarGHI

NarG spectrum typical of a species with an S = 3/2 ground state

and a redox potential of −55 mV.14
NarG belongs to the dimethyl sulfoxide (DMSO) reduc-
tase family containing the Mo-bisMGD cofactor. Several
structures from this family have emerged in the recent The molybdenum catalytic center
years,23–27 showing a common architecture. NarG ‘core’
presents the four conserved α–β domains organized around The Mo-bisMGD cofactor elongates to 34 Å within NarG,
the catalytic center where nitrate is reduced to nitrite with one half, MGD-P (nomenclature as in Schindelin
(Figure 5). Domains II and III form a cleft where the et al.),24 coordinated by domain II and the other half,
Mo-bisMGD cofactor is located. As observed previously in MGD-Q, coordinated by domain III. During the catalytic
NarH, several extra motifs are present (AA 1–40, 115–150, cycle, the Mo metal center cycles through the +4,
339–472, 616–638, 667–690, and 843–990) and mostly +5, and +6 oxidation states. The NarGHI crystals10
located at the surface of the enzyme toward the cytoplasm. were obtained aerobically and most likely Mo is in the
These extra motifs decorate the narrow funnel also, which +6 oxidated state. In the structure, Mo is coordinated
leads to the active site. by six ligands: four cis-thiolate sulfur atoms (Mo–S
Unexpectedly, NarG contains an additional [4Fe–4S] distance ∼2.4 Å) belonging to the two halves MGD-P
cluster that was not detected by EPR spectroscopy prior to and MGD-Q and both side-chain oxygens of the residue
the determination of the structure.13 This cluster, known Asp222 (Mo-OD1 1.9 Å and Mo-OD2 2.4 Å). Aspartic acid
as FS0, is coordinated by three cysteines (Cys54, Cys58, coordination is observed for the first time in a Mo-bisMGD
and Cys93) and one histine (His50). In the light of this enzyme, where only serine, cysteine, or selenocysteine
finding, FS0 has been restudied and characterized by EPR were previously reported.28 Asp222 is absolutely conserved
spectroscopy and redox potentiometry, revealing an EPR among NarG of gram-positive and gram-negative bacteria
and Archaea.29 The crystal structure of the soluble E. coli
NarGH domain revealed ASP coordination.29 However,
the coordination sphere around the Mo center shows
significant differences (Figure 6): the bidentate Asp222
interaction is substituted by the interaction of only one
carboxylate oxygen of Asp222 and an additional oxo group
Domain II (Mo–oxo 1.8 Å). The second carboxylate oxygen of Asp222
Domain III
is hydrogen bonded to His546. The authors suggest an
‘Asp pendulum hypothesis’: ASP could oscillate between
FeS0
single and bidentate interactions with Mo, according to
the protonation state of His546. It was previously shown
C Domain I that the enzyme activity is pH dependent and controlled
Domain IV by pK of ∼8.12,30 The observed differences might also be
because of structural flexibility at the active site or different
N
(a) oxidation states in the two structures. Further investigation
is required to gain deeper insight into the molecular details
Domain II
5 h11 h10
of nitrate reduction.
26 30
4 6 23
22
h8
29
31
31
An additional peculiarity revealed by the NarGHI
h6 h7
28 30 structure10 is the novel bicylic dihydropterin structure
27
7 6 25 25 24 24 23 7 8 8 9 19 22 20 21
29
of MGD-Q, rather than the typical tricyclic pyranopterin
h9
D222
28
Domain III structure of MGD-P, as well as all of other MGD moieties
32
26 MGD-P 16
21 19 in the solved crystal structures (Figure 6). The authors
27 15
Mo 33 9 18 18 speculate a possible involvement in the catalytic mecha-
32 C92H49 FeS0
h1 3 20 17
C53 34 14 nism. The following point mutations were introduced in
MGD-Q

3 17
35 14 13
4
N 5
C57 h2
10
15 NarGHI: NarG-S719A, NarG-H1163A, and the double
36 35 38 33 34 10 16
1 2 37
13 11
h5 h4h3 mutation (RA Rothery, personal communication, 2008).
Domain I 11
36
12 Both residues are in the proximity of MGD-Q and in
1 2 39 12
40 C particular His1163 is hydrogen bonded to the oxygen in
Domain IV
the open ring of MGD-Q. In NarGHI carrying H1163A
(b)
and the double mutation, a drop in the overall enzyme
Figure 5 Structure of NarG and its domain organiza- potential is observed: from about +135 mV, which is the
tion. (a) Ribbon representation and (b) topology diagram. Each average value of the Mo (+6/+5) and Mo (+5/+6) conver-
β-strand is represented by an arrow and each α-helix by a cylinder. sions, to approximately +50 mV. A concomitant drop in

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DOI: 10.1002/0470028637.met235
The membrane-bound nitrate reductase A from Escherichia coli: NarGHI
Asp222
oxo
Mo
MGD-P
Figure 6 Ball-and-stick representation of the active site of NarGHI (blue) superimposed on FdnGHI (yellow). Red arrows indicate the
dihydropterin form observed in MGD-Q.
the enzyme activity is also observed. An attractive expla-
nation for these observations is that MGD-Q is in the
closed pyranopterin state in a NarG-H1163A mutant and
its enzyme activity is retained at reduced level (RA Rothery,
personal communication, 2008).
FUNCTIONAL ASPECTS
The reaction catalyzed by NarGHI is a quinol:nitrate
oxidoreduction. Quinone is oxidized and the redox
cofactors (two b-hemes and five iron–sulfur clusters)
transfer electrons across a potential range from −100
and +100 mV, depending on the reductant quinone,
menaquinol, or ubiquinol respectively, to +420 mV
(nitrate/nitrite at pH 7.0) for the final reduction of nitrate.
The two sites catalyzing these substrate redox reactions
are connected by a series of prosthetic groups with poten-
tials not consistently organized in a thermodynamically
‘downhill’ direction. For example, a fairly large thermo-
dynamic barrier exists for electron transfer between the
FS3 cluster (Em = −55 mV) and FS2 (Em = −420 mV),
with
Em of −365 mV. Such large energy barriers are
also observed in other respiratory chain enzymes, including
succinate: ubiquinone oxidoreductase (complex II) and the
membrane-bound E. coli DMSO reductase (DmsABC). The
presence of such energy barriers in electron transfer relays
remains unexplained.
Another intriguing aspect of the electron transfer relay
of NarGHI is its length. The membrane-extrinsic NarH
subunit coordinates a chain of four [Fe–S] clusters
(FS4–FS1) that is supplemented by a fifth cluster in NarG
(FS0). It represents an archetype of a subunit architecture
found in a large family of prokaryotic enzymes. Amongst
the other well characterized [Fe–S] cluster containing elec-
tron transfer relays, most contain only three clusters (e.g.
complex II). The prominent exception to this simplifying
trend is exemplified by the membrane-extrinsic arm of the
Thermus thermophilus nicotinamide adenine dinucleotide
(NADH): ubiquinone oxidoreductase (complex I), whose
Handbook of Metalloproteins, Online © 2006–2008 John Wiley & Sons, Ltd.
This article is © 2008 John Wiley & Sons, Ltd.
This article was published in the Handbook of Metalloproteins in 2008 by John Wiley & Sons, Ltd.
DOI: 10.1002/0470028637.met235
His546
MGD-Q
His1163
electron transfer relay comprises nine [Fe–S] clusters, seven
of which appear to be on a logical electron transfer
relay connecting its NADH-binding active site to the as
yet structurally uncharacterized membrane-intrinsic arm of
complex I.31
The precise mechanism of nitrate reduction has
confounded oversimplification. For example, a simplified
redox cycle for the enzyme would envision an oxo-transfer
reaction in which an oxo group on the oxidized Mo +6
metal center is lost as OH− /H2 O when the cofactor is
reduced to the Mo +4 state. Nitrate would then bind to
the reduced state and is reduced to nitrite, which is released
leaving behind a nitrato oxygen to regenerate the oxo group
on the Mo +6 species.
Recent protein film voltammetry studies have indicated
that while the oxo-transfer aspects of this mechanism are
valid, substrate binding appears to occur to the intermediate
Mo +5 state of the cofactor, and that this binding
is enhanced by the protonation of an ionizable moiety
within the protein with a pKa of approximately 7.8.32
Interestingly, with low-nitrate concentrations (<250 µM),
increasing the thermodynamic driving force (more negative
electrode potentials) decreases catalytic activity. These
results suggest that a redox transition (a reduction)
occurring in the vicinity of the cofactor inhibits nitrate
reduction. It is tempting to speculate that the ‘regulating’
species undergoing reduction is the FS0 cluster of NarG,
but it could also represent an as-yet-uncharacterized redox
transition associated with one of the pterins of the cofactor.
The Mo center of NarGHI was scrutinized extensively
by EPR, ranging from studies carried out in the 1970s12
to more recent studies that also incorporated site-directed
mutants and enzymology.33 In each case, a common theme
is the observation of hyperfine splittings in the Mo +5 EPR
associated with a pKa very similar to that discussed above.
A strong correlation exists between catalytic activity and
protonation of the enzyme. Furthermore, at pH 5.85, it was
observed that Mo (+5/+6) transition is not readily observed
in potentiometric titrations,33 increasing the availability of
the catalytically competent Mo +5 state at high redox
6
 The membrane-bound nitrate reductase A from Escherichia coli: NarGHI
potentials. It should be noted that both the protonated and
unprotonated forms of the enzyme active site appear to be
catalytically competent, with catalysis being quicker via the
protonated form.
The precise details of the protonation and the func-
tionality responsible for it will likely be delineated by
mutagenesis of conserved residues within the enzyme
active site. An obvious candidate is His546 identified
by Moura and coworkers28 as important in the ‘Asp
pendulum’ hypothesis.
FUNCTIONAL DERIVATIVES
The X-ray structure of NarGHI in complex with PCP
Previous biochemical, kinetic, and biophysical studies
showed the presence of at least one quinol binding and
oxidation site (Q-site) in NarI, in proximity to the periplasm
and the distal heme bD (QD ).21,34–37 These data were further
supported by the 2.0-Å resolution structure of NarGHI in
complex with the quinol binding inhibitor PCP.38 PCP is
structurally related to the physiological quinol substrates of
NarGHI and it was shown to strongly inhibit the enzyme
activity and to alter the spectroscopic properties of the
heme bD .38 PCP binds into a small hydrophobic pocket
delimited by helices II and III and located in proximity to
the periplasmic side of the membrane (Figure 7). Most of
the residues lining the pocket (Gly65, Gly69, Ala90, and
Gly94) are highly conserved among the diverse bacterial
NarI subunits. The hydroxyl group of PCP forms two
hydrogen bonds, with one of the propionate groups of
bD
His66
Thr143
PCP
Ser147
Lys86
Gln87
Figure 7 A detailed view of PCP binding to NarI. Hydrogen
bonds are indicated in red.
Handbook of Metalloproteins, Online © 2006–2008 John Wiley & Sons, Ltd.
This article is © 2008 John Wiley & Sons, Ltd.
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DOI: 10.1002/0470028637.met235
bD (2.8 Å) and with the Nε of His66, which coordinates
bD (2.8 Å). The edge-to-edge distance between PCP and bD
is 2.8 Å, which is well within the physiological limit for
electron transfer.20 In light of the structure and further
modeling and mutagenesis investigation, it was proposed
that the PCP pocket mimics the physiological QD -site. The
conserved Lys86 was suggested to be an essential residue
in defining the QD -site and in facilitating the binding
of the electron donors.38 A recent study has supported
this hypothesis, showing that Lys86 is required for the
stabilization of a semiquinone radical which is located
at the QD site and most likely involved in the catalytic
mechanism.39 Although the crystal structures of Q sites
characterized to date show no structural similarities, two
other respiratory enzymes, the bD quinol oxidase40,41 and
the E. coli fumarate reductase,42,43 possess a lysine residue
involved in quinol oxidation.
Quinol oxidation is coupled to proton translocation.21
Does the NarGHI-PCP crystal structure suggest a possible
proton pathway from the Q site to the periplasm? A
network of water molecules can be observed in the 9-Å path
between the propionate groups of bD and the perisplasmic
space. The four water molecules are the only ones buried
within NarI; they are highly ordered and stabilized by
extensive hydrogen bonding among themselves and the
surrounding residues. ‘Proton wires’ formed by water
molecules were already proposed in the E. coli FdnGHI27,44
and the yeast cytochrome bc1 complex.44
ATOMIC SNAPSHOTS OF THE
Fdn G H I/Nar G H I R E D O X L O O P
The E. coli nitrate respiratory chain is formed by two
membrane-bound enzymes, FdnGHI and NarGHI. This
respiratory chain is the paradigm for the ‘proton motive
force redox loop’, which was proposed by Mitchell in
his chemiosmotic theory.7 Proton movement across the
membrane, which generates proton motive force, is driven
by a redox loop between the two distinct enzymes.
Now the high-resolution crystal structures of the two
protein complexes have been resolved as well as their
functional derivatives. FdnGHI (1.6-Å resolution)27 and its
redox counterpart NarGHI (1.9-Å resolution)10 are highly
similar. They are both formed by three subunits with a
soluble domain, FdnGH and NarGH, and a TM anchor,
FdnI, and NarI. Despite differences in the fold of the TM
subunits, NarI and FdnI, NarG superimposes on FdnG
with root-mean-square (rms) deviation of 2.46 Å over 634
Cα atoms and NarH superimposes on FdnH with an rms
deviation of 1.8 Å over 158 Cα atoms. Both enzymes form
oligomers, trimers of FdnGHI, and dimers of NarGHI,
which are oriented in opposite directions with respect to
the membrane: toward the periplasm FdnGHI and toward
the cytoplasm NarGHI. Their different topology is the key
7
The membrane-bound nitrate reductase A from Escherichia coli: NarGHI
Nitrate reduction
NarGHI
Electron
transfer
Cytoplasm
Q site
Q site
FdnGHI
Formate oxidation
Figure 8 Atomic view of the FdnGHI–NarGHI redox loop and formation of proton motive force across the membrane.
for energy-efficient proton motive force generation. Fdn
and Nar possess eight redox cofactors: two b-type hemes,
five iron–sulfur clusters, and one Mo-bisMGD. By X-ray
crystallography, the Q sites in FdnGHI and NarGHI were
characterized, located at the cytoplasmic and periplasmic
sides of the membrane, respectively.10,27 Functional and
structural information provide today an atomic framework
of the nitrate redox loop (Figure 8). Formate is oxidized
in the periplasm by FdnGHI to CO2 and H+ , releasing
two electrons, which are channeled through the redox
cofactors to the Q site, where menaquinone is reduced to
menaquinol taking up two protons from the cytoplasm.
Then, the menaquinol diffuses within the lipid bilayer and
binds to the NarGHI Q site, where it undergoes oxidation,
releasing two protons in the periplasm and transferring
two electrons to the nitrate reduction site in NarGHI.
Nitrate is reduced to nitrite in the cytoplasm, consuming
two cytoplasmic protons. Energy is conserved and proton
motive force is generated.
ACKNOWLEDGEMENTS
I thank R. A. Rothery for critical reading and scientific
discussions. I thank N. C. Strynadka for great supervision
and support when I solved the NarGHI structure in her lab
at University of British Columbia, Vancouver, Canada.
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This article is © 2008 John Wiley & Sons, Ltd.
This article was published in the Handbook of Metalloproteins in 2008 by John Wiley & Sons, Ltd.
DOI: 10.1002/0470028637.met235
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Handbook of Metalloproteins, Online © 2006–2008 John Wiley & Sons, Ltd. 9

This article is © 2008 John Wiley & Sons, Ltd.
This article was published in the Handbook of Metalloproteins in 2008 by John Wiley & Sons, Ltd.
DOI: 10.1002/0470028637.met235