Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)
Daniel Schneider, Thomas Pohl, Julia Walter1, Katerina Dörner, Markus Kohlstädt, Annette Berger2,
Volker Spehr3, Thorsten Friedrich⁎
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104 Freiburg, Germany
a r t i c l ei n f o a b s t r a c t
Received 31 January 2008
Accepted 3 March 2008
Available online 15 March 2008
The proton-pumping NADH:ubiquinone oxidoreductase is the firstof the respiratorychain complexes in many
bacteria and the mitochondria of most eukaryotes. In general, the bacterial complex consists of 14 different
subunits. In addition to the homologues of these subunits, the mitochondrial complexcontains approximately
31 additional proteins. While it was shown that the mitochondrial complex is assembled from distinct
intermediates, nothing is known about the assembly of the bacterial complex. We used Escherichia coli
mutants, inwhich the nuo-genes coding the subunits of complex I were individually disrupted byan insertion
of a resistance cartridge to determine whether theyare required for the assembly of a functional complex I. No
complex I-mediated enzyme activity was detectable in the mutant membranes and it was not possible to
extract a structurally intact complex I from the mutant membranes. However, the subunits and the cofactors
of the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of some of the
nuo-mutants. It is discussed whether this fragment represents an assembly intermediate. In addition, a
membrane-bound fragment exhibiting NADH/ferricyanide oxidoreductase activity and containing the iron–
sulfur cluster N2 was detected in one mutant.
© 2008 Elsevier B.V. All rights reserved.
The proton-pumping NADH:ubiquinone oxidoreductase, also
called respiratory complex I, is the least understood of all the re-
spiratory enzyme complexes due to its enormous complexity [1–7].
The eucaryotic complex consists of 45 different subunits, seven of
which are encoded by mitochondrial DNA [1,2,8]. The bacterial com-
plexI representsa structuralminimalformof theproton-translocating
NADH:ubiquinone oxidoreductase generally consisting of 14 different
subunits [5,7,9]. In Escherichia coli the genes are named nuoA to nuoN
with nuoC and nuoD fused to one gene [10–12]. Electron microscopy
revealed that complex I consists of a peripheral and a membrane arm
[13–15]. The six peripheral subunits NuoB, CD, E, F, G, and I build
the peripheral arm and contain the known redox groups, namely
one flavin mononucleotide (FMN) and nine iron–sulfur (Fe–S) clusters
complex I was solved at 3.3 Å resolution [7,17]. The remaining seven
subunits are hydrophobic proteins predicted to fold into 61 α-helices
across the membrane, a value supported by electron microscopy .
Thesesubunitscomprisethemembrane arm.Little isknownaboutthe
function of the seven hydrophobic subunits, but they are most likely
involved in ubiquinone reduction and proton translocation [9,20].
The assembly of the mitochondrial complex I from eukaryotes has
been investigated in Neurospora crassa and human cells lines. It was
shown that the peripheral arm and the membrane arm assemble
independently from each other and are fused en bloc to build the
holoenzyme [21–23]. The membrane arm itself is assembled from two
intermediates with the participation of chaperones [23–25]. However,
a different, more complicated model was proposed for the assembly of
the human complex I . Nothing is known about the assembly of
the bacterial complex. Phylogenetic analyses have shown that the
bacterial complex evolved from preexisting modules for electron
transfer and proton translocation [3,9,27,28]. The soluble NADH de-
hydrogenase module comprises the electron input module of complex
I. It is connected to the hydrophilic part of an amphipatic hydrogenase
module, which is also present in a familiy of multisubunit membra-
neous hydrogenases . The hydrophobic part of the hydrogenase
module is connected to the so-called transporter module containing
homologues of multisubunit monovalent cation/proton antiporters
[3,28,30]. It is reasonable to assume that assembly intermediates of
the bacterial complex possibly resemble these modules.
Fragments of the complex reminiscent to the modules described
above have been obtained by splitting the isolated E. coli complex
[31,32]. The soluble NADH dehydrogenase fragment comprises the
subunits NuoE, F and G and harbors the FMN, the binuclear Fe–S
clusters N1a and N1b, and the tetranuclear Fe–S clusters N3, N4, N5,
and N7 [16–18]. The NADH dehydrogenase fragment corresponds
to the above mentioned NADH dehydrogenase module concerning
Biochimica et Biophysica Acta 1777 (2008) 735–739
⁎ Corresponding author. Tel.: +49 761 203 6060; fax: +49 761 203 6096.
E-mail addresses: Thorsten.Friedrich@uni-freiburg.de,
Thorsten.Friedrich@ocbc.uni-freiburg.de (T. Friedrich).
1Present address: Freie Universität Berlin, Institut für Immunologie und Moleku-
larbiologie, Philippstraße 13, 10115 Berlin, Germany.
2Present address: Berger Consulting, Meringer Str. 20, 86511 Schmiechen, Germany.
3Present address: Intervet Innovation GmbH, Drug Discovery, Zur Propstei, 55270
0005-2728/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
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subunit and cofactor composition. This fragment is also obtained by
overexpression of the genes nuoB-G [7,11]. Expression of nuoB and
nuoCD is essential for the assembly of the fragment, but the subunits
are not contained in the preparation .
Here, we report that the deletion of any of the nuo-genes resulted
in a loss of complex I activity in the membrane. However, the NADH
dehydrogenase fragment was assembled and equipped with the co-
factors in the cytoplasm of several nuo-mutants. The possible role of
this fragment as an assembly intermediate is discussed in the light of
the evolution of the bacterial complex I. A membrane-bound fragment
with a yet unknown subunit composition was found in one mutant.
2. Loss of a functional complex I in nuo-mutants from
To exemplarily determine whether the nuo-genes that code for
subunits of the hydrogenase module and the transporter module are
necessary for the assembly of a functional complex I in E. coli, the
genes nuoB, H, I, and N on the chromosome of E. coli strain AN387 
were disrupted by insertion of a kanamycin cartridge. In brief, a
fragment from the E. coli nuo-operon containing nuoA and B and one
containing nuoE to N  were cloned in pT7T319U using the PstI and
the SmaI/PstI restriction sites, respectively. For the construction of the
plasmid pTnuoB::Kmrthe Km cartridge was cut from pCHK20 and
inserted into nuoB, leaving 1.3 kb and 1.0 kb flanking regions from the
E. coli DNA, respectively. For the construction of the plasmid pTnuoH::
Kmrthe Km cartridge was inserted into nuoH, leaving 3.1 kb and 3.6 kb
flanking regions from the E. coli DNA, respectively. For the construc-
tion of the plasmid pTnuoI::Kmr, a BamHI/ClaI fragment was ligated in
pT7T3 19U and the Klenow-treated Km cartridge cut with BamHI was
inserted into the blunt ended KpnI restriction of nuoI, leaving 1.0 kb
and 1.1 kb flanking regions, respectively. For the construction of
pTnuoN::Kmra HincII/PstI fragment was ligated in pT7T3 19U and the
Km cartridge was inserted into the BamHI restriction site of the nuoN
gene, leaving 1.1 kb and 1.6 kb flanking regions, respectively.
Restriction endonuclease mapping of the plasmids confirmed the
same direction of the KmRgene and of the nuo-genes (data not
shown). E. coli strain JC7623 was transformed with these plasmids
individually and chromosomal mutants were selected on kanamycin
plates. Double crossovers replacing the corresponding chromosomal
nuo-gene by the respective nuo::Km constructions were identified
by screening Km transformants for ApS. The chromosomal nuo::Kmr
constructions were transferred from strain JC7623 into strain AN387
by bacteriophage P1 transduction. Transductants were selected for
KmRand screened for ApS. The correct integration into the chromo-
some was confirmed by southern blot analysis (data not shown). The
resulting mutant strains were named ANN021 (nuoB::Km), ANN081
(nuoH::Km), ANN091 (nuoI::Km) and ANN141 (nuoN::Km).
The growth rate of the nuo-mutant strains was approximately 1.5
times less than that of the parental strain in M9-minimal medium
supplemented with mannitol. To examine the presence of a functional
complex I, the d-NADH:decyl-ubiquinone oxidoreductase activity of
the cytoplasmic membranes from the parental strain and the nuo-
mutants was determined. The titration was performed with the arti-
ficial substrate d-NADH due to the presence of the alternative NADH
dehydrogenase, which has a low affinity to but a high turnover
withNADH. Complex I hasthe sameaffinitytod-NADHand NADH, but
d-NADH is a poor substrate for the alternative NADH dehydrogenase
. Membranes from the parental strainrevealed biphasickinetics as
reported . At high d-NADH concentrations the activity of the
alternative NADH dehydrogenase was measured marked by its low
affinity of approximately KM
concentrations the activity of complex I is measured as indicated by
the extrapolated KM
kinetics resulted from the overlap of the activities of the two
NADH dehydrogenases. The membranes of the nuo-mutant strains
exhibited a monophasic titration curve with an KM
mately 100 µM, indicating that complex I is not active in the mutant
The proteins of the cytoplasmic membrane of the parental strain
and the strains ANN021, ANN081, ANN091, and ANN141 were
solubilized with dodecyl-maltoside and were applied to sucrose gra-
dient centrifugation as described . The artificial NADH/ferricya-
nide oxidoreductase activity of the fractions of the gradient mediated
by the NADH-binding domain of complex I was measured. It is well
known that complex I sediments through two thirds of the gradient
under the given conditions [31,35,36]. While this was the case with
the detergent extract of the membranes from the parental strain, no
activity peak was detected at the corresponding position in the extract
from the nuo-mutant strains (data not shown). Neither was an
additional peak at a different position observed. Thus, a structurally
intact complex I or a fragment of the complex exhibiting NADH/fer-
ricyanide oxidoreductase activity was not detectable in detergent
extracts of the nuo-mutant strains.
Cytoplasmic membranes of strains ANN021, ANN081, ANN091, and
ANN141 were adjusted to 50 mg/ml protein and investigated by EPR
spectroscopy to detect whether Fe–S clusters were possibly present in
fragments of the complex assembled in the mutant membranes. The
samples were reduced by NADH and spectra were recorded at 13 K.
Under these conditions, the gx,ysignal of Fe–S cluster N2 located on
subunit NuoB [17,37] was detected at g=1.91 in the EPR spectrum of
the membranes from the parental strain (Fig. 2). This signal was
missing in the membranes obtained from the nuo-mutant strains
(Fig. 2). This was in agreement with the fact that we did not detect
subunit NuoB in the cytoplasmic membranes of the nuo-mutant
strains by immunological means (data not shown).
d-NADHof 100 µM (Fig.1). At lower d-NADH
d-NADHof approximately 10 µM (Fig.1). The biphasic
Fig. 1. Double-reciprocal plots of the kinetic data of complex I and the alternative NADH dehydrogenase in E. coli cytoplasmic membranes. Plots of 1/V against 1/[d-NADH] using
membranes from parental strain (square) and strain ANN141 (circle). Titrations with membranes from strains ANN021, ANN081, and ANN091 gave similar data as derived from the
strain ANN141. 150 µg protein were applied and the concentration of decyl-ubiquinone was 20 µM.
D. Schneider et al. / Biochimica et Biophysica Acta 1777 (2008) 735–739
The cytoplasm of the nuo-mutant strains was searched for the
presence of soluble complex I subunits by immunological means.
Using antibodies directed against NuoB, CD and I, we were not able to
detect the subunits in the cytoplasm of any of the mutants (data not
shown). Therefore, we attempted to detect the Fe–S clusters of the
NADH dehydrogenase fragment by means of EPR spectroscopy in the
cytoplasm of the nuo-mutants. We have shown that the binuclear Fe–
S clusters N1a and N1b are readily detected in the cytoplasm of E. coli
strains containing this fragment, which is made up of the subunits
NuoE, F, and G [11,16]. The cytoplasm of the mutant and parental
strains was concentrated 10-fold and one aliquot was reduced by
NADH while another aliquot was diluted with the same volume buffer.
As expected, the EPR difference spectra of the NADH-reduced minus
the air-oxidized cytoplasm of the parental strain did not show the
signals attributed to clusters N1a and N1b. The signals were also not
present in the EPR difference spectra of the strains ANN021, ANN081
and ANN091, but the clusters N1a and N1b were detected in the
cytoplasm of strain ANN141 (Fig. 3). It was impossible to detect the
tetranuclear Fe–S clusters N3 and N4 of this fragment due to their
spectral overlap with Fe–S clusters of other proteins (data not shown).
The presence of N1a and N1b in the cytoplasm of strain ANN141
indicated that the NADH dehydrogenase fragment is fully assembled
in this mutant.
3. Detection of complex I fragments in nuo-mutants from
While we constructed the chromosomal nuo-mutants from strain
AN387, the ‘Keio collection’ of the Nara Institute of Science and
Technology, Japan  was open to the public. This splendid and most
helpful collection provides researchers with E. coli strains containing
an insertion of a kanamycin resistance cartridge in every known gene
and ORF. This collection is based on E. coli strain BW25113, a derivative
of E. coli K-12 BD792 . Fora systematic investigation of the effectof
disrupting the nuo-genes on the presence of a functional complex I,
we obtained all 13 nuo-mutants from the Keio collection.
As with AN387 derivatives, the growth rate of nuo-mutant strains
of E. coli BW25113 was decreased by approximately 1.5 times and
reached approximately half of the final optical density compared to
the parental strain. The presence of a functional complex was
examined by measuring the physiological NADH oxidase activity of
the cytoplasmic membranes. To determine the contribution of com-
plex I to the total NADH oxidase activity, the sensitivity of this activity
towards annonin VI, a specific complex I inhibitor not affecting the
alternative NADH dehydrogenase, was measured . Approximately
half of the specific NADH oxidase activity was inhibited by an addition
of annonin VI, indicating that half of the NADH oxidase activity of
strain BW25113 is mediated by complex I. The specific NADH oxidase
activity of all nuo-mutant strains was completely insensitive to
annoninVI, demonstrating thatit derived entirely from the alternative
NADH dehydrogenase. Thus, all nuo-genes are needed for the pro-
duction and assembly of a functional complex I.
The NADH/ferricyanide oxidoreductase activity of the cytoplasmic
membranes is an indication of the amount of both NADH dehydro-
genases present in the membranes. This artificial activity was ap-
proximately halved in the mutant membranes corroborating the loss
of complex I. To search for membrane-bound fragments of complex I
containing the NADH dehydrogenase fragment, the ratio between the
NADH oxidase and NADH/ferricyanide oxidoreductase activity of the
mutants membranes was compared. The complete loss of complex I
has no significant impact on the ratio. However, if a fragment of the
complex exhibiting NADH/ferricyanide oxidoreductase activity but
not NADH:ubiquinone oxidoreductase activity is enriched in the mu-
tant membranes, the ratio slightly decreases. The ratio was reduced by
nearly one quarter in the membranes of the nuoL mutant strain.
According to this criterion, a membrane-bound complex I fragment
with NADH/ferricyanide oxidoreductase activity was expected to be
present in the mutant. To examine this possibility, the cytoplasmic
membranes from both the parental and the nuoL mutant strain were
analyzed by EPR spectroscopy for the presence of Fe–S cluster N2 as
describedabove.Indeed,an EPR signalat g=1.91, which was attributed
to N2, was detected in both strains .
The stability and approximate molecular mass of the complex I
fragment present in the nuoL mutant strain was examined by sucrose
gradient centrifugation of dodecyl-maltoside extracts obtained from
cytoplasmic membranes. The fully assembled E. coli complex I of the
parental strain sedimented through two thirds of the gradient as
observed with other strains (Fig. 4). No activity peak was detected at
the corresponding position in the extract from all nuo-mutant strains
indicating that none of the mutants contained a fully assembled
complex I as expected from the insensitivity of their NADH oxidase
activity to the specific complex I inhibitor annonin VI. However, a
Fig. 2. EPR spectra of NADH-reduced cytoplasmic membranes from E. coli strains
ANN141 (A) and AN387 (B). The protein concentration was adjusted to 50 mg/mL.
Spectra similar to (A) were obtained from the membranes of strains ANN021, ANN081,
and ANN091. The signal attributed to cluster N2 at g=1.91 is indicated by an arrow. The
spectra were recorded at 13 K and 10 mW. Other EPR conditions were: microwave
frequency, 9.44 GHz; modulation amplitude, 0.6 mT; time constant, 0.124 s; scan rate,
Fig. 3. EPR difference spectra of the cytoplasm fromvarious E. coli strains. (A) shows the
spectrum of the cytosol from BL21(DE3) pET-24/nuoB–G, overproducing the NADH
dehydrogenase fragment , (B) spectrum from strain ANN141, and (C) spectrum from
strain AN387. The spectra were recorded at 40 K and 1 mW. The protein concentration
was adjusted to 40 mg/mL. Other EPR conditions were as given in Fig. 2. The signals of
cluster N1a (gx, y, z=1.92, 1.94, and 2.00) and N1b (g//,⊥=2.03, 1.94) are indicated. A
radical signal overlaps the signal at g=2.00 in (A). The amount of cluster N1b relative to
N1a is reduced in (B).
D. Schneider et al. / Biochimica et Biophysica Acta 1777 (2008) 735–739
prominent peak of the NADH/ferricyanide oxidoreductase activity was
detected in the extract from the nuoL mutant in the middle of the
gradient (Fig. 4). According to its position, the peak most likely
represents a fragment of complex I missing subunit NuoL, which has a
molecular mass of 66.4 kDa, but no other subunits.
As described above, the cytoplasm of the nuo-mutants from strain
BW25113 was searched for the presence of the binuclear Fe–S clusters
N1a and N1b of the NADH dehydrogenase fragment. The signals of
these clusters were detected in the difference spectra of the cytoplasm
of the nuoCD, nuoH, nuoI, nuoJ, nuoK, and nuoN mutant strains. This
indicated that the NADH dehydrogenase fragment is fully assembled
not only in the nuoN mutant as observed with AN387 strain. It was not
possible to isolate the fragment from the mutant strains due to its
instability. To provethat subunitsNuoE, F, and G comprisingthe NADH
dehydrogenase fragment are assembled in one complex, we separated
the cytoplasmic proteins by BN-PAGE and the position of any NADH
dehydrogenase was determined by staining the gel with NADH and
NBT . A sharp band with an apparent molecular mass of
approximately 200 kDa was detected. The subunits NuoE (18.6 kDa),
NuoF (49.3 kDa), and NuoG (100.2 kDa) were detected by means of
Western blot analysis using specific antibodies at the same position in
the gel and at the same height as the band stained with NBT. The
molecular massof the NADH dehydrogenase fragment as derivedfrom
the DNA sequence of the individual subunits is 170 kDa, which is in
good agreement with the mass determined by BN-PAGE.
4. Implications for the assembly of the respiratory complex I
in E. coli
although the structure of the peripheral arm of the complex was
of the assembly of this huge and modular machinery may shed light on
the function of the individual modules and their interaction within the
complex. Our data show that inactivation of any of the E. coli nuo-genes
the assembly of a functional complex I. This was demonstrated by the
loss of any complex I-mediated activity in the mutant membranes and
the failure to detect the fully assembled complex in detergent extracts
cartridge did not cause a strong polar effect because we were able to
complement the nuoB and nuoF mutants with the wild-type gene in
trans . The slightly reduced complex I activity measured in all these
experiments is most likely due to a mild polar transcriptional effect of
the integration mutagenesis. The marginal polar effect of the inserted
kanamycin resistance cartridge has also been reported for the
mutagenesis of the nuo-operon in Rhodobacter capsulatus .
The EPR signals of the binuclear Fe–S clusters N1a and N1b of the
NADH dehydrogenase fragment were detected in the cytoplasm of the
nuoCD, nuoH, nuoI, nuoJ, nuoK, and nuoN mutants of strain BW25113,
This might indicate that the stability of the fragment is different in the
two strains. Therefore, we cannot completely rule out the possibility
nuo-mutants, although we were neither able to detect the binuclear
Fe–S clusters nor the subunits NuoE, F, and G in the cytoplasm of the
nuoA, nuoB, nuoL, and nuoM mutant strains. The absence of the NADH
dehydrogenase fragment in the nuoE, nuoF, and nuoG mutants is
cytoplasmic proteins of the nuoN mutant by BN-PAGE, a single band
was detected consisting of the subunits NuoE, F, and G and showing
NADH dehydrogenase activity . The position of the band cor-
responds to a protein of a molecular mass of approximately 200 kDa
indicating that no other complex I subunits are associated with the
fragment. Thus, the NADH dehydrogenase fragment with the FMNand
at the least two binuclear Fe–S clusters is assembled in the cytoplasm
of the nuoN mutant strain. We assume that the fragment also contains
the tetranuclear clusters, which were, however, not detectable in the
proteins. We propose that the NADH dehydrogenase fragment might
represent an assembly intermediate of complex I in E. coli, which
would be compatible with both evolutionary schemes that have been
dehydrogenase fragment is the latest acquisition of the progenitor of
today's complex I of bacteria and eukaryotes. However, we cannot
exclude the possibility that it is a stable fragment resulting from the
decay of a larger assembly intermediate present in the corresponding
nuo-mutant strains. To discriminate between these two possibilities,
we arecurrentlystudyingtheassemblyof the E. colicomplex bypulse-
Most surprisingly, a membrane-bound complex I fragment with-
out a detectable NADH oxidase activity was found in the nuoL mutant
strain . Due to its instability we were not able to characterize its
subunit composition. As this fragment exerts NADH/ferricyanide
oxidoreductase activity and contains cluster N2 it should at least
comprise subunits NuoB, E, F, and G. From its position after sucrose
gradient centrifugation it should be made up of all complex I subunits
with the exception of NuoL. It was shown by electron microscopy that
NuoL and NuoM are located at the distal part of the membrane arm of
the complex and can be individually removed from the entire complex
while retaining the other complex I subunits [20,43]. It is discussed
that NuoL is involved in proton translocation, due to its homology to
subunits of multisubunit monovalent cation/proton antiporters. Thus,
NuoL is of central importance to the enzyme's mechanism [3,15]. In
order to understand the function of the complex, it will be crucial to
determine, whether this fragment exerts any electron transfer or
proton translocation activity at all. Due to its lacking abundance in the
mutantmembrane,itis possible thatthere is residual electron transfer
activity in the fragment, which is obscured by the activity of the
alternative NADH dehydrogenase. Currently, our aim is to overexpress
the nuo-operon lacking nuoL from the nuo-expression plasmid
developed recently in our laboratory  and to characterize the
properties of the purified fragment.
Thiswork in theauthorslaboratorywassupportedbythe Deutsche
Forschungsgemeinschaft and the Volkswagen Stiftung. We thank Drs.
Fig. 4. Sucrose gradient centrifugation from dodecyl-maltoside solubilized cytoplasmic
membranes from strainsBW25113 (circle) and BW25113 nuoL::nptI (square). Membrane
proteins were separated by means of gradients of 5–30% (w/v) sucrose in 50 mM MES/
NaOH, pH 6.0, 50 mM NaCl and 0.1% dodecyl-maltoside. The activities were stan-
dardized to 30 mg protein loaded on each gradient. Fractions of the gradients
(numbered 1–20 from top to bottom) were collected and analyzed for NADH/ferri-
cyanide oxidoreductase activity.
D. Schneider et al. / Biochimica et Biophysica Acta 1777 (2008) 735–739
Takao Yagi and Akemi Matsuno-Yagi for the kind gift of the specific Download full-text
antibodies directed against NuoE, NuoF, and NuoG. We thank the Keio
collection, Nara Institute, Japan, for providing us with nuo-mutant
strains. We are grateful to Linda Williams for her help in preparing the
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