From the Institut für Org. Chemie und
Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse
21, D-79104 Freiburg, Germany and the ¶ Institut für
Biophysik, Johann-Wolfgang-Goethe-Universität,
Theodor-Stern-Kai 7, Haus 75, 60590 Frankfurt/M.,
Germany
Received for publication, August 29, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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The proton-pumping NADH:ubiquinone oxidoreductase
(complex I) couples the transfer of electrons from NADH to ubiquinone
with the translocation of protons across the membrane. Electron
transfer is accomplished by FMN and a series of iron-sulfur clusters.
Its coupling with proton translocation is not yet understood. Here, we
report that the redox reaction of the FeS cluster N2 located on subunit
NuoB of the Escherichia coli complex I induces a
protonation/deprotonation of tyrosine side chains. Electrochemically
induced FT-IR difference spectra revealed characteristic tyrosine
signals at 1,515 and 1,498 cm The NADH:ubiquinone oxidoreductase, also known as respiratory
complex I, links the electron transfer from NADH to ubiquinone with the
translocation of protons across the membrane. By this means, complex I
establishes a proton motive force required for energy consuming
processes (1-3). Homologues of the complex are present in archaea,
bacteria, and eukaryotes (4, 5). Complex I from bacteria generally
consists of 14 different subunits (4-6). Seven of these are peripheral
proteins including those subunits that bear all known redox groups of
complex I, namely one FMN and up to nine iron-sulfur
(FeS)1 clusters. The
remaining 7 subunits are hydrophobic proteins predicted to fold into 54 The genes of the Escherichia coli complex I are named
nuoA to nuoN (7). nuoC and
D are fused in E. coli giving rise to 13 different subunits assembling the complex (8). The preparation of the
E. coli complex I contains one non-covalently bound FMN, two
binuclear (N1a and N1b), and five tetranuclear (N2, N3, N4, N6a, and
N6b) FeS clusters (Refs. 9, 10, and
11).2 The tetranuclear
cluster N5 present in complex I from other species has not yet been
detected in this preparation. However, it should be present because of
the preservation of the corresponding binding motif (7, 12, 13). The
isolated complex can be split into a so-called NADH dehydrogenase
fragment, a connecting fragment, and a membrane fragment (8, 9). The
soluble NADH dehydrogenase fragment harbors the FMN and the
EPR-detectable FeS clusters N1a, N1b, N3, and N4 (Refs. 9, 14, and
15).2 The amphipathic connecting fragment contains the FeS
clusters N2, N6a, and N6b (9, 11), while a chromophore with a yet unknown chemical structure, has been detected in the membrane fragment
(16-18).
The so-called isopotential FeS clusters N1a, N1b, N3, N4, N6a, and N6b
show similar midpoint potentials ranging from If cluster N2 is involved in proton pumping, its redox reaction should
be coupled to a protonation/deprotonation of the cluster itself and/or
of surrounding amino acids. Recently, we were able to show that the
reduction of N2 is coupled with a deprotonation of an Asp or Glu side
chain by means of electrochemically induced FT-IR difference spectra
(18, 21). In this study, we show that the reduction of cluster N2 is
associated with the protonation of two tyrosine side chains. Mutants of
the three conserved tyrosines on NuoB were generated by complementation
of a chromosomal nuoB deletion strain with expression of
nuoB from a plasmid. Complementation of the nuoB
deletion strain with either wild-type or mutated nuoB resulted in a fully assembled, active, and stable complex I. EPR spectroscopy of complex I isolated from the mutants revealed a shift in
the signal of cluster N2. While the single mutants exhibited wild type
NADH oxidase activity, the Y114C/Y139F double mutant showed an
80% reduced activity. FT-IR spectroscopy revealed that tyrosines 114 and 139 on NuoB are both involved in the protonation reaction coupled
with the reduction of N2.
Materials and Strains--
E. coli strains DH5 Site-directed Mutagenesis and Expression of the nuoB
Gene--
E. coli genomic DNA was prepared with the Genomic
DNA purification kit (MBI). The entire nuoB gene was
amplified from E. coli genomic DNA as template and cloned
into pSTBlue-1. Mutations in nuoB were created using either
the QuickChange or ExSite PCR-based site-directed mutagenesis kit (both
from Stratagene) and are given in Table
I. Sequences of all plasmids were
confirmed by DNA sequencing. pSTBlue-1 containing nuoB was
digested with KpnI/SacI, and the resulting 860-bp
fragment was cloned into the expression vector pBAD33. The resulting
plasmids were named pBAD-Btype with
type defining either the wild type gene or the corresponding mutation. The plasmids carrying the mutation were used for
transformation of the deletion strain ANN023. Transformants were grown
in a 10-liter culture of LB medium at 37 °C. Arabinose was added to
a final concentration of 0.2% (w/v) at an OD600 of
approximate 0.5. Three hours after induction, 30-40 g of cells were
harvested by centrifugation for 10 min at 4,000 × g.
Cells were washed with 50 mM MES/NaOH, pH 6.0 and stored at
Isolation of Complex I from NuoB Mutants--
In principle,
complex I was isolated by means of a procedure developed for a strain
overproducing complex I (10). However, the membranes of the mutant
strains were not washed with NaBr to remove ATP synthase as described
due to the lower amount of complex I. Therefore, the preparations
contained detectable amounts of ATP synthase, which, however, did not
interfere with our assays. All steps were carried out at 4 °C.
60 g of cells were resuspended in 150 ml of 50 mM
MES/NaOH, 0.1 mM phenylmethanesulfonyl fluoride, pH 6.0, with 10 µg/ml DNase I and disrupted by a single pass through a French
Pressure cell (SLM Aminco) at 110 MPa. Cell debris was removed by
centrifugation for 20 min at 36,000 × g, and
cytoplasmic membranes were obtained by centrifugation for 1 h at
250,000 × g. Membrane proteins were extracted by
adding dodecyl maltoside to a final concentration of 3%. The solution
was gently homogenized and centrifuged for 1 h at 250,000 × g. The supernatant was applied to a 60-ml Source 15Q
(Amersham Biosciences) column equilibrated in 50 mM
MES/NaOH, 50 mM NaCl, and 0.1% dodecyl maltoside, pH 6.0. The column was eluted with a 700-ml linear gradient of 50-350 mM NaCl in 50 mM MES/NaOH, 0.1% dodecyl
maltoside, pH 6.0 at a flow rate of 4 ml/min. Fractions containing
NADH/ferricyanide reductase activity were combined, concentrated by
precipitation with 9% (w/v; final concentration) polyethylene glycol
4,000 and dissolved in 2 ml of 50 mM MES/NaOH, 50 mM NaCl, and 0.1% dodecyl maltoside, pH 6.0. The protein
was subjected to size-exclusion chromatography on a 180-ml Ultrogel AcA
34 (Serva) column in 50 mM MES/NaOH, 50 mM
NaCl, and 0.1% dodecyl maltoside, pH 6.0, at a flow rate of 7 ml/h.
Peak fractions were pooled and applied to a 10- ml Source 15Q (Amersham
Biosciences) equilibrated as described above and eluted with a 100-ml
linear gradient of 50-350 mM NaCl in the same buffer at a
flow rate of 2 ml/min. Fractions with NADH/ferricyanide reductase
activity were pooled and stored at Electrochemistry--
The ultrathin layer spectroelectrochemical
cell for UV/Vis and IR was used as previously described (25).
Sufficient transmission in the 1,800 cm FT-IR Spectroscopy--
FT-IR difference spectra as a function
of the applied potential were obtained simultaneously from the same
sample with a setup combining an IR beam from the interferometer
(modified IFS 25, Bruker, Germany) for the 4,000 cm EPR Spectroscopy--
Low temperature EPR measurements were
conducted with a Bruker EMX 1/6 spectrometer. The sample temperature
was controlled with an Oxford Instruments ESR-9 helium flow cryostat.
Complex I at 10 mg/ml was reduced with a 1,000-fold molar excess of
NADH in the presence of redox mediators as described (9). The EPR tube
was frozen in 5:1 isopentane/methylcyclohexane (v/v) at 150 K. The
magnetic field was calibrated using a strong pitch standard.
Enzyme Activity--
The NADH:decylubiquinone reductase activity
of complex I was measured with a Perkin Elmer 156 dual-wavelength
spectrophotometer using the wavelength 340 and 400 nm and Other Analytical Procedures--
Protein concentration was
measured with the biuret method using bovine serum albumin as standard.
Sucrose gradient centrifugation in the presence of 0.2% dodecyl
maltoside was performed as described (9). Following SDS-PAGE protein
bands were either stained with Coomassie Blue or electroblotted onto
0.45-µm pore size nitrocellulose membrane (Schleicher und
Schüll) according to Ref. 31. A rabbit polyclonal antibody raised
against NuoB was used for detection.
FT-IR Spectroscopy of Wild Type Complex
I--
Electrochemically-induced FT-IR difference spectra of complex I
contain contributions from redox-dependent changes in the protein structure and protonation states of cofactors and individual amino acids. In order to detect changes associated with the redox reaction of FeS cluster N2 we recorded FT-IR spectra of complex I at
the potential step from
The double difference spectrum (Fig. 1A, c)
showed spectral contributions from rearrangement of the polypeptide
backbone and contributions of individual amino acids such as the
protonation/deprotonation of aspartic or glutamic side chain(s) as
reported (21). In addition, the spectrum showed contributions at 1,515 and 1,500 cm Complementation of the nuoB Deletion--
Approximately 70% of
nuoB was deleted from the chromosome of wild type strain
AN387 without interrupting the reading frame.3 The
resulting strain ANN023 was complemented with the wild type nuoB on the plasmid pBAD33, under control of the inducible
pBAD promotor. This strain was called
ANN023/pBAD33-BWT.
The presence of NuoB in the strains AN387, ANN023, and
ANN023/pBAD33-BWT was determined by reaction of
a specific antibody with blotted membrane proteins after SDS-PAGE (Fig.
2). NuoB was detected in the membranes of
the strains AN387 and ANN023/pBAD33-BWT while it
was missing in the membranes of strain ANN023.
We tested whether NuoB expressed from the plasmid was able to assemble
an intact complex I with the residual complex I subunits encoded on the
chromosome by sucrose gradient centrifugation (Fig. 3). Dodecyl maltoside extracts of
cytoplasmic membranes from strains ANN023 and
ANN023/pBAD33-BWT were applied on a 12-ml
sucrose gradient. Complex I from E. coli typically sediments
two thirds of the way through the gradient as detected by its
NADH/ferricyanide activity (9, 10). No NADH/ferricyanide activity was
detectable in the corresponding fractions of strain ANN023 indicating
that complex I was not assembled in the absence of NuoB (Fig. 3).
Instead, fractions in the first third of the gradient exhibited
NADH/ferricyanide activity, which stemmed from the alternative,
non-proton pumping NADH dehydrogenase. The expression of this
alternative NADH dehydrogenase is increased in the absence of complex
I. The sedimentation profile of the extract obtained from strain
ANN023/pBAD33-BWT showed NADH/ferricyanide
activity in the fractions corresponding to the wild type enzyme (Fig.
3). Western blot analysis confirmed the presence of NuoB in the
corresponding fractions (data not shown). Again, the alternative NADH
dehydrogenase was detected in the first third of the gradient but in
smaller amounts. These data showed that the assembly of complex I was
restored by complementation of nuoB on a plasmid.
The amount of complex I in the cytoplasmic membranes of these strains
was estimated from the (d-) NADH/ferricyanide activity as well
as from the (d-) NADH oxidase activity (Table
2). The artificial substrate d-NADH was
used to discriminate the two membrane-bound NADH dehydrogenases of
E. coli (30, 36). While NADH can be used by both NADH
dehydrogenases, d-NADH preferently reacts with complex I. The
d-NADH/ferricyanide activity as well as the d-NADH oxidase activity was
nearly abolished in strain ANN023 lacking an assembled complex I. These
activities were restored to wild type level in strain
ANN023/pBAD33-BWT (Table 2). The
NADH/ferricyanide and NADH oxidase activity of strain ANN023 was
decreased to about one-third of the wild type activity due to that of
the alternative NADH dehydrogenase. Two-thirds of the NADH oxidase
activity was inhibited by 10 µM piericidin A in AN387 and
ANN023/pBAD33-BWT. The NADH oxidase activity of
strain ANN023 was virtually not inhibited by piericidin A,
demonstrating the lack of complex I in this strain.
Site-directed Mutagenesis of nuoB--
Among all
subunits present in complex I from different sources, NuoB shows the
highest degree of sequence conservation (4, 7). Alignment of the
homologues of NuoB showed the presence of three tyrosine residues being
conserved within all three domains of life (Fig.
4).
Involvement of the conserved tyrosines in the
protonation/deprotonation induced by the redox reaction of cluster N2
was advanced by constructing site-directed mutants. The mutants Y114C,
Y139C, Y154H, and Y114C/Y139F of NuoB were expressed using the system described above. To determine whether complex I was assembled in the
mutant strains, the (d-) NADH/ferricyanide reductase activity and the
(d-) NADH oxidase activity of cytoplasmic membranes were measured as
described above. The strains carrying a single mutation exhibited
activities with d-NADH as substrate comparable to the wild type strain
AN387 and the complemented strain
ANN023/pBAD33-BWT (Table 2) indicating that
complex I in the mutants was able to transfer electrons from NADH to
ubiquinone. The NADH oxidase activity in these mutant strains was
inhibited by about two-thirds by 10 µM piericidin A. Thus, the amount and the activity of complex I in the membranes was not
influenced by the mutations introduced. The d-NADH/ferricyanide
activity of the strain carrying the Y114C/Y139F double mutation was
50% reduced compared with wild type and this strain showed 20% of the
d-NADH oxidase activity. NuoB was detected in the cytoplasmic membranes
from this mutants by Western blot analysis (Fig. 2). These data
suggested that half of the amount of complex I with a strongly reduced
activity is present in this strain.
Isolation of Complex I from the Mutant Strains--
For
spectroscopic characterization complex I was isolated from the mutants
Y114C, Y139C, Y154H, and Y114C/Y139F. All steps were performed in the
presence of 0.1% dodecyl maltoside at pH 6.0. As an example, the
preparation of the complex from the mutant Y114C is shown in Fig.
5. The other preparations showed similar elution profiles. The enzyme eluted from both anion-exchange
chromatography on Source Q-15 at 280 mM NaCl and from the
Ultrogel AcA34 size-exclusion column at 70 ml (Fig. 5). Thus, the
mutant complexes showed chromatographic properties comparable to the
wild type complex (10). Approximately 1-2 mg of complex I were
obtained from 60 mg of cells of the single mutant strains (Table
3), while less than 1 mg was obtained
from the double mutant strain.
Upon SDS/PAGE the preparation was resolved into the 13 complex I
subunits plus some protein bands due to the presence of ATP synthase
(Fig. 5). The presence of NuoB in the preparations was confirmed by
Western blot analysis (Fig. 2). Complex I isolated from the single
mutants catalyzed electron transfer from NADH to ubiquinone with a rate
of 2.1-2.3 µmol of NADH/min·mg in the presence of 50 µM NADH and 25 µM decylubiquinone. Complex
I isolated from the Y114C/Y139F double mutant showed no
NADH:decylubiquinone activity. Under the same conditions the wild type
exhibits an activity of 2.4 µmol of NADH/min·mg.
EPR Spectroscopic Analysis of the NuoB Mutants--
NuoB contains
three conserved cysteines which are bona fide ligands of
cluster N2 (8, 14, 37). The fourth ligand is not yet known. To
investigate whether one of the conserved tyrosines is the missing
fourth ligand, we determined the amount and spectral properties of the
FeS clusters in complex I by EPR-spectroscopy. The samples were reduced
by an excess NADH in the presence of redox mediators. The EPR spectra
recorded at 40 K were nearly identical, indicating no changes in the
microenvironment of the binuclear FeS clusters N1a and N1b (data not
shown). The spectra recorded at 13 K revealed the presence of the
EPR-detectable clusters N2, N3, and N4 (Fig.
6). The amount of cluster N2 measured as signal amplitude was diminished by about one-fifth in all single mutants. A small but clear shift of the gz
signal of N2 from 2.049 in the wild type to 2.051 in the mutant Y154H
and to 2.052 in the mutants Y114C and Y139C was detected (Fig. 6). The
gx,y signal of N2 did not change. The EPR
spectrum from the double mutant Y114C/Y139F showed no change in the
gz signal of N2 with respect to its spectral
position and amplitude. However, its gx,y signal was decreased by ~50% (Fig. 6) This spectral region was hard to evaluate due to the spectral overlap with signals from cluster N3 and
N4. From these data it was evident that neither of the three tyrosines
was a ligand of cluster N2, but that they were in close vicinity.
FT-IR Spectroscopic Analysis of the NuoB
Mutants--
The possible participation of the conserved tyrosines in
the proton pathway coupled with the redox reaction of cluster N2 was
investigated by means of FT-IR spectroscopy (21). FT-IR spectra of the
isolated complexes were recorded in an electrochemical cell for the
potential step from
In order to depict possible variations of the modes typical for the
tyrosine residues, double difference spectra were obtained for the
critical spectral range from 1,530 to 1,480 cm
The difference spectra of wild type complex I displayed a prominent
signal at 1,515 cm Knowledge of the mechanism of complex I is rather limited due to
its enormous complexity in combination with the lack of structural data. The mitochondrial complex I is made up of at least 43 different subunits (38) with seven of them being encoded on the mitochondrial genome. Together these subunits constitute a molecular mass of about 1 MDa (1, 2). Although the bacterial complex shows a simpler composition
of 14 different subunits, this number is still quite large (4-6, 39).
In addition, mutagenesis of individual subunits turned out to be very
laborious as they have to be introduced as unmarked mutations in the
chromosome. Insertion of a resistance cartridge in the nuo
operon coding the complex I subunits disturbed the assembly of the
complex.4 So far, an easy to
manage extrachromosomal system containing the 16-kb
nuo-operon is not available. Therefore, we constructed a
strain with an in-frame deletion of the complex I gene of interest, namely nuoB. The mutant ANN023 is devoid of 70% of the
nuoB reading frame. Neither NuoB nor a smaller fragment of
it was detected in the strain ANN023 (Fig. 2). The absence of NuoB was
sufficient to prohibit the assembly of the entire complex as the
membranes of strain ANN023 did not exhibit any d-NADH oxidase activity
and a strongly reduced d-NADH/ferrricyanide activity (Table 2). When the cells were supplied with nuoB on the pBAD33 expression
plasmid, complex I was rescued (Table 2). The amount of complex I in
membranes was restored to 90% compared with the wild type as judged by
the d-NADH oxidase activity and the specific d-NADH/ferricyanide
reductase activity of cytoplasmic membranes (Table 2), as well as by
the specific NADH/ferricyanide reductase activity of the membrane extract after sucrose gradient centrifugation (Fig. 3). Using this
system we were able to introduce point mutations in NuoB and to
investigate the participation of individual amino acids in proton
translocation of complex I.
The FeS cluster N2 is located on NuoB as indicated by site-directed
mutagenesis of complex I from E. coli, Yarrowia
lypolytica, and Neurospora crassa (8, 19, 37). Although
N2 is a tetranuclear FeS cluster, NuoB lacks a canonical motif for the
coordination of such a cluster (7, 12, 13). Two of four of the
conserved cysteines on NuoB are located in the vicinity.
Although it cannot be excluded, it is unlikely that both cysteines are
ligands to the same cluster. The fourth ligand of this cluster remains
to be detected. Here, we detected that the reduction of cluster N2 was
linked with the protonation of tyrosine residues (Fig. 1). It could be
possible that one of these tyrosine residues is the fourth ligand of
N2. EPR spectra of complex I isolated from the NuoB tyrosine mutants
showed the presence of N2 in almost the same amount as in the wild type
(Fig. 6). Complex I isolated from the mutants showed a shift of the
signals of N2 (Fig. 6). However, the change from a tyrosine to a
cysteine, phenylalanine, or histidine ligand would have caused a more
severe change of the cluster signal or even a loss of the cluster as it
has been reported for the E. coli and the N. crassa complex (8, 19). Therefore, the tyrosine residues on NuoB
investigated in this study were located close to but did not ligate N2.
The mutations of the conserved tyrosine residues resulted in
minor structural rearrangements of the protein backbone.
This was evident from the enzymatic activity, which was close to wild type level (Table 2) as well as from the EPR and FT-IR spectra (Figs. 6
and 7). These were the only effects detected in the mutant Y154H (and
Y154F, data not shown). However, irrespective of the general changes in
the spectra, the tyrosine signals at 1,515 and 1,498 cm The redox driven proton pump within complex I is suggested to be linked
with the redox reaction of cluster N2 due to pH dependence of its
midpoint potential (20). The pH dependence of N2 exhibits a slope of
It is not unusual that a single mutation within a proton
pathway does not correlate with a loss of electron transfer activity. Previous detailed studies of other proton pumping enzymes such as
cytochrome c oxidase showed this effect. There are two
possible explanations for this phenomenon. First, the electron transfer may be uncoupled from the proton translocation and second, single mutations in a large proton pathway do not affect turnover (Refs. 40
and 42 and references within). Double or triple mutants are needed in
the latter case for a complete inhibition of coupled electron/proton
transfer. This was demonstrated for example in the case of the highly
conserved arginine side chains bridging the propionates of hemes
a/b and
a3/o3, respectively, in
cytochrome oxidase from different organisms (42, 43) as well as by
several residues forming the D-path of cytochrome oxidase
(40, 41). Our study could represent another example where the mutation
of both tyrosine residues is needed to influence the enzymatic activity.
Mutations from a tyrosine residue to a cysteine, phenylalanine, and
histidine residue, as presented here, still allow hydrogen bonding to
be established. Therefore, it would still be possible to transfer
protons in a large proton path as found for example in cytochrome
oxidase. We note that a deletion of a putative hydrogen bonding would
have induced strong but nonspecific structural variations. This would
have prohibited a discussion of the spectroscopic data.
As a working hypothesis we speculate that tyrosine residues
114 and 139 on NuoB are protonated upon reduction of N2. The proton being released from the tyrosine residues upon oxidation of N2 is
transferred via yet unknown groups to an acidic amino acid on NuoB,
which is subsequently protonated (21). Thus, the redox reaction of
cluster N2 would be linked with the uptake of protons by tyrosine
residues and a release of protons to an acidic amino acid. This could
initiate a directed proton movement given the right spatial orientation
of the tyrosine residues and the acidic amino acid(s) around cluster N2.
1 for the protonated and
deprotonated form, respectively. Mutants of three conserved tyrosines
on NuoB were generated by complementing a chromosomal in-frame deletion
strain with nuoB on a plasmid. Though the single mutations
did not alter the electron transport activity of complex I, the EPR
signal of cluster N2 was slightly shifted. The tyrosine signals
detected by FT-IR spectroscopy were roughly halved in the mutants Y114C
and Y139C while only minor changes were detected in the Y154H mutant.
The enzymatic activity of the Y114C/Y139F double mutant was 80%
reduced, and FT-IR difference spectra of the double mutant revealed a
complete loss the modes characteristic for protonation reactions of
tyrosines. Therefore, we propose that tyrosines 114 and 139 on
NuoB were protonated upon reduction of cluster N2 and were thus
involved in the proton-transfer reaction coupled with its redox reaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices across the membrane (1, 2). They are most likely involved
in ubiquinone reduction and proton translocation. The
mitochondrial complex I of eukaryotes contains at least 29 extra
proteins in addition to the homologues of the 14 prokaryotic complex I
subunits (1, 2).
0.28 to
0.24 mV.
Together with the FMN (Em,7 =
0.26 mV) they form the
electron input part of complex I involved in NADH oxidation (4).
Electrons are transferred further to the high potential cluster N2
(Em,7 =
0.22 mV). Cluster N2 is located on
NuoB (11, 19). This subunit contains solely three conserved cysteines as possible ligands (7). The fourth ligand of cluster N2, which may
even reside on another subunit, remains to be established. The midpoint
potential of FeS cluster N2 is, in contrast to most other clusters,
pH-dependent (20). Therefore, it has been proposed that N2
is the electron donor to quinone and directly involved in proton
translocation (13, 20).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(22), AN387 (23), and ANN0233
and the plasmids pSTBlue-1 (Novagen) and pBAD33 (24) were used. Strain ANN023 is a derivative of strain AN387 and contains a chromosomal in-frame deletion of nuoB. When required for maintenance of
plasmids, chloramphenicol was added to 20 mg/liter and ampicillin to
100 mg/liter. All enzymes used for recombinant DNA techniques were from
Roche Molecular Biochemicals. All other chemicals were from Merck
(Darmstadt), Serva (Heidelberg), or Sigma.
80 °C.
Oligonucleotides used for genomic amplification and site-directed
mutagenesis
80 °C. Complex I from wild type
was prepared with the same procedure. The NADH dehydrogenase fragment
of the complex was isolated as described (15). For electrochemistry,
the preparations were concentrated by ultrafiltration (Centricon 100, Amicon) to ~0.2 mM.
1 to 1,000 cm
1 range, even in the region of strong water absorbance
around 1,645 cm
1, was achieved with the cell pathlength
set to 6-8 µm. To avoid protein denaturation, the gold grid-working
electrode was chemically modified by a 2 mM cysteamine
solution as reported (25). In order to accelerate the redox reaction,
the following mediators were used at a final concentration of 45 µM each: ferrocenyltrimethylammoniumiodide (+645 mV),
1,1'-dicarboxylferrocene (+644 mV),
diethyl-3-methylparaphenylenediamine (+367 mV), ferricyanide (+424 mV),
dimethylparaphenylenediamine (+371 mV), 1,1'-dimethylferrocene (+341
mV), tetramethylparaphenylenediamine (+270 mV), tetrachlorobenzoquinone
(+280 mV), 2,6-dichlorophenolindophenol (+217 mV), ruthenium hexamine
chloride (200 mV), 1,2-naphthoquinone (
145 mV), trimethylhydroquinone
(100 mV), menadione (
12 mV), 2-hydroxy-1,4-naphtoquinone (
125 mV),
anthraquinone-2-sulfonate (
225 mV), neutral red (
307 mV), benzyl
viologen (
360 mV), and methyl viologen (
446 mV). At the given
concentrations and with the pathlength below 10 µm, no spectral
contributions from the mediators in the visible and IR range used were
detected in control experiments with samples lacking the protein.
Approximately 6-7 µl of the protein solution were sufficient to fill
the spectroelectrochemical cell.
1 to
1,000 cm
1 range and a dispersive spectrometer for the
400-900-nm range as reported previously (26, 27). First, the protein
was equilibrated with an initial potential at the electrode and single
beam spectra in the visible and IR range were recorded. Then a
potential step toward the final potential was applied and
single beam spectra of this state were again recorded after
equilibration. Difference spectra were calculated from the two single
beam spectra with the initial single beam spectrum taken as reference.
No smoothing or deconvolution procedures were applied. The
equilibration process for each applied potential was followed by
monitoring the electrode current and by successively recording spectra
in the visible range until no further changes were observed. The
equilibration generally took less than 8 min for the full potential
step from
0.5 V to 0.0 V. Typically, 128 interferograms at 4 cm
1 resolution were coadded for each single beam IR
spectrum and Fourier-transformed using triangular apodization. Up to
5 × 20 difference spectra have been averaged. The presence of
atmospheric water vapor was avoided as described (21).
of 6.3 mM
1 cm
1. The assay contained 5 mM MES/NaOH, 0.1 mM NaCl, pH 6.0, and 50 µM NADH and 25 µM decylubiquinone. The
reaction was started by addition of 10 µg of complex I (28). The
NADH/ferricyanide reductase activity, and the NADH oxidase activity
were measured as described (29, 30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.5 to 0.0 V (Fig.
1A, a). The
difference spectrum of complex I obtained for this potential step was
compared with difference spectra of the NADH dehydrogenase fragment of complex I for the same potential step (Fig. 1A,
b).
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Fig. 1.
FT-IR spectroscopic detection of tyrosine
residues involved in the redox reaction of complex I. A,
oxidized reduced difference FT-IR spectra of complex I
(a), the NADH dehydrogenase fragment of complex I
(b), both for the potential step from
0.5 to 0.0 V, and
double difference FT-IR spectra of the difference spectra of complex I
minus the difference spectra of NADH dehydrogenase fragment
(c). B, absorbance spectra of tyrosine (Fluka)
in situ protonated upon solution in 1 M HCl
(a) and deprotonated upon solution in 1 M NaOH
(b). The contributions of the solvent were interactively
subtracted. The signals at 1,515 and 1,498 cm
1 discussed
to specifically indicate the protonation/deprotonation of tyrosine side
chain(s) are marked with arrows.
1, which were tentatively attributed to
tyrosine side chains. From model spectra of the protonated tyrosine
(Fig. 1B, a) the prominent signal at 1,518 cm
1 was attributed to the
19(CC) ring
mode. At 1,249 cm
1 a signal composed of the
7'a(CO) vibration and the
(COH) vibration was
expected, the position being sensitive to the hydrogen-bonding environment. For deprotonated tyrosine in solution (Fig. 1B,
b) the
8a/8b(CC) ring mode was
identified at 1,560 cm
1 and the
19(CC)
ring mode at 1,499 cm
1, thus reflecting the sensitivity
of the ring modes to the protonation state of the phenol group. The
7'a(CO) mode was present at 1,269 cm
1
(32-35). From direct comparison of the signals in the double
difference spectrum and in the model compound spectra, a protonation of
one or more tyrosine side chains was suggested to be coupled with the
reduction of the enzyme. We set out to confirm this tentative assignment by site-directed mutagenesis of the conserved tyrosines on NuoB.
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Fig. 2.
Western blot of membranes of the
E. coli strains AN387 (a),
ANN023 (b), ANN023/pBAD33-BWT
(c), ANN023/pBAD33-BY114C
(d), ANN023/pBAD33-BY114C/Y139F
(e), and of complex I isolated from strain
ANN023/pBAD33-BY114C (f). Western
blotting was performed with an antiserum raised against NuoB.
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Fig. 3.
Sucrose gradient centrifugation of detergent
extracts of cytoplasmic membranes of strains ANN023 (closed
squares) and ANN023/pBAD-BWT (closed
circles). Cytoplasmic membranes were extracted with 3%
dodecyl maltoside (w/v) and separated by means of gradients of 5-25%
(w/v) sucrose in 50 mM MES/NaOH, pH 6.0, 50 mM
NaCl, and 0.2% dodecyl maltoside. Each gradient was loaded with 4 mg
of protein. Fractions of the gradients (numbered 1-18 from
top to bottom) were collected and analyzed for
NADH/ferricyanide reductase activity.
Enzyme activities of the membrane-bound NADH dehydrogenases of E. coli
wild type and various NuoB mutants
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Fig. 4.
Sequence alignment of NuoB homologues.
The protein sequences from various organisms were aligned using
ClustalX (44). Identical amino acids are marked by
asterisks. Conserved cysteines are shaded in
gray, the position of the conserved tyrosines are marked
with arrows (E. coli numbering).
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Fig. 5.
Isolation of E. coli complex
I from strain ANN023/pBAD-BY114C. Chromatography on
Source 15Q (a); chromatography on Ultrogel AcA 34 (b); chromatography on Source 15Q (c); absorbance
at 280 nm ( ); NADH/ferricyanide reductase activity (
); NaCl
gradient (
). SDS/PAGE of the indicated fractions of the second anion
exchange chromatography (d).
Isolation of E. coli complex I from strain
ANN023/pBAD33-BY114C
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Fig. 6.
EPR spectra of complex I isolated from wild
type (a) and the mutants Y114C (b),
Y139C (c), Y154H (d), and Y114C/Y139F
(e). The spectra were recorded at 13 K and 5 milliwatts. The signals of the FeS clusters N1a, N1b, N2, N3, and N4
are indicated. The position of the gz signal of
cluster N2 is indicated by the dotted line for a better
comparison. The asterisks denote the radical signal of the
redox mediators. Other EPR conditions were: microwave frequency, 9.44 GHz; modulation amplitude, 0.6 mT; time constant, 0.124 s; scan rate,
17.9 mT/min.
0.5 to 0.0 V (Fig.
7A). Complex I isolated from
the mutant strains was more unstable during the electrochemical
measurements than the complex from wild type. This resulted in a lower
signal to noise ratio of the corresponding spectra. Whereas the
difference spectra of the Y154H mutant revealed only small variations
in comparison to wild type spectra, strong variations were evident for
the Y114C, Y139C, and Y114C/Y139F mutants in the amide I range, where
the contributions of the polypeptide backbone are included. These
shifts indicated structural rearrangement of the protein upon mutation
of the tyrosine residues in NuoB. In addition, smaller variations for
the full spectral range were seen.
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Fig. 7.
FT-IR difference spectra of complex I from
wild type and NuoB mutants for the potential step from 0.5 to 0.0 V. A, difference spectrum of oxidized
reduced
spectra of complex I isolated from wild type (a),
ANN023/pBAD-BY139C (b),
ANN023/pBAD-BY154H (c),
ANN023/pBAD-BY114C (d), and
ANN023/pBAD-BY114C/Y139F. B, double difference
spectra of difference spectra of complex I minus difference spectra of
NADH dehydrogenase fragment. The designation of the spectra is the same
as in A. An enlargement of the spectral region from 1,530 to
1,480 cm
1 is shown. The absorptions of the tyrosine side
chains are marked with dotted lines.
1, by
subtracting the spectra of the mutant enzymes from the corresponding spectra of the NADH dehydrogenase fragment (Fig. 7B). The
absoption of the tyrosine residues proved to be located as shoulders of a positive peak at 1,506 cm
1 in complex I (Fig.
7B).
1, which we interpreted as the
protonation of one or more tyrosine residues due to the reduction of
cluster N2 as well as the mode at 1,498 cm
1, which we
interpreted as the deprotonation of the corresponding tyrosine side
chain(s) due to the oxidation of cluster N2 (Figs. 1 and 7). The
decrease of both signals observed for the mutant Y154H was
significantly smaller from the extinction coefficients observed in
model compound spectra than expected. This excluded tyrosine side chain
154 to be addressed by the redox reaction. The introduced mutation
might have an influence on nearby residues or the involvement of low
amide II signals. The strongest decrease of the modes at 1,515 and
1,498 cm
1 were observed for the Y114C and Y139C mutants.
The amplitude of the signal was roughly halved in both mutants,
demonstrating that Y114 and Y139 were protonated upon reduction of
cluster N2. A residual signal of the characteristic tyrosine modes
accounting for another tyrosine residue was present in both single
mutants suggesting that no other tyrosines were involved in the signals discussed here. This conclusion was supported by the electrochemically induced FT-IR difference spectra of the double mutant Y114C/Y139F. Clearly any signal that was attributed to the tyrosines was absent.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
of complex I isolated from strains Y114C and Y139C were approximately halved in each mutant and absent in the double mutant (Fig. 7). Our
data implied that tyrosine residues 114 and 139 of NuoB were part of
the proton pathway coupled with the redox reaction of cluster N2. It is
noteworthy that the structural rearrangements upon electron transfer
reported for the wild type enzyme (21) were strongly reduced in the
Y114C/Y139F double mutant (Fig. 7). The proton pump mechanism of
complex I might be coupled to conformational changes of the complex (6,
28). The loss of the conformational flexibility of the complex from the
double tyrosine mutant is in line with the possible involvement of the
two tyrosine residues in proton pumping.
60 mV/pH (20) implying that the redox Bohr group associated with N2
receives a proton in the reduced state and becomes deprotonated with
the oxidation of N2. The protonation/deprotonation of tyrosine residues
114 and 139 of NuoB were coupled with the redox reaction of N2 in such
a way that they might be discussed for such a redox Bohr group.
However, the electron transport activity of the complex from the single
mutants was comparable to the one of the wild type demonstrating an
uncoupling of electron transfer involving N2 and protonation of the
tyrosine residues. Thus, the individual tyrosine residues 114 and 139 on NuoB cannot represent the redox Bohr group associated with N2.
However, complex I of the Y114C/Y139F double mutant exhibited only 20%
of the wild type d-NADH oxidase activity in the membrane (Table 2), and
the isolated complex had no enzymatic activity. This implies the
presence of a cluster of protonable groups including tyrosine residues
114 and 139 that sense the redox state of cluster N2.
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Acknowlegments |
---|
We thank Helga Lay and Franz Butz for excellent technical assistance, Stefan Stolpe for help in enzyme preparation, and Linda Böhm for help in preparing the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this study.
To whom correspondence should be addressed. Tel.:
49-(0)761-203-6060; Fax: 49-(0)761-203-6096; E-mail:
tfriedri@uni-freiburg.de.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M208849200
2 M. Uhlmann and T. Friedrich, unpublished data.
3 V. Spehr, T. Bischof, and T. Friedrich, unpublished results.
4 A. Berger, V. Spehr, and T. Friedrich, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: FeS, iron-sulfur; EPR, electron paramagnetic resonance; d-NADH, deamino-NADH; FT-IR, fourier-transform infrared; MES, 2-(N-morpholino)-ethanesulfonic acid; decyl-ubiquinone, 2,3-dimethoxy-5-methyl-6-decylbenzoquinone; T, Tesla; WT, wild type.
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REFERENCES |
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