From the Laboratoire de Chimie Bactérienne and
§ Laboratoire de Bioénergétique et
Ingénierie des Protéines, Institut de Biologie
Structurale et Microbiologie, Centre National de la Recherche
Scientifique, 31 chemin Joseph Aiguier, BP 71, 13402 Marseille Cedex 20, France
Received for publication, September 28, 2000
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ABSTRACT |
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Reduction of trimethylamine N-oxide
(E'0(TMAO/TMA) = +130 mV) in Escherichia
coli is carried out by the Tor system, an electron transfer chain
encoded by the torCAD operon and made up of the periplasmic
terminal reductase TorA and the membrane-anchored pentahemic
c-type cytochrome TorC. Although the role of TorA in the
reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapC c-type cytochrome
family based on homologies of its N-terminal tetrahemic domain
(TorCN) to the cytochromes of this family, but TorC
contains a C-terminal extension (TorCC) with an additional
heme-binding site. In this study, we show that both domains are
required for the anaerobic bacterial growth with TMAO. The intact TorC
protein and its two domains, TorCN and TorCC,
were produced independently and purified for a biochemical
characterization. The reduced form of TorC exhibited visible absorption
maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the
heme centers of the purified components identified two negative
midpoint potentials ( Bacterial respiration allows cells to grow in a changing
environment (1). Under anaerobic conditions, several bacteria can use
diverse electron acceptors such as nitrate, Me2SO or
trimethylamine N-oxide
(TMAO)1 (2, 3) for the
oxidation of organic substrates. To reduce TMAO, Escherichia
coli synthesizes two homologous systems, but one, the TorCAD
system, is strongly induced by TMAO (4, 5), whereas the other, TorYZ,
is expressed at a low level (6). Each of these systems comprises a
periplasmic terminal reductase and a membrane-anchored
c-type cytochrome. The membranous Me2SO reductase, which is constitutively expressed in anaerobiosis, is also
capable of TMAO reduction (7).
The TorCAD system is encoded by the torCAD operon, and its
expression is under the control of the TorS/TorR two-component regulatory system, which mediates the response to the presence of TMAO
in the medium (8). The torA gene encodes the periplasmic molybdoreductase TorA (5). According to sequence homologies, TorA
belongs to the Me2SO reductase family, a group of
periplasmic enzymes that contain a molybdenum cofactor as a single
prosthetic group and are capable of utilizing TMAO and/or
Me2SO as electron acceptors (9). In the past few years,
structures have been published for several members of this family
(10-13). They are organized in four domains surrounding a
bismolybdopterin guanine dinucleotide cofactor that is consequently
buried in a deep depression of the protein surface. The insertion of
the molybdenum cofactor into the TorA apoprotein is a cytoplasmic event
(14) that probably involves the TorD cytoplasmic chaperone (15).
It was previously shown that the first gene of the tor
operon, torC, encodes protein TorC, a 46-kDa pentahemic
c-type cytochrome (5, 16). TorC is anchored to the inner
membrane by a sequence of about 20 hydrophobic residues, while a
globular domain containing five hemes faces the periplasm. Heme binding
to the five consensus CXXCH motifs takes place in the
periplasm by a mechanism involving the c-type cytochrome
maturation machinery encoded by the ccm genes (17, 18). From
sequence comparisons, TorC has been included in the NirT/NapC class of
membrane-anchored multiheme c-type cytochromes. This family
originally was composed of tetrahemic c-type cytochromes of
about 20 kDa that were involved in periplasmic nitrite and nitrate
reduction (19). The N-terminal region of TorC, which is homologous to
NirT and NapC, possesses the four heme binding sites, whereas the
C-terminal part, which is present specifically in the
Me2SO/TMAO respiratory systems, carries the fifth heme motif (5). Recently, two members of the NirT/NapC family, NapC and the
TorC homologue DorC, have been characterized, and the redox potential
values determined for the four hemes of NapC and the five hemes of DorC
were all negative (20, 21).
In TMAO and Me2SO reduction systems, pentahemic cytochromes
proved to be essential for the electron transfer and thus appeared to
be the intermediate components between the membrane quinone pool and
the terminal reductase (6, 22). Since it has been shown in E. coli that TMAO reduction involves menaquinones (23), TorC should
receive the electrons from the menaquinone pool
(E'0(menaquinol/menaquinone) = Interestingly, a recent genetic study showed that TorC in its
unprocessed form is a negative regulator of the torCAD
operon (18). Indeed, apo-TorC seems to inhibit the kinase activity of
the TMAO sensor TorS by an unknown mechanism. This negative autoregulation probably means that the maturation of TorC is the limiting step for the Tor system biogenesis.
This paper provides in vivo and in vitro evidence
that the mature form of TorC is directly involved in the electron
transfer to the terminal reductase, TorA. We also show that TorC
exhibits four negative redox potentials and a positive one
corresponding to the fifth heme. The latter group is responsible for
the direct electron transfer to the catalytic site of TorA. In
addition, TorC binds TorA, and this interaction involves the N-terminal domain of TorC.
Bacterial Strains, Plasmids, Media, and Growth Conditions
The bacterial strains and plasmids used in this work are listed
in Table I. To maintain selection for plasmids, antibiotics were
added at the following concentrations: ampicillin, 100 µg/ml; chloramphenicol, 10 µg/ml. For the biochemical study, cells were grown anaerobically at 37 °C on Luria broth medium (24). The concentration of arabinose or glucose, added to the growth medium, is
detailed for each experiment under "Results." The growth kinetics studies were performed as described by Gon et al. (6).
Constructions Leading to TorC, TorCN, and
TorCC Production
To synthesize the membrane His tag TorC, the complete
torC coding sequence was amplified from MC4100 chromosomal
DNA by polymerase chain reaction, using an oligonucleotide (C5Hc) that
corresponds to an EcoRI site followed by the 5' coding
sequence of torC and a primer (Cct) that corresponds to a
SmaI site followed by a sequence encoding a C-terminal
His6 tag and the complementary sequence of the
3'-end of torC (Fig. 1A).
To synthesize the N-terminal domain of TorC (TorCN,
positions 1-194 relative to TorC) that includes the first four heme
binding sites of TorC (Fig. 1B), the corresponding coding
sequence was amplified by polymerase chain reaction using
oligonucleotide C5Hc and a primer that corresponds to a SmaI
site followed by a His6 tag coding sequence and the
appropriate torC internal sequence.
To synthesize the periplasmic C-terminal domain of TorC
(TorCC, from position 198 to 390) that contains the fifth
heme of TorC (Fig. 1C), the corresponding torC
coding sequence was amplified by polymerase chain reaction using an
oligonucleotide that contains an EcoRI site followed by the
TorT signal peptide (18 codons) coding sequence (28) and the
appropriate torC sequence and primer Cct. Detailed
information on the primer sequences is available on request.
The purified polymerase chain reaction products were digested by the
appropriate restriction enzymes and ligated into the corresponding
cloning sites of the expression vector pBAD24 (27) downstream from the
arabinose-inducible PBAD promoter to give pBC,
pBCN, and pBCC (Table
I). All of the polymerase chain reaction products and the fusion sites were confirmed by sequencing. DNA preparations were carried out with high pure DNA isolation kits from
Roche Diagnostics. Transformations were performed according to the
method of Chung and Miller (29). Plasmids pBC, pBCN, and
pBCC were digested with NheI and
HindIII, and the resulting DNA fragments encoding the
modified His tag TorC proteins were cloned just downstream of
torD, into the compatible sites XbaI and
HindIII of plasmid pTorAD (15), leading to plasmids
pTorADC, pTorADCN, and pTorADCC,
respectively.
177 and
98 mV) localized in the tetrahemic
TorCN and one positive midpoint potential (+120 mV) in the
monohemic TorCC. In agreement with these values, the in vitro reconstitution of electron transfer between TorC,
TorCN, or TorCC and TorA showed that only TorC
and TorCC were capable of electron transfer to TorA.
Surprisingly, interaction studies revealed that only TorC and
TorCN strongly bind TorA. Therefore, TorCC
directly transfers electrons to TorA, whereas TorCN, which probably receives electrons from the menaquinone pool, is involved in
both the electron transfer to TorCC and the binding to TorA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
74 mV) and
transfer them to the active site of TorA, where TMAO reduction
(E'0(TMAO/TMA) = +130 mV) occurs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and plasmids used in this study
Purification of TorA, TorC, TorCN, and TorCC
The purification of the TMAO reductase (TorA) was performed from the periplasmic fraction of cells DSS401/pEC86. TorA protein enrichment was achieved as follows. The periplasmic fraction of strain DSS401/pEC86, grown anaerobically in the presence of TMAO (30 mM), was prepared by the lysozyme-EDTA procedure (30) and precipitated with ammonium sulfate 60% (w/v). The pellet was resuspended into Tris-HCl 40 mM buffer (pH 7.6) and then loaded on a Mono Q HR 5/5. The TMAO reductase active fractions, obtained from a 50-500 mM NaCl gradient were then dialyzed, pooled, concentrated by 60% (w/v) ammonium sulfate precipitation, and loaded on a Superdex G200 HR 10/30 column. At this stage, the active fractions contained 3 mg of protein, and the purity of the preparation was checked on Coomassie Blue-stained gel.
Purification of TorC and TorCN was performed from the membrane fraction of cells MC4100, containing both plasmids pEC86 and either pBC or pBCN, grown anaerobically in the presence of 0.0005% arabinose. Membrane fraction, prepared as described by Silvestro et al. (4), was incubated for 1 h at 4 °C in 20 mM phosphate buffer (pH 7.4) with 500 mM NaCl containing 2% Triton X-100. After centrifugation at 14,000 rpm for 30 min at 4 °C, the fraction containing the solubilized cytochrome c, TorC or TorCN, was diluted two times with the same buffer containing 0.2% Triton X-100 and 2 mM imidazole and loaded onto a HiTrapTM chelating Ni2+ column (Amersham Pharmacia Biotech). The proteins were eluted with a step gradient of imidazole from 20 to 500 mM. Both TorC and TorCN cytochromes were eluted with 150 mM imidazole.
TorCC, the soluble C-terminal domain of TorC, was purified from the periplasmic fraction of cells MC4100 containing both pEC86 and pBCC grown under anaerobic conditions in the presence of 0.1% arabinose. The periplasm was diluted two times into 20 mM phosphate buffer (pH 7.4) with 500 mM NaCl, 2 mM imidazole, and the purification onto a HiTrapTM chelating Ni2+ column was performed as described for TorC and TorCN.
Analytical Procedure
Protein analysis was carried out using 12.5 or 10% SDS-PAGE. After electrophoresis, the presence of hemes within TorC and derivatives was revealed by staining the gel for peroxidase activity using 3,3',5,5'-tetramethylbenzidine as described by Thomas et al. (31); then Coomassie Blue staining of total proteins was performed (32). Protein concentrations were estimated using the technique of Lowry et al. (33).
Spectroscopy and Mediated Redox Potentiometry
Optical difference spectra and redox titration were performed on
a Kontron Uvikon 932 UV-visible spectrophotometer. The UV-visible spectra of TorC, TorCN, and TorCC were carried
out at 16 °C in 20 mM phosphate buffer (pH 7.4) with
Triton X-100 0.05%. Optical redox titrations of TorC (5 µM) and TorCC (10 µM) were
performed according to Dutton (34), and samples were kept at 20 °C
under argon atmosphere. The following redox mediators were present
at 5 µM: 1,4-benzoquinone (E'0 = +280 mV), DCIP (E'0 = +217 mV), 2,5-dimethyl
benzoquinone (E'0 = +180 mV), 1,2-naphtoquinone
(E'0 = +145 mV), 1,4-naphtoquinone
(E'0 = +60 mV), duroquinone
(E'0 = +5 mV), 2-methyl, 1,4-naphtoquinone
(E'0 = 0 mV), pyocyanine (E'0 = 34 mV), 2,5-dihydroxybenzoquinone
(E'0 =
60 mV), indigocarmine (E'0 =
125 mV) 1,4-dihydroxynaphtoquinone
(E'0 =
145 mV), antraquinone 2-sulfonate
(E'0 =
225 mV), safranine T
(E'0 =
289 mV), and neutral red
(E'0 =
325 mV). All potentials quoted are with
respect to the normal hydrogen electrode. The absorbance change at 552 nm was plotted against redox potential, and theoretical Nernstian curves were fitted to the data using Sigma Plot.
Kinetics of Electron Transfer
The kinetic assays were achieved using anaerobic cuvettes,
typically filled with phosphate buffer (20 mM, pH 7.4),
TMAO (50 mM), and either TorCC (10 µM), TorCN (5 µM), or TorC (5 µM) previously reduced by dithionite. Before adding TMAO
reductase (350 units·ml1), the cuvette
mixture was flushed with argon (10 min). The oxidation rate of the
cytochrome was determined from the absorption band at 552 nm.
Interaction Experiments
Interaction Studies on PAGE under Native Conditions-- TorA (5 µM) and either TorC (5 µM), TorCN (5 µM), or TorCC (10 µM) were incubated for 30 min at room temperature in 20 mM phosphate buffer (pH 7.4), Triton X-100 0.05%. The interactions were analyzed by 10 or 12.5% PAGE under native conditions. For identification, the proteins were either stained for heme with 3,3',5,5'-tetramethylbenzidine or were transferred to a HybondTM ECLTM nitrocellulose membrane. The protein complexes were immunodetected with anti-His antibodies (Invitrogen). The ECL-Western blotting system was used as recommended by the supplier (Amersham Pharmacia Biotech).
Interaction Studies by Biosensor Experiments--
The BIAcore
apparatus (Amersham Pharmacia Biotech) was used to analyze, in real
time, the binding between TorA (Mr 94,000) and
TorC (Mr 46,000), TorCC
(Mr 23,000), or TorCN
(Mr 24,500), by using surface plasmon resonance.
All experiments were carried out at 25 °C. TorA, in 10 mM sodium acetate buffer (pH 5), was immobilized on a CM5
sensor chip (BIAcore) through amine coupling (3000 resonance units).
TorC, TorCN, and TorCC were diluted in 20 mM phosphate buffer (pH 7.4), 0.05% Triton X-100 and
injected using a constant flow rate of 10 µl/min. The resulting
sensorgrams were evaluated using the biomolecular interaction analysis
evaluation software (BIAcore) to calculate the kinetic constants of the
complex formation.
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RESULTS |
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The Intact TorC Protein Is Required for TMAO Respiration-- To establish that the c-type cytochrome TorC is required for electron transfer to the terminal enzyme TorA, we cloned the torAD genes alone (15) or together with torC under the control of the PTAC promoter of the plasmid pJF119. The resulting pTorAD and pTorADC plasmids were introduced into strain LCB504, which is unable to reduce TMAO. Indeed, this strain carries an interposon in the beginning of the first gene of the torCAD operon and a deletion of the entire dmsABC operon (6). The strains carrying either pJF119 or pTorAD grew very slowly and at the same rate under anaerobic conditions in the presence of TMAO (Fig. 2). In contrast, when plasmid pTorADC was introduced into strain LCB504, the recombinant strain exhibited a significant growth rate in the presence of TMAO (Fig. 2) and produced the characteristic odor of volatile TMA, which results from reduction of TMAO. These results confirmed the hypothesis that TorC is essential for TMAO reduction.
The TorC protein belongs to the NapC/NirT family of c-type
cytochrome because its tetrahemic N-terminal domain (hereafter called
TorCN; Fig. 1B) is
homologous to the NapC protein and contains four heme-binding sites. In
addition, TorC contains a C-terminal domain (hereafter called
TorCC; Fig. 1C) with a single heme-binding motif. The C-terminal extension is found exclusively in TorC/DorC systems (35). To determine whether one or both of the two domains are
required for the TMAO reduction process, we cloned DNA fragments encoding either one of these domains behind torAD. The
resulting plasmids pTorADCN and pTorADCC were
introduced into strain LCB504, and the growth rate was monitored. The
recombinant strains containing pTorADCN did not grow
significantly in the presence of TMAO (Fig. 2), and the cells containing
pTorADCC grew very slowly only after 36 h of
incubation. These results strongly suggest that both domains of the
TorC protein are required for the TMAO respiratory process.
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Overproduction and Purification of TorC and of Its N- and C-terminal Domains-- To study the biochemical characteristics of the TorC protein, we constructed recombinant plasmids that encode either TorC; TorCN, which corresponds to the N-terminal domain of TorC; or TorCC, which corresponds to the second half of the TorC protein (Fig. 1). DNA fragments encoding TorC, TorCN, and TorCC were cloned into the pBAD24 vector, giving rise to plasmids pBC, pBCN, and pBCC with His tags added at the C-terminal extremity of each protein. Moreover, since the heme incorporation occurs in the periplasm, we introduced the TorT signal peptide in front of TorCC to assure its periplasmic localization.
Overproduction of mature c-type cytochrome, and especially
multiheme c-type cytochromes, is difficult in E. coli, due to the limited capacity for synthesis of the
c-type cytochrome of this organism. To overcome this
problem, we used a strain containing the plasmid pEC86 that carries the
c-type cytochrome maturation ccm genes and thus
improves production of c-type cytochromes (17). Nevertheless, the amount of mature cytochrome produced is rather low
even in the presence of this plasmid, and the expression of cytochrome-encoding genes has to be controlled to prevent saturation of
the maturation machinery. Accordingly, the TorC genes were cloned into
the pBAD24 vector under the control of the PBAD promoter for controlled expression of TorC proteins. Plasmids pBC,
pBCN, and pBCC were then introduced into strain
MC4100 carrying the compatible plasmid pEC86. TorC and
TorCN expression was induced with 0.0005% arabinose
because concentrations higher than 0.001% apparently overloaded the
heme delivery machinery and led to degraded proteins. Higher levels of
the mature TorCC protein were obtained by using 0.1%
arabinose, probably because TorCC is a monoheme protein.
Heme-staining proteins of 47 and 24 kDa were present in membrane
extracts of the strains containing pBC and pBCN,
respectively, indicating that TorC and TorCN were matured
(data not shown). Since these proteins were exclusively located in the
membrane fraction (data not shown), detergent Triton X-100 was used to solubilize them. A 23-kDa heme-staining protein was detected in the
periplasmic extract of the strain containing pBCC (data not shown). The detergent-solubilized fractions containing TorC or TorCN or the periplasmic fraction containing
TorCC were loaded onto Ni2+ columns, and the
His-tagged proteins were eluted from the column at about 150 mM imidazole. Analysis by SDS-PAGE revealed the presence of
TorC, TorCN, and TorCC as a single major band
on gels stained for heme-dependent peroxidase activity
(Fig. 3A). No other
c-type cytochrome copurified with the proteins and no
apparent degradation occurred. The same gels stained with Coomassie
Blue (Fig. 3B) revealed one major band; moreover, this band
cross-reacted with anti-His antibodies in Western blots (data not
shown), confirming that His-tagged derivatives of TorC had been
purified.
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Characterization of TorC by Optical Spectroscopy Shows That TorC
Exhibited Positive and Negative Heme Redox Potentials--
The
UV-visible spectrum of purified TorC shows a Soret peak at 411 nm in
the oxidized state and peaks at 417 (Soret), 523, and 552 nm in the
dithionite reduced form (Fig.
4A). These peaks are
characteristic of c-type cytochromes and confirm that TorC is a c-type cytochrome as predicted by the presence of
c-type heme binding sites (CXXCH) in the amino
acid sequence. TorC was totally reduced by dithionite, but it was only
partially reduced by ascorbate (Fig. 4A). These results
indicate that at least one of the TorC hemes exhibits a positive redox
potential, whereas the remaining hemes display more negative redox
potentials. Similar UV-visible spectra were obtained with membrane
extracts from strain DSS401 containing native TorC after reduction with
ascorbate or dithionite (Fig. 4B), indicating that the
purified His tag TorC cytochrome has spectral properties similar to
those of the unmodified native TorC protein.
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To determine which domain of TorC contains a positive redox potential center, we carried out UV-visible spectra with the purified TorCN and TorCC domains (Fig. 4, C and D). Fig. 4C shows that the hemes of TorCN are totally reduced by dithionite but not by ascorbate. In contrast, Fig. 4D shows that the unique heme of TorCC is totally reduced by both ascorbate and dithionite. These findings strongly suggest that the fifth heme of TorC has a positive potential, whereas the first four hemes exhibit negative potentials.
Determination of the Redox Potentials of the TorC Protein--
To
define more precisely the heme potentials, a mediated redox
potentiometry was carried out on the purified TorC protein. This
analysis revealed three distinct redox type centers, one at 177 mV
(two hemes), one at
98 mV (two hemes), and one at +114 mV (one heme)
(Table II). These potentials are in
agreement with the previous spectral evidence for one positive heme
potential in the TorCC domain and four negative heme
potentials in the TorCN domain. Mediated redox
potentiometry of TorCC (Table II) yielded a value of +120
mV, in complete agreement with the spectral evidence for a positive
heme in the TorCC domain of TorC (Fig. 4D) and with the heme potential values obtained with the intact TorC
protein.
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Transfer of Electrons from TorC to the TMAO Reductase TorA--
In
the model of the Tor electron transfer pathway, TorC transfers
electrons from the menaquinones, which are embedded in the cytoplasmic
membrane, to the periplasmic TMAO reductase TorA. To demonstrate
directly that TorC can act as an electron shuttle from the inner
membrane to TorA, we studied electron transfer between purified TorC
and TorA. TorC was previously reduced by dithionite under anaerobic
conditions (Fig. 4A). Oxidation of TorC, followed at 552 nm
(Fig. 5) was specifically dependent upon TMAO reductase, since no oxidation occurred before the addition of TorA
(data not shown).
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Based on the redox potentials of TorCN (177 and
98 mV)
and TorCC (+120 mV) and on
E'0(TMAO/TMA) (+130 mV), we hypothesized that
electrons are transferred from TorCN to TorCC
and from TorCC to TorA. In this model, TorCC is
the direct electron donor to TorA. Purified TorCN and
TorCC were tested as electron donors for TMAO reductase.
The monoheme TorCC domain was oxidized by the addition of
TorA, whereas the tetrahemic TorCN domain was not (Fig. 5).
Although oxidation of TorCC was slower than that of TorC,
these results indicate that the electron donor for TorA is most
probably located in the C-terminal part of TorC. When a mixture of
purified TorCN and TorCC was tested, the
electron transfer was slightly higher than that of TorCC
alone but slower than that of TorC. These results indicate that
TorCN also plays a role in the electron transfer pathway.
Binding of TorC to TorA Mainly Involves the TorC N-terminal
Domain--
The above results with purified components imply a direct
interaction between these proteins. To characterize the binding between
TorC and TorA, the formation of a TorC-TorA complex was studied under
nondenaturing conditions by native PAGE and by BIAcore experiments.
TorA and TorC were mixed together and loaded in the same conditions
onto two polyacrylamide gels. After electrophoresis under native
conditions, one gel was stained for heme (Fig.
6A, lanes
1 and 2), and a Western blot with anti-His
antibodies was performed with the second gel (Fig. 6A,
lanes 3 and 4). Significantly, the
migration of TorC was retarded in the presence of TorA (Fig. 6A, compare lanes 1 and 3 with lanes 2 and 4, respectively).
This result suggested that TorC can bind to TorA. To confirm this
result, we studied the interaction between TorA and TorC by using the BIAcore technique (surface plasmon resonance). For this purpose, purified TorA protein was coupled to the dextran matrix of a sensor chip, and TorC was injected into the TorA-containing sensor chip. The
sensorgram reflected the association and dissociation of two proteins,
indicating that TorC directly interacts with TorA (Fig. 7A) in agreement with the
above result.
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The interaction of the individual domains with TorA was also studied by the same two approaches. Surprisingly, no binding between TorCC and TorA was detected on a native gel stained for heme or by Western blot with anti-His antibodies. Indeed, whatever the conditions used, TorCC migrated at exactly the same position in the presence and in the absence of TorA (Fig. 6B, compare lanes 1 and 3 with lanes 2 and 4, respectively). Similarly, no binding between TorCC and TorA was detected by the BIAcore procedure (Fig. 7B). As a control, we coupled TorCC to the dextran matrix and injected TorA, and again, no formation of a complex corresponding to the association of TorCC and TorA was detected (data not shown). When TorCN and TorA were mixed and loaded onto a polyacrylamide gel under nondenaturing conditions, TorCN migration was retarded by the presence of TorA (Fig. 6C), suggesting that the N-terminal domain of TorC is sufficient for TorA binding. The fact that TorCN can bind to TorA was confirmed by using the BIAcore technique. Indeed, an increase in the amount of recovered resonance units was observed when the TorCN protein was injected (Fig. 7C). These results clearly show that the TorCN domain interacts with TorA.
The association rate constants (kon) and the
dissociation rate constant (koff) of TorC and
TorCN to TorA were determined at four different
concentrations between 2.5 and 10 µM (data not shown).
TorCN exhibited a Kd value of
4.5·108 M (Table
III). Significantly, the sensorgram of
intact TorC binding to TorA exhibits at least two steps leading to the
determination of two constants, Kd1 = 1.7·10
8 M and
Kd2 = 3.0·10
6
M (Table III). These two steps could correspond to the
fixation of TorCN and TorCC, but
TorCN is mainly responsible for the binding of intact TorC
to TorA because TorCC alone does not bind TorA. The low
affinity between TorCC and TorA could also explain the poor
catalytic efficiency in electron transfer to TorA exhibited by
TorCC compared with that of the intact TorC protein (Fig.
5).
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DISCUSSION |
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This study has revealed that TorC is made up of two domains of similar size, corresponding to its N- and C-terminal half (TorCN and TorCC, respectively) (Figs. 1 and 3). As the other members of the NirT/NapC family with which it shares sequence homologies, TorCN contains a membrane anchor segment followed by four heme-binding sites (5, 20). Production of the isolated TorCN domain resulted in a stable mature protein. Unfortunately, and in contrast to what was described previously for NapC (20), the removal of the membrane anchor of TorC and TorCN led to an unstable protein (data not shown). The second domain, TorCC contains the fifth heme-binding motif of TorC (Fig. 1C) and presents sequence homologies with the corresponding region of pentahemic cytochromes specifically involved in TMAO/Me2SO respiration of various bacteria (6, 35). Although the fifth heme binding site is located at the C-terminal extremity (positions 329-333 relative to TorC), we were unable to produce a stable protein starting from position 285 of TorC, i.e. just upstream from the fifth heme binding site (data not shown). The region located between the fourth and the fifth heme binding sites probably plays an essential role in the stability and/or the folding of the TorCC domain.
The comparison between the midpoint redox potential values obtained for
the soluble form of the Paracoccus denitrificans NapC (n = 1, 235,
207,
181, and
56 mV) (20) and
TorCN, the N-terminal domain of TorC (n = 2,
177 and
98 mV) (Table II), highlights that they are all negative
and in the same range. This result confirms that NapC and
TorCN are highly related. It is also striking that, for
TorCN, each of the midpoint redox potentials corresponds to
two heme centers. This feature could be representative of a symmetry in
the N-terminal domain of TorC. This hypothesis agrees with the previous
proposal of Roldan et al. (20), that the tetrahemic domain
of NirT/NapC c-type cytochromes comes from a gene
duplication leading to two related diheme subdomains. If true, then the
redox potential of the first and third hemes of TorC or that of the second and the fourth ones should be very similar.
The midpoint redox potential described by Shaw et al. (21)
for purified DorC, a TorC pentahemic homologue, is in the same range of
values (276,
185,
184,
128 mV, and
34 mV) as those detailed
above for NapC. Surprisingly, all the potentials exhibited by DorC are
negative, while a positive one has been detected for TorC. Indeed, we
have clearly shown that the redox potential of the fifth heme is about
+120 mV, and the presence of a positive heme center has been observed
in both the isolated TorCC domain and the intact TorC
protein (Table II). Moreover, this result has been confirmed by a study
performed directly on native TorC using membrane extracts (Fig.
4B). These results are also in agreement with the fact that
ascorbate reduced partially TorC and TorCC completely (Fig.
4). Positive midpoint redox potentials are often associated to
His-Met-ligated hemes (36). The comparison of the sequences surrounding
the fifth heme-binding site of the pentahemic cytochrome family reveals
the presence of one highly conserved residue of methionine (position
353 in the TorC amino acid sequence) (35). This residue is a
good candidate for the axial ligand, although a conserved histidine
residue (position 340) might also play such a role.
As shown by the in vitro electron transfer experiments, the TorCC domain donates the electrons directly to TorA (Fig. 5). This finding fits well with the positive midpoint redox potential exhibited by the TorCC heme (E'0 = +120 mV) and that of the reduction reaction of TMAO (E'0(TMAO/TMA) = + 130 mV). The rate of electron transfer is slightly but significantly enhanced by the presence of the tetrahemic domain TorCN in the reconstitution system. The enhanced rate in the presence of TorCN probably means that TorCN transfers the electrons to TorCC, since TorCN alone cannot directly feed TorA with electrons (Fig. 5). This result, together with the fact that the presence of both TorCN and TorCC domains are required for bacterial growth with TMAO as a sole exogenous electron acceptor (Fig. 2), support a model in which TorCN receives the electrons from the menaquinone pool and then transfers them to TorCC, which gives them to TorA. In the Nir and Nap respiratory systems, the TorCN homologues (NirT and NapC) and their associated diheme cytochromes (NirB and NapB, respectively) constitute distinct proteins (19, 37). In the case of TorC, the TorCN tetrahemic domain is fused to the TorCC monohemic domain, and it is clear from our experiments that the intact TorC protein is more efficient than a mixture of the two isolated domains.
Although the simplest model of interaction between TorC and TorA would
have been that TorCC binds TorA, since it transfers the
electrons to TorA, we have shown by two different approaches that
TorCC does not significantly bind TorA, whereas
TorCN binds TorA efficiently (Figs. 6 and 7). The apparent
equilibrium dissociation constant obtained by the analysis of the
BIAcore data is similar for TorCN and TorC, although a
second Kd with a higher value can also be calculated
for TorC (Table III). This latter might correspond to a weak
interaction between TorCC and TorA that takes place after
the TorCN-TorA binding, allowing the electron transfer.
Based on these findings, we proposed a model in which the tetrahemic
domain of TorC transfers the electrons to TorCC and binds
TorA in such a way that TorCC is correctly positioned to
transfer them to TorA (Fig. 8). This may
also explain why TorCN and TorCC are fused.
Finally, the binding of the TorCN domain to TorA raises the
question of a possible binding of DorC and also of the tetrahemic
homologues of TorC to their associated terminal reductase.
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ACKNOWLEDGEMENTS |
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We are indebted to J. Demoss for critical reading of this manuscript. We also thank J.-C. Patte, F. Bayman, W. Nitschke, and B. Schoepp for fruitful discussions.
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FOOTNOTES |
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* This work was supported by grants from the Center National de la Recherche Scientifique, the Université de la Méditerranée, and an Ministère de l'Education Nationale, de la Recherche et de la Technologie fellowship (to S. G.).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.
¶ To whom correspondence should be addressed. Tel.: 33-4-91-16-44- 27; Fax: 33-4-91-71-89-14; E-mail: iobbi@ibsm.cnrs-mrs.fr.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008875200
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ABBREVIATIONS |
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The abbreviations used are: TMAO, trimethylamine N-oxide; TMA, trimethylamine; PAGE, polyacrylamide gel electrophoresis.
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