From the Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
Received for publication, October 14, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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The aerobic respiratory chain of the
Gram-positive Corynebacterium glutamicum involves a
bc1 complex with a diheme cytochrome c1 and a cytochrome aa3
oxidase but no additional c-type cytochromes. Here we show
that the two enzymes form a supercomplex, because affinity
chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome
aa3 always resulted in the copurification of
the subunits of the bc1 complex (QcrA, QcrB,
QcrC) and the aa3 complex (CtaD, CtaC, CtaE).
The isolated bc1-aa3
supercomplexes had quinol oxidase activity, indicating functional
electron transfer between cytochrome c1 and the
CuA center of cytochrome aa3.
Besides the known bc1 and
aa3 subunits, few additional proteins were
copurified, one of which (CtaF) was identified as a fourth subunit of
cytochrome aa3. If either of the two
CXXCH motifs for covalent heme attachment in cytochrome c1 was changed to SXXSH, the
resulting mutants showed severe growth defects, had no detectable
c-type cytochrome, and their cytochrome b level
was strongly reduced. This indicates that the attachment of both heme
groups to apo-cytochrome c1 is not only
required for the activity but also for the assembly and/or stability of the bc1 complex.
Corynebacterium glutamicum is a non-pathogenic aerobic
soil bacterium that has gained considerable interest because of its use
in large scale biotechnological production of L-glutamate and L-lysine (1) and because of its emerging role as a
model organism for the Gram-positive bacteria with high G+C content (2), which include a number of important pathogens, in particular Corynebacterium diphtheriae and Mycobacterium
tuberculosis. In this context, the respiratory chain of C. glutamicum was also analyzed in recent years, both genetically and
biochemically. It is composed of several dehydrogenases that transfer
electrons to an intramembrane pool of menaquinone-9 (3) and at least two branches for reoxidation of menaquinol, one consisting of a
cytochrome bd-type quinol oxidase (4) and the second one consisting of menaquinol:cytochrome c oxidoreductase
(cytochrome bc1 complex) and cytochrome
aa3 oxidase (5-7). The latter one is of primary
importance for growth, because mutants lacking either the
bc1 complex or cytochrome
aa3 have severe growth defects (5) (see also
Fig. 2).
The dehydrogenases include a non-proton-pumping NADH dehydrogenase
encoded by the ndh gene (8, 9), malate:quinone
oxidoreductase encoded by the mqo gene (8, 10), and
succinate dehydrogenase encoded by the sdhCAB genes
(Cgl0370, Cgl0371, Cgl0372). Succinate oxidase activity was shown to be
inhibited by an uncoupler, indicating that electron transfer from
succinate to menaquinone requires the electrochemical proton potential
across the cytoplasmic membrane (11).
The cytochrome bd oxidase was purified and shown to consist
of two subunits of 56 and 42 kDa encoded by cydA and
cydB, respectively. It was proposed that this oxidase is
predominant during the stationary phase of growth (4). The cytochrome
bc1 complex is encoded by the qcrCAB
genes (Fig. 1) for cytochrome
c1, Rieske iron-sulfur protein, and cytochrome
b, respectively (5, 6). The protein sequences deduced from
these genes revealed a number of differences to classical
representatives of the bc1 complex, such as an
extension of about 120 amino acids at the C terminus of cytochrome
b and the presence of three putative transmembrane helices
in the N terminus of the Rieske iron-sulfur protein rather than only
one. Most remarkably, cytochrome c1 was found to
have two CXXCH motifs for covalent heme attachment,
suggesting that it is a diheme c-type cytochrome (5, 6).
Purification of cytochrome c1 confirmed the
presence of two heme groups in the protein (6). Upstream of
qcrC, the genes encoding subunit II (ctaC) and
III (ctaE) of cytochrome aa3 oxidase
were identified (5, 6), as was as an additional open reading frame
located in between these two genes, which was designated
ctaF in the course of this work (Fig. 1). Compared with
"classical" subunit II representatives, CtaC of C. glutamicum contained an insertion of about 30 amino acid residues
in the substrate binding domain, which was proposed to play a role in
the interaction with cytochrome c1 (7). The gene
encoding subunit I of cytochrome aa3 was found
to be located separately at a different genomic site (5, 7). Cytochrome aa3 oxidase was isolated by conventional
chromatographic techniques as a complex consisting of CtaD, CtaC and
CtaE (7). HPLC1 and mass
spectrometry of the isolated heme of subunit I indicated that it is
presumably heme aS, in which the farnesyl group
(C15H25
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) of heme a is replaced by
a geranylgeranyl side chain (C20H33
). Subunit
II contains a lipoprotein signal sequence, and in fact Cys-29, whose
thiol group might be diacylglycerated, was identified as the N-terminal
residue of the mature protein (7).
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Fig. 1.
Physical map of the C. glutamicum
genome region harboring the genes for the
bc1 complex and cytochrome
aa3 oxidase except
ctaD. The DNA regions deleted in strains
13032 qcr and 13032
ctaF are indicated, as
well as the fragments present in plasmid
pJC1-qcrBSt and derivatives and in plasmid
pBM20-QXA.
Staining of proteins separated by SDS-PAGE for covalently bound heme
indicated that there is only a single c-type cytochrome with
an apparent mass of 31 kDa present in C. glutamicum wild type (5, 6). This protein was missing in the mutant strain 13032qcr, which lacks the qcrCAB genes,
confirming that it represents cytochrome c1 (5).
The absence of additional c-type cytochromes in C. glutamicum indicated that the second heme group of cytochrome c1 takes over the function of a separate
cytochrome c in electron transfer to cytochrome
aa3 oxidase. Such a function would require an
intimate contact between cytochrome c1 and the
CuA electron entry site in subunit II of cytochrome
aa3, and therefore we suggested that the
bc1 complex and cytochrome
aa3 might form a supercomplex (5). In this work,
we were able to prove the existence of such a supercomplex by using a
very gentle method for its purification, i.e. affinity
chromatography with the StrepTag II/StrepTactin system (12). Moreover,
a fourth subunit of cytochrome aa3 oxidase was
identified, and it was shown that incorporation of both heme groups
into cytochrome c1 is essential for the assembly
and/or stability of the entire bc1 complex.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Culture Conditions--
C.
glutamicum strain ATCC 13032 (13) and derivatives were cultivated
aerobically in Erlenmeyer flasks at 120 rpm and 30 °C in brain heart
infusion medium (BHI; Difco) with 2% (w/v) glucose or in CGXII minimal
medium with 4% (w/v) glucose as carbon and energy source (14). When
appropriate, 25 µg of kanamycin/ml was added. The C. glutamicum strains and the plasmids used in this study are listed
in Table I. For all cloning
purposes, Escherichia coli DH5 (Invitrogen) was
used and routinely grown at 37 °C in LB medium (15). When
appropriate, 50 µg of kanamycin/ml or 100 µg of ampicillin/ml was
added.
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Recombinant DNA Work-- All enzymes for recombinant DNA work were obtained either from Roche Diagnostics or New England Biolabs. The oligonucleotides used in this study were obtained from MWG Biotech (Ebersberg, Germany) and are listed in Table II. Standard methods were used for the cloning procedures (15).
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For the purification of the cytochrome bc1
complex, a QcrB derivative with a C-terminal StrepTag II (12) was
constructed as follows. The entire ctaE-qcrCAB gene cluster
including the putative promoter region was amplified by PCR (Expand
high fidelity PCR system; Roche Diagnostics) using a reverse primer
that included the codons for the StrepTag II (WSHPQFEK) preceded by two
alanine codons. The resulting 5.0-kb fragment was cloned into the
E. coli-C. glutamicum shuttle vector pJC1 using
the XbaI and SalI restriction sites introduced by
the primers. The resulting plasmid pJC1-qcrBSt encoded a QcrB derivative with ten additional residues at the C
terminus (calculated mass, 61.1 kDa). A CtaD derivative with a
C-terminal StrepTag II for the purification of the cytochrome aa3 complex was constructed similarly except
that only the monocistronic ctaD gene with its native
promoter was amplified by PCR. The resulting 2.0-kb fragment was cloned
via XbaI restriction sites into pJC1 yielding
pJC1-ctaDSt. The modified CtaD protein contained
ten additional residues at the C terminus (calculated mass, 66.3 kDa). Each of the two plasmids was transferred into the C. glutamicum strains 13032ctaD and
13032
qcr by electroporation as described (16). For the
synthesis of cytochrome c1 derivatives defective in covalent binding of either the N-terminal or the C-terminal heme
group, site-directed mutagenesis of qcrC was performed by a
two-step PCR procedure according to Higuchi et al. (17). For that purpose, a 2.0-kb XbaI-ApaI fragment from
pJC1-qcrBSt was cloned into pUC-BM20. The
resulting plasmid pBM20-QXA served as template for mutagenesis with the
universal primers M13-forward/-reverse and the mutagenic primers
C67S-forward/-reverse and C177S-forward/-reverse, respectively. The
products obtained after crossover PCR were digested with
XbaI and ApaI and cloned into pUC-BM20, yielding
pBM20-QXA-C67S and pBM20-QXA-C177S. The presence of the desired
mutations and the absence of additional mutations were confirmed by DNA
sequence analysis (18). The mutated 2.0-kb
XbaI-ApaI fragments were exchanged against the
corresponding wild-type fragment of pJC1-qcrBSt
yielding pJC1-qcrBSt-C67S and
pJC1-qcrBSt-C177S, respectively, which were transferred into C. glutamicum strain
13032
qcr. In the cytochrome c1
variants encoded by these plasmids, residues Cys-67 and Cys-70 or
Cys-177 and Cys-180 are replaced by serine residues. In-frame deletion mutants of C. glutamicum were constructed as
described previously using crossover PCR and the suicide vector
pK19mobsacB (5). The deletions were verified by PCR and by
Southern blot analysis (data not shown).
Preparation of Cell Membranes--
Cells (10 g wet weight) were
suspended in 15 ml of 100 mM Tris-HCl buffer, pH 7.5, containing 5 mM MgSO4 and 10 mg/ml lysozyme. After 45 min of incubation at 37 °C, 1 mM of the
protease inhibitor phenylmethanesulfonyl fluoride was added, and the
cells were disrupted by five passages at 207 mega Pascals
through a French pressure cell (SLM Aminco). Cell debris was removed by
centrifugation at 27,000 × g for 20 min, and the
supernatant was ultracentrifuged at 150,000 × g for 90 min. The pellet containing the cytoplasmic membrane fraction was washed
in 100 mM Tris-HCl, pH 7.5, and centrifuged again at
150,000 × g for 90 min. Then the membranes were
resuspended in a small volume of the same buffer containing 10% (v/v)
glycerol and stored at 20 °C.
Purification of the Strep-tagged Protein Complexes--
Washed
membranes were adjusted to a protein concentration of 5 mg/ml in 100 mM Tris-HCl, pH 7.5, containing 50 µg/ml egg white avidin
(Sigma). The membrane proteins were solubilized by adding n-dodecyl--D-maltoside (Biomol, Hamburg,
Germany) from a 10% (w/v) aqueous solution to a final ratio of 2 g of dodecyl maltoside/g of protein. After 45 min of incubation on ice
with slow stirring the sample was ultracentrifuged at 180,000 × g for 20 min. The supernatant was applied to a
StrepTactin-Sepharose column with a bed volume of 2 ml (IBA,
Göttingen, Germany) equilibrated with 100 mM Tris-HCl
buffer, pH 7.5, containing 0.025% (w/v) dodecyl maltoside. The column
was washed with 9 ml of a buffer containing 100 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM
MgSO4, and 0.025% (w/v) dodecyl maltoside. Specifically
bound proteins were eluted with the same buffer supplemented with 2.5 mM D-desthiobiotin (Sigma) and 10% (v/v) glycerol.
Protein Identification by Peptide Mass Fingerprinting-- Proteins separated by SDS-PAGE and stained with Coomassie Blue were identified as described previously (19) by peptide mass fingerprinting after in-gel digestion with trypsin (Promega) or with cyanogen bromide (20). If required for reliable identification, post-source decay analysis of selected peptides was carried out (19). Peptide mass lists were used to search a local digest data base of 3312 C. glutamicum proteins, provided by the Degussa AG (Frankfurt, Germany).
Enzyme
Assays--
N,N,N',N'-Tetramethyl-p-phenylenediamine
(TMPD) oxidase activity was measured spectrophotometrically at 562 nm
in air-saturated 100 mM Tris-HCl buffer, pH 7.5, containing
200 µM TMPD, at 25 °C. For the calculation, an
extinction coefficient of 10.5 mM1·cm
1 was used (7). One
unit of activity is defined as 1 µmol of TMPD oxidized per min.
Quinol oxidase activity was measured as oxygen consumption in a
magnetically stirred 2-ml chamber with a Clark-type oxygen electrode
(Rank Brothers, Cambridge, United Kingdom). The chamber was
thermostatted at 25 °C and filled with 1 ml of 50 mM
air-saturated sodium phosphate buffer, pH 6.5, supplemented with 200 µM dimethylnaphthoquinol (DMNH2). After
recording the rate of autoxidation, the measurement was started by
adding the protein sample. One unit of activity refers to 1 µmol of
O2 reduced per min. Dimethylnaphthoquinone (DMN) was
obtained initially as a gift from A. Kröger (Frankfurt, Germany)
and later synthesized by mild oxidation of 2,3-dimethylnaphthaline with
chromium(VI) oxide as described by Kruber (21). DMNH2 was
formed by adding a few grains of sodium borohydride and sodium
dithionite to a 5 mM solution of DMN in 50% ethanol.
Cytochrome c oxidase activity was measured
spectrophotometrically at 550 nm with bovine heart cytochrome
c (Sigma) or yeast cytochrome c (Sigma) as
described (7). One unit of activity refers to 1 µmol of cytochrome
c oxidized per min.
Difference Spectroscopy--
Dithionite-reduced minus
ferricyanide-oxidized difference spectra were recorded at room
temperature with a Jasco V560 spectrophotometer. For turbid samples
(intact cells and membranes) a special silicon photodiode detector was
used (22). Heme contents were calculated from reduced minus oxidized
spectra using the following wavelength pairs and absorption
coefficients (mM1cm
1): heme
a,
630-600 nm = 11.6 (23); heme
b,
562-577 nm = 22, and heme
c,
552-540 nm = 19.1 (24).
Miscellaneous--
Protein concentrations were determined with
the bicinchoninic acid protein assay (25) using bovine serum
albumin as the standard. SDS-PAGE was carried out as described
(26) except that the samples were incubated at 40 °C for 30 min
before loading. Staining of c-type cytochromes in
polyacrylamide gels was performed with 3,3',5,5'-tetramethylbenzidine
(27). For Western blotting, proteins were separated by Tricine-SDS-PAGE
(28) and electroblotted onto a polyvinylidene difluoride membrane
(Immobilon P; Millipore) using the semidry method according to
Schägger and von Jagow (29). Strep-tagged QcrB was detected
using streptavidin-alkaline-phosphatase conjugate and CDP-star (Roche
Diagnostics, Mannheim, Germany) as described (12).
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RESULTS |
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Construction and Functional Analysis of Strep-tagged Variants of
Cytochrome b (QcrB) and of Subunit I (CtaD) of Cytochrome
aa3 Oxidase--
To purify the cytochrome
bc1 complex and cytochrome
aa3 oxidase by affinity chromatography, plasmids
pJC1-qcrBSt and
pJC1-ctaDSt were constructed encoding QcrB and
CtaD proteins elongated with a C-terminal StrepTag II, respectively.
Plasmid pJC1-qcrBSt contained the entire
ctaE-qcrCAB gene cluster under control of its presumed native promoter and was able to complement the severe growth defect of
C. glutamicum strain 13032qcr, which contains
a deletion of the chromosomal qcrCAB genes (Fig.
2). Plasmid
pJC1-ctaDSt contained the ctaD gene
with its promoter region and complemented the growth defect of C. glutamicum strain 13032
ctaD (Fig. 2). Reduced minus oxidized difference spectra of the complemented strains
Q-BSt (13032
qcr with plasmid
pJC1-qcrBSt) and
C-DSt
(13032
ctaD with plasmid
pJC1-ctaDSt) revealed a wild-type-like pattern
with cytochromes of the a-, b-, and
c-type (data not shown) whereas those of strains 13032
qcr and 13032
ctaD lacked cytochrome
c and cytochrome a, respectively (5). Thus,
pJC1-qcrBSt and
pJC1-ctaDSt allowed the synthesis of a
functional bc1 complex and of a functional cytochrome aa3 oxidase, respectively, and the
presence of the StrepTag II did not interfere with the activity of the
two respiratory complexes.
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Isolation of a Cytochrome bc1-aa3
Supercomplex--
For the purification of the
bc1 complex and cytochrome
aa3 oxidase, membranes of the complemented
strains Q-BSt and
C-DSt were isolated,
and the proteins obtained after solubilization with dodecyl maltoside
were subjected to affinity chromatography on StrepTactin-Sepharose.
After washing, specifically bound proteins were eluted with
desthiobiotin and analyzed by SDS-PAGE. Surprisingly, the protein
pattern observed in the eluates from strains
Q-BSt (Fig.
3, lane 3) and
C-DSt (Fig. 3, lane 2) were highly similar and contained eight protein bands of identical apparent mass. The
protein of 24 kDa (P24) was not only copurified with
QcrBSt but also appeared in some preparations obtained with
CtaDSt (data not shown). The identity of the proteins
indicated in Fig. 3 except for the 17-kDa protein (P17) was determined
by peptide mass fingerprinting using in-gel digestion with trypsin or
cyanogen bromide and matrix-assisted laser desorption ionization-time
of flight mass spectrometry. Because the bands with an apparent mass of
52 and 29 kDa were found to consist of two different proteins at a
time, 10 proteins were identified in total. Besides the known subunits
of the bc1 complex (QcrA, QcrB, QcrC) and of
cytochrome aa3 oxidase (CtaC, CtaD, CtaE), the
four additional proteins with an apparent mass of 29 kDa
(P29), 24 kDa (P24), 20 kDa (P20) and 19 kDa (P19) were assigned to the
hitherto hypothetical proteins Cgl2664, Cgl2226, Cgl2017, and Cgl2194,
respectively.
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The successful protein identification clearly showed that the eluate
both of strain Q-BSt (Fig. 3, lane 3) and of
strain
C-DSt (Fig. 3, lane 2) contained the
three subunits of the bc1 complex (QcrA, QcrB,
and QcrC) and the three subunits of cytochrome aa3 oxidase (CtaD, CtaC, and CtaE). The fact
that these proteins were copurified irrespective of whether the
purification was performed via QcrBSt or CtaDSt
strongly indicated that the bc1 complex and cytochrome aa3 oxidase form a supercomplex in
C. glutamicum.
Heme Contents and Enzymatic Activities of the
bc1-aa3 Supercomplex--
Reduced
minus oxidized difference spectra of the supercomplex purified either
via QcrBSt or via CtaDSt showed that both
preparations contained cytochromes of the a-, b-,
and c-type but in different ratios (Fig.
4). The calculated contents of heme
a, heme b, and heme c were 1.6, 6.1, and 2.8 µmol/g of protein in the QcrBSt complex and 4.2, 2.6 and 3.0 µmol/g in the CtaDSt complex. These values
cannot be fit into a simple ratio of small integers, indicating that
the preparations are stoichiometrically heterogeneous. In both cases
the strep-tagged subunit was most abundant, i.e. cytochrome b in the QcrBSt complex and cytochrome
a in the CtaDSt complex. Thus, the
bc1-aa3 supercomplex was
partially dissociated despite the gentle method used for
purification.
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A functional association of bc1 complex and
cytochrome aa3 oxidase should possess quinol
oxidase activity. Using the menaquinol analogon DMNH2 as
substrate, such an activity could be measured polarographically not
only with membrane fractions but also with the purified supercomplexes
as summarized in Table III. The turnover number decreased during the purification, most likely because of the
partial dissociation of the supercomplex indicated above. Besides
quinol oxidase activity of 1.5-1.7 units/mg protein, the preparations
also possessed TMPD oxidase activity of 0.8 units/mg (QcrBSt complex) and 1.0 units/mg (CtaDSt
complex) corresponding to turnover numbers
(TMPD/aa3 s1) of 16.1 and 7.6, respectively.
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Purification of the bc1 Complex and of Cytochrome
aa3 Oxidase as Single Complexes--
To purify the
bc1 complex as a single complex rather than as a
supercomplex, plasmid pJC1-qcrBSt was
transferred to C. glutamicum 13032ctaD. The
resulting strain
C-BSt had the same growth defect as
strain 13032
ctaD because of the absence of CtaD and
formed both wild-type and strep-tagged QcrB. Purification of
strep-tagged proteins from dodecyl maltoside-solubilized membranes by
StrepTactin affinity chromatography resulted in two dominant proteins
that were identified as cytochrome b (QcrB) and Rieske
iron-sulfur protein (QcrA; Fig. 3, lane 4). In addition,
minor amounts of P24 were enriched. Most remarkably, cytochrome
c1 (QcrC) was not present in this preparation,
indicating that the interaction between QcrC and the two other subunits
of the bc1 complex is quite weak.
For the purification of cytochrome aa3 as a
single complex, a similar approach was applied as described above for
the bc1 complex, i.e. plasmid
pJC1-ctaDSt was transferred into strain 13032qcr. The resulting strain
Q-DSt had
the same phenotype as strain 13032
qcr and formed both
wild-type and strep-tagged CtaD. The eluate obtained after StrepTactin
affinity chromatography of dodecyl maltoside-solubilized membranes
contained four proteins (Fig. 3, lane 1), which were
identified as CtaD, CtaC, CtaE, and P19. The TMPD oxidase activity of
the cytochrome aa3 oxidase preparation was 0.34 units/mg, corresponding to a turnover number of 1.1 TMPD oxidized/aa3 s
1. The 10-fold
decreased TMPD oxidase activity compared with the supercomplexes is
because of the absence of cytochrome c1. The cytochrome c oxidase activity with bovine heart cytochrome
c and yeast cytochrome c was 0.35 and 0.28 units/mg, respectively. This corresponds to turnover numbers of 1.2 and
0.9 cytochrome c oxidized/aa3 s
1.
Evidence for a Fourth Subunit of Cytochrome aa3
by Phenotypic Analysis of Mutants Lacking P29, P24, P20, or
P19--
To determine the relevance of proteins P29, P24, P20, and P19
for respiration and formation of the
bc1-aa3 supercomplex, the corresponding genes were deleted in-frame from the chromosome of
C. glutamicum, resulting in strains Cg2664,
Cg2226,
Cg2017, and
Cg2194, respectively. The former three strains showed
no obvious phenotype regarding growth in rich medium and the formation of a-, b-, and c-type cytochromes
(data not shown). Apparently, proteins P29, P24, and P20 are not
essential for the formation and activity of the
bc1-aa3 branch of the
respiratory chain, and the functional significance of the interaction
between these proteins and the
bc1-aa3 supercomplex
remains to be elucidated.
In contrast, deletion of the gene Cgl2194 encoding P19 led to a similar
phenotype observed previously for the 13032ctaD strain. Growth on rich medium agar plates was strongly impaired (data not
shown), cytochrome a was almost absent in the spectrum of dithionite-reduced cells, and the level of cytochrome
c1 was markedly lower than in the wild-type
(Fig. 5). Consequently, the P19 protein is essential for the formation of an active cytochrome
aa3 oxidase. P19 was enriched with the
supercomplex and the isolated cytochrome aa3
oxidase, but not with the isolated bc1 complex
(Fig. 3), showing that copurification is because of an interaction with
the cytochrome aa3 subunits. Based on these
data, P19 has to be regarded as a fourth subunit of the C. glutamicum cytochrome aa3 oxidase.
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Protein P19 is composed of 143 amino acids and has a predicted mass of
15.5 kDa. It contains three hydrophobic regions extending from residues
7-27, 40-60, and 97-130, which presumably form three or four
transmembrane helices. The first transmembrane helix may be part of a
signal peptide. As shown in the alignment in Fig. 6, the primary sequence is well
conserved in other species of the actinomycetales including
C. diphtheriae (68% identity), mycobacteria (38-39%),
Streptomyces coelicolor (39%), and Thermobifida
fusca (33%). In all these organisms the corresponding gene is
located immediately downstream of ctaC or a ctaCD
gene cluster in the case of S. coelicolor and T. fusca and presumably is cotranscribed with these genes. This
further supports the previous suggestion that the P19 homologues
represent a fourth subunit of cytochrome aa3
oxidase in the actinomycetes. Therefore, the corresponding genes were
named ctaF.
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Necessity of Heme Incorporation into Cytochrome c1
for Assembly and/or Stability of the
bc1-aa3
Supercomplex--
According to our previous proposal that the second
heme group of the C. glutamicum diheme cytochrome
c1 is involved in electron transfer from the
first heme group of c1 to the CuA
center of cytochrome aa3 oxidase, both heme
groups should be essential for the activity of the
bc1-aa3 branch of the
respiratory chain. To test this assumption, both cysteine residues in
each of the two CXXCH heme binding motifs of QcrC were
converted to serine residues by site-directed mutagenesis of plasmid
pJC1-qcrBSt. The effects of these mutations were
analyzed after transformation of strain 13032qcr with the
resulting plasmids pJC1-qcrBSt-C67S and
pJC1-qcrBSt-C177S, respectively. Both mutant
strains showed strongly impaired growth similar to strain
13032
qcr (Fig. 7),
indicating the absence of a functional bc1
complex. The membranes of the two strains did not contain
c-type cytochromes as judged by heme staining of SDS gels
(Fig. 8A) and reduced minus
oxidized difference spectra of membranes (data not shown). Obviously,
both heme groups of cytochrome c1 are essential
for respiration via the
bc1-aa3 branch of the respiratory chain, and no stable monoheme intermediate can be formed
during the maturation of QcrC if the incorporation of the other heme
group is blocked.
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Western blot analysis with streptavidin-alkaline phosphatase conjugate
was performed to check whether the disturbed cytochrome c1 maturation in the mutant strains also
influences the QcrBSt content of the cytoplasmic membranes.
As shown in Fig. 8B, both QcrC mutant strains had strongly
decreased QcrBSt levels of less than 10% compared with
strain Q-BSt as estimated from the signal intensities.
This indicated that the presence of holo-cytochrome c1 is highly important for the assembly and/or
stability of the entire bc1 complex. Besides
QcrBSt, which was unequivocally identified with a sample of
the purified QcrBSt complex, additional proteins were
detected by the streptavidin-alkaline phosphatase conjugate (Fig. 8),
which represented the biotinylated proteins pyruvate carboxylase (not
shown) (30) and the
-subunit of acyl CoA carboxylase (AccBC; see
Ref. 31). The cytochrome a level of the cytochrome c1 mutants was unchanged compared with the
control strain
Q-BSt (data not shown), indicating that
synthesis of cytochrome aa3 is independent of an
intact bc1 complex.
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DISCUSSION |
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Identification of a Cytochrome bc1-aa3 Supercomplex in C. glutamicum-- In the present study we show that the bc1 complex and cytochrome aa3 oxidase of C. glutamicum are organized in a supercomplex with quinol oxidase activity. The approach used to isolate this supercomplex involved the modification of one subunit with a StrepTag II and subsequent affinity purification with StrepTactin-Sepharose. Similar approaches were used previously, e.g. to systematically define protein complexes in yeast (32, 33). Although this procedure can lead to the accidental copurification of proteins, this was not the case here, because all subunits of the bc1 complex and of cytochrome aa3 were isolated both with strep-tagged QcrB and with strep-tagged CtaD. The lack of evidence for a supercomplex in the previous purification of either cytochrome c1 (6) or cytochrome aa3 oxidase (7) shows that the interactions are relatively weak and require a very gentle purification procedure for preservation. Although we could also isolate the bc1-aa3 supercomplex using a hexahistidine-tagged QcrB and Ni2+-chelate affinity chromatography (data not shown), the StrepTag II/StrepTactin system proved to be superior in our hands.
The formation of a bc1-aa3 supercomplex with quinol oxidase activity is not unique to C. glutamicum. In fact, such complexes were purified from several bacteria, i.e. Paracoccus denitrificans (34), the thermophilic Bacillus PS3 (35), or the thermoacidophilic archaeon Sulfolobus sp. strain 7 (36). In Bradyrhizobium japonicum, a bc1-cM-aa3 complex was isolated from aerobically grown cells but not characterized for its quinol oxidase activity (37). From bacteroids of B. japonicum a complex of cytochrome bc1 and a cb-type cytochrome oxidase, most probably cytochrome cbb3 (38, 39), was isolated (40). It displayed cytochrome c oxidase and TMPD oxidase activity but no quinol oxidase activity, presumably because of the lack of the Rieske iron-sulfur protein.
A supramolecular organization of complexes III and IV was also shown in yeast and bovine mitochondria (41, 42). Thus, quinol oxidase supercomplexes were detected in Gram-positive and Gram-negative eubacteria, in archaea, and in eukaryotes, indicating that this highly organized state is a general feature rather than a specific character of certain species.
Identification of Subunit IV of Cytochrome aa3 Oxidase-- In contrast to the previous purification of cytochrome aa3 oxidase from C. glutamicum by conventional column chromatography, which resulted in the isolation of subunits I, II, and III (7), our preparation contained an additional protein (CtaF) encoded by the gene downstream of ctaC (Fig. 3). The identification of this protein as a fourth subunit rests on the observation that a C. glutamicum mutant lacking ctaF showed the same growth defect as a ctaD deletion mutant, and like in this strain cytochrome a was almost undetectable. Although CtaF is essential for the formation of a functional cytochrome aa3 oxidase, it is presumably not required for catalytic activity, because the turnover numbers of the four-subunit complex were in the same range as those of the three-subunit complex (7). Therefore, CtaF is probably involved in the assembly and/or stabilization of cytochrome aa3 oxidase.
The composition of four subunits is common within the heme-copper family of bacterial terminal oxidases. Subunit IV (CtaH) of cytochrome aa3 oxidase from P. denitrificans consists of a single transmembrane helix residing in a cleft between subunits I and III (43). Deletion of the ctaH gene had no consequences for the integrity of the complex and its spectral and enzymatic properties (44). Subunit IV (CyoD) of the bo-type ubiquinol oxidase from E. coli consists of three transmembrane helices and is located between subunits I and III. The third helix is in contact with helix VII of subunit I in the vicinity of the CuB-heme a3 binuclear center (45). Deletion analyses indicated that subunit IV is essential for the synthesis of the functional bo3 oxidase complex and for the CuB binding to the binuclear center, although it can be removed in vitro without a loss of the enzymatic activity (46). Subunit IV (QoxD) of the cytochrome aa3 menaquinol oxidase from B. subtilis, like CyoD of E. coli, consists of three transmembrane helices. A mutant lacking the qoxD gene was reported to have decreased respiratory activity and proton pumping activity (47).
CtaF of C. glutamicum shows no significant sequence similarity to CtaH of P. denitrificans, CyoD of E. coli, QoxD of B. subtilis, and CtaF of B. subtilis, and homologs of these proteins were absent in the C. glutamicum genome. In contrast, all actinomycetes with known genome sequence contain homologs of C. glutamicum CtaF (Fig. 6), and the corresponding genes are always clustered with ctaC. Thus, CtaF represents the first member of subunit IV of cytochrome aa3 oxidase in this group of bacteria.
Electron Transfer between the bc1 Complex and Cytochrome aa3-- The identification of a bc1-aa3 supercomplex with quinol oxidase activity supports the assumption that the second heme group of the diheme cytochrome c1 transfers electrons from the first heme group to the CuA center in subunit II of cytochrome aa3 oxidase. The question whether further proteins are involved in this process remains open at present. The proteins P29, P24, and P20 can certainly be excluded, because C. glutamicum mutants lacking these proteins showed no obvious growth defects. In the case of subunit IV (CtaF) of cytochrome aa3 oxidase, a role in electron transfer is also very unlikely (see above). However, the copurified protein P17 could not yet be identified, and therefore the effect of its absence on the formation and activity of the supercomplex could not be tested.
Besides two covalently bound heme groups, cytochrome c1 of C. glutamicum has another unusual property, i.e. its weak interaction with the Rieske iron-sulfur protein and cytochrome b. Neither of these two proteins was copurified during the isolation of cytochrome c1 by conventional chromatographic methods (6), and vice versa, the preparation isolated via strep-tagged cytochrome b lacked cytochrome c1 (Fig. 3, lane 4). The presence of QcrC in the supercomplex, purified either via strep-tagged QcrB or via strep-tagged CtaD, therefore must be because of an interaction with cytochrome aa3 oxidase or requires interaction with both the bc1 complex and cytochrome aa3 oxidase. Further studies are needed to discriminate between these possibilities.
Formation of Holo-cytochrome c1 Is Essential for
Assembly and/or Stability of the bc1
Complex--
Mutation of either the N-terminal or the C-terminal
CXXCH motif in cytochrome c1 to
SXXSH led to a severe growth defect in the corresponding
mutants similar to strain 13032qcr (Fig. 7), which was
because of the absence of holo-cytochrome c1 and
drastically reduced levels of cytochrome b. This shows that
if any monoheme cytochrome c1 is formed, it must
be rapidly and completely degraded. Similar results have been reported
for the diheme cytochrome c subunit (FixP) of the
cbb3 oxidase in B. japonicum (48).
The strong effect of the cytochrome c1 mutations
on the cytochrome b level showed that holo-cytochrome
c1 is not only required for electron transfer
but also for the maturation and/or stabilization of the entire
bc1 complex. The purification of an apparently
stoichiometrical complex of cytochrome b and Rieske
iron-sulfur protein from strain
C-BSt argues against a
role of cytochrome c1 in stabilization but
certainly does not exclude this possibility.
The data from C. glutamicum are in accordance with results
from other bacteria, i.e. that deletion of the cytochrome
c1 gene in P. denitrificans (49),
Rhodobacter capsulatus (50), and B. japonicum
(51), as well as mutation of the heme binding site of B. japonicum cytochrome c1 (37), caused
degradation of cytochrome b and, if tested, also of the
Rieske iron-sulfur protein. According to these data the current model
for bc1 complex maturation predicts that
formation of holo-cytochrome c1 is an early and
essential requirement for assembly of the whole complex (52).
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FOOTNOTES |
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* 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.: 49-2461-615515;
Fax: 49-2461-612710; E-mail: m.bott@fz-juelich.de.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M210499200
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high pressure liquid chromatography; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; DMN, 2,3-dimethyl-1,4-naphthoquinone.
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