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J. Biol. Chem., Vol. 278, Issue 6, 4339-4346, February 7, 2003

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Purification of a Cytochrome bc₁-aa₃ Supercomplex with Quinol Oxidase Activity from Corynebacterium glutamicum

IDENTIFICATION OF A FOURTH SUBUNIT OF CYTOCHROME aa₃ OXIDASE AND MUTATIONAL ANALYSIS OF DIHEME CYTOCHROME c₁^*

Axel Niebisch and Michael Bott

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 Cu_A 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 a_S, in which the farnesyl group (C₁₅H₂₅) 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).

View larger version (7K): [in this window] [in a new window]   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 13032qcr and 13032ctaF 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 Cu_A 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

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).

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-qcrB_St 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-ctaD_St. 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 13032qcr 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-qcrB_St 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-qcrB_St yielding pJC1-qcrB_St-C67S and pJC1-qcrB_St-C177S, respectively, which were transferred into C. glutamicum strain 13032qcr. 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·cm¹ 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 (mM¹cm¹): 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-qcrB_St and pJC1-ctaD_St were constructed encoding QcrB and CtaD proteins elongated with a C-terminal StrepTag II, respectively. Plasmid pJC1-qcrB_St 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-ctaD_St contained the ctaD gene with its promoter region and complemented the growth defect of C. glutamicum strain 13032ctaD (Fig. 2). Reduced minus oxidized difference spectra of the complemented strains Q-B_St (13032qcr with plasmid pJC1-qcrB_St) and C-D_St (13032ctaD with plasmid pJC1-ctaD_St) revealed a wild-type-like pattern with cytochromes of the a-, b-, and c-type (data not shown) whereas those of strains 13032qcr and 13032ctaD lacked cytochrome c and cytochrome a, respectively (5). Thus, pJC1-qcrB_St and pJC1-ctaD_St 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.

View larger version (112K): [in this window] [in a new window]   Fig. 2.   Complementation of the growth defects of the C. glutamicum strains 13032ctaD and 13032qcr by the plasmids pJC1-ctaDSt and pJC1-qcrBSt encoding strep-tagged CtaD and QcrB, respectively. The C. glutamicum strains were cultivated for 2 days at 30 °C on brain heart infusion agar plates containing 0.5 M D-sorbitol and 25 µg/ml kanamycin. A, ATCC13032 (wild-type)/pWK0; B, 13032qcr/pJC1; C, 13032qcr/pJC1-qcrBSt (Q-BSt); D, 13032ctaD/ pJC1-ctaDSt (C-DSt); E, 13032ctaD/pWK0.

Isolation of a Cytochrome bc₁-aa₃ Supercomplex-- For the purification of the bc₁ complex and cytochrome aa₃ oxidase, membranes of the complemented strains Q-B_St and C-D_St 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-B_St (Fig. 3, lane 3) and C-D_St (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 QcrB_St but also appeared in some preparations obtained with CtaD_St (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.

View larger version (78K): [in this window] [in a new window]   Fig. 3.   SDS-polyacrylamide gel analysis of cytochrome bc1 and cytochrome aa3 single complexes and supercomplexes from C. glutamicum purified by affinity chromatography on StrepTactin-Sepharose via QcrBSt or CtaDSt. The proteins were denatured, separated on an 8-16% Tris-HCl gradient gel (Bio-Rad), and stained with Coomassie Blue. Lane 1, cytochrome aa3 oxidase (7.2 µg of protein) purified from strain Q-DSt; lane 2, cytochrome bc1-aa3 supercomplex purified from strain C-DSt (4.7 µg); lane 3, cytochrome bc1-aa3 supercomplex purified from strain Q-BSt (5.7 µg); lane 4, cytochrome "bc1" complex purified from strain C-BSt (4.3 µg).

The successful protein identification clearly showed that the eluate both of strain Q-B_St (Fig. 3, lane 3) and of strain C-D_St (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 QcrB_St or CtaD_St 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 QcrB_St or via CtaD_St 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 QcrB_St complex and 4.2, 2.6 and 3.0 µmol/g in the CtaD_St 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 QcrB_St complex and cytochrome a in the CtaD_St complex. Thus, the bc₁-aa₃ supercomplex was partially dissociated despite the gentle method used for purification.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Reduced minus oxidized difference spectra of the bc1-aa3 supercomplex purified from the C. glutamicum strains C-DSt and Q-BSt. The supercomplexes purified by affinity chromatography on StrepTactin-Sepharose were reduced with dithionite or oxidized with ferricyanide before recording the spectra at room temperature (protein concentration 0.5 mg/ml).

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 (QcrB_St complex) and 1.0 units/mg (CtaD_St complex) corresponding to turnover numbers (TMPD/aa₃ s¹) of 16.1 and 7.6, respectively.

View this table: [in this window] [in a new window]   Table III Purification of a cytochrome bc1-aa3-supercomplex from membranes of the C. glutamicum strains Q-BSt and C-DSt The quinol oxidase activity was determined with DMNH2 as substrate by measuring the oxygen consumption rate with a Clark-type oxygen electrode. One unit corresponds to 1 µmol of O2 consumed per min. The turnover numbers (TN) are calculated as electrons transferred per cytochrome aa3 per second, except for the number in parentheses, which was calculated as electrons transferred per cytochrome b per second. ND, not determined.

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-qcrB_St was transferred to C. glutamicum 13032ctaD. The resulting strain C-B_St had the same growth defect as strain 13032ctaD 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-ctaD_St was transferred into strain 13032qcr. The resulting strain Q-D_St had the same phenotype as strain 13032qcr 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 s¹. 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 s¹.

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.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Absolute spectra of reduced intact cells of the C. glutamicum strains ATCC13032 (wild-type, WT), 13032ctaD, and 13032ctaF. Cells were resuspended in 100 mM Tris-HCl buffer, pH 7.5, to an A600 nm of 200 and reduced by the addition of dithionite before recording the spectra at room temperature. The relevant differences in the cytochrome pattern are indicated by arrows. The spectra of strains 13032ctaD and 13032ctaF are drawn with an offset of 0.2 and 0.4, respectively.

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.

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Sequence alignment of CtaF proteins from different species of actinomycetes. Putative transmembrane helices are indicated by lines and are numbered. Amino acids identical in at least five sequences are shaded in black, and conservative exchanges are in gray. The bacteria have been abbreviated as follows: Cgl, C. glutamicum (Cgl2194); Cdi, C. diphtheriae (NC_002935); Mtu, M. tuberculosis (Rv2199c); Mbo, M. bovis (NC_002945); Mle, M. leprae (ML0876); Sco, S. coelicolor (NP_626410); Tfu, T. fusca (Tfus_p_278).

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 Cu_A 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-qcrB_St. The effects of these mutations were analyzed after transformation of strain 13032qcr with the resulting plasmids pJC1-qcrB_St-C67S and pJC1-qcrB_St-C177S, respectively. Both mutant strains showed strongly impaired growth similar to strain 13032qcr (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.

View larger version (118K): [in this window] [in a new window]   Fig. 7.   Effect of defective heme binding sites in cytochrome c1 on growth of C. glutamicum. The strains were cultivated for 2 days at 30 °C on brain heart infusion agar plates containing 0.5 M D-sorbitol and 25 µg/ml kanamycin. A, ATCC13032 (wild-type)/pWK0; B, 13032qcr/pJC1; C, 13032qcr/pJC1-qcrBSt-C67S; D, 13032qcr/pJC1-qcrBSt-C177S; E, 13032qcr/pJC1-qcrBSt.

View larger version (43K): [in this window] [in a new window]   Fig. 8.   Analysis of cytochrome c1 (QcrC) and strep-tagged cytochrome b (QcrBSt) in mutant strains of C. glutamicum. A, membrane proteins (200 µg per lane) of the strains were separated by SDS-PAGE, and the gels were stained with tetramethylbenzidine for covalently bound heme in cytochrome c1. B, Western blot analysis of QcrBSt in membranes of the strains indicated below. The membrane proteins (50 µg per lane) were separated by SDS-PAGE on a 10% Tris-Tricine gel and subsequently blotted onto a polyvinylidene difluoride membrane. Detection was performed with a streptavidin-alkaline phosphatase conjugate and the CDP-Star substrate (both from Roche Diagnostics). Lane 1, 13032qcr/pJC1-qcrBSt; lane 2, 13032qcr/pJC1-qcrBSt-C177S; lane 3, 13032qcr/pJC1-qcrBSt-C67S; lane 4, 13032qcr/pJC1; lane 5, purified cytochrome bc1-aa3 supercomplex (4.6 µg of protein) from C. glutamicum Q-BSt.

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 QcrB_St content of the cytoplasmic membranes. As shown in Fig. 8B, both QcrC mutant strains had strongly decreased QcrB_St levels of less than 10% compared with strain Q-B_St 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 QcrB_St, which was unequivocally identified with a sample of the purified QcrB_St 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-B_St (data not shown), indicating that synthesis of cytochrome aa₃ is independent of an intact bc₁ complex.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-c_M-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 Cu_B-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 Cu_B 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 Cu_A 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-B_St 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).

	FOOTNOTES

^* 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

	ABBREVIATIONS

The abbreviations used are: HPLC, high pressure liquid chromatography; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; DMN, 2,3-dimethyl-1,4-naphthoquinone.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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