Multi-step Assembly Pathway of the cbb3-type Cytochrome c Oxidase Complex

doi:10.1016/j.jmb.2005.11.039

J. Mol. Biol. (2006) 355, 989–1004

Multi-step Assembly Pathway of the cbb3-type Cytochrome c Oxidase Complex Carmen Kulajta1,2, Jo¨rg Oliver Thumfart3, Sybille Haid1, Fevzi Daldal4 and Hans-Georg Koch1* 1

Institute for Biochemistry and Molecular Biology, Faculty of Medicine, University of Freiburg, D-79104 Freiburg Germany 2

Faculty of Biology, University of Freiburg, D-79104 Freiburg Germany 3

Physiology Institute, Faculty of Medicine, University of Freiburg, D-79104 Freiburg Germany 4

Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia PA 19104-6018, USA

The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa subcomplex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: assembly of membrane proteins; maturation of c-type cytochrome complexes; Rhodobacter capsulatus; respiration; photosynthesis

Introduction Heme-copper oxidases are universally conserved integral multisubunit membrane proteins that Abbreviations used: BN-PAGE, blue-native polyacrylamide gel electrophoresis; cbb3–Cox, cbb3-type cytochrome c oxidase; cyt/cyts, cytochrome(s); DDM, n-dodecylmaltoside; ICM, intracytoplasmic membranes; NADI reaction, (N 0 -N 0 -dimethyl-p-phenyldiamine (DMPD)CO2(indophenol blueCwater); TMBZ, 3 0 ,3 0 ,5 0 ,5 0 -tetramethylbenzidine. E-mail address of the corresponding author: [email protected]

catalyse the four-electron reduction of dioxygen to water, the terminal reaction of respiratory electron transport chains in mitochondria and aerobic bacteria.1,2 Bacterial respiratory chains usually contain multiple terminal oxidases, which allow bacteria to adapt to different environmental O2 concentrations during their life cycle. These terminal oxidases are divided into two sub-families, depending on the use of either cytochrome (cyt) c or hydroquinone as electron donors. A major difference between the cyt c oxidases and the quinol oxidases is the presence of a binuclear copper centre (CuA) located in the hydrophilic domain of subunit II of the cyt c oxidases, which is absent from

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

990 the corresponding subunit of the quinol oxidases. A subclass of cyt c oxidases, called the cbb3-type cyt c oxidases (cbb3–Cox) is present in proteobacteria and probably also in the CFB (Cytophaga, Flexibacter, Bacteroides) group of bacteria.3,4 These enzymes use a c-type cyt as a substrate, but instead of a CuA-containing subunit, they contain membranebound c-type cyt subunits.5,6 Due to their high oxygen affinity,7 cbb3–Cox are believed to support efficient respiration even under microaerobic conditions. They consist of three or four subunits and their corresponding genes were designated originally fixNOQP in Bradyrhizobium japonicum8 to reflect their important role during symbiotic nitrogen fixation. However, as the cbb 3–Cox complexes are not restricted to nitrogen-fixing bacteria, the fixNOQP genes are referred to as ccoNOQP for many bacteria. The catalytic subunit I (CcoN) contains six conserved histidine residues that are diagnostic for the heme-copper oxidase superfamily.3,9 These residues ligate a low-spin heme b and a high-spin heme b3–CuB binuclear centre, where oxygen reduction takes place. The subunits II (CcoO) and III (CcoP) of the cbb3–Cox are membrane-anchored c-type cyts required for transferring electrons from a donor cyt c to the catalytic binuclear centre of CcoN. Many but not all cbb3–Cox operons include a fourth gene, ccoQ, which presumably encodes a small single-membrane spanning protein with only limited homology to proteins of known function.4 Although CcoQ is not essential for the activity of cbb3–Cox,10,11 it might be involved in its stabilization under aerobic conditions.12 The frequent occurrence of cbb3–Cox in many pathogenic proteobacteria and the observation that it is the only terminal oxidase recognized in the genomes of Helicobacter pylori, Campylobacter jejuni and Neisseria meningitides, has raised the question of whether the cbb3–Cox and their specific assembly factors are important determinants for pathogenicity.13 Currently, very limited information is available on the assembly process of the cbb3–Cox.10,14 Interestingly, in all organisms characterized so far, the ccoNOQP operon is located upstream of the ccoGHIS gene cluster, which encodes four putative integral membrane proteins. Since the formation and stability of an active cbb3–Cox is affected in the absence of the ccoGHIS gene products, they seem to be involved specifically in the biogenesis of the cbb3–Cox. On the other hand, other b-type or c-type cyts containing membrane complexes remain unimpaired by ccoGHIS mutations.15,16 In this study, we have analysed the assembly pathway of cbb3–Cox in the facultative photosynthetic proteobacterium Rhodobacter capsulatus, which is commonly used as a model organism for studying microbial energy transduction.17 By combining blue-native polyacrylamide gel electrophoresis with activity staining using wild-type and mutant membranes, we show here for the first time that the cbb3–Cox subunits assemble via an inactive

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210 kDa (CcoNOQ) sub-complex into the final 230 kDa (CcoNOQP) active complex. Our data indicate that this transition involves the recruitment of CcoH-stabilized monomeric CcoP into a likewise CcoHS-stabilized 210 kDa sub-complex. Moreover, in this work we provide the first evidence for a direct interaction between the proposed assembly proteins CcoH and CcoS and the cbb3–Cox subunits during its assembly in R. capsulatus membranes.

Results The cbb3-type cytochrome c oxidase assembles into an active 230 kDa complex To elucidate the assembly process of cbb3–Cox, the molecular mass, composition and oligomeric state of this multi-subunit membrane protein complex in R. capsulatus membranes were examined by blue-native polyacrylamide gels (BN-PAGE).18 An enzymatically active cbb3–Cox was revealed by using the NADI reaction (N 0 -N 0 dimethyl-p-phenyldiamine (DMPD) CO2/indophenol blueCwater) in situ on BN-PAGE. Although this reaction is commonly used to detect cyt c oxidase activities in intact cells,14,19 to our knowledge, it has never been applied as an in-gel activity staining method prior to this work. Using this staining method with dodecyl maltoside (DDM)-solubilized native membranes, a NADIpositive membrane protein complex of about 230 kDa was recognized (Figure 1(a)). This complex was absent from the cbb3–Cox knockout strain GK3214 and was much stronger in membranes of a cbb 3–Cox over-expressing strain (Figure 1(a)). In all three strains, several bacteriochlorophyll-containing high molecular mass complexes were also visible. One of them, located immediately above the NADI-stainable band, was used as an internal reference to monitor the solubilization efficiency and electrophoretic properties of the 230 kDa complex. As the visualization of membrane protein complexes by BN-PAGE depends largely on the nature of the detergent, and the protein/detergent ratio used,20 different detergents and various ratios of protein to detergent were tested. A 230 kDa NADI-stainable complex was also detected in membranes solubilized in Triton X-100 and digitonin, but not in octylglycoside (data not shown). Since the CcoO and CcoP subunits of cbb3–Cox are membrane-bound c-type cyts containing covalently attached heme groups, they can be visualized via their intrinsic peroxidase activity using 3 0 ,3 0 ,5 0 ,5 0 -tetramethylbenzidine (TMBZ) in the presence of H2O2.21 If the 230 kDa NADI-positive complex corresponds to cbb3–Cox, then it should be TMBZ-stainable, which was indeed the case (Figure 1(b)). The TMBZstainable complex was readily visible in the cbb3–Cox over-producing strain but detectable only weakly in wild-type membranes (Figure 1(b)), probably due

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Figure 1. cbb3–Cox assembles into an active 230 kDa complex in R. capsulatus native membranes. Intracytoplasmic membranes (ICMs, 200 mg total protein) from a wild-type (WT), a cbb3–Cox overproducer strain (WT pCW25) and a cbb3–Cox deletion strain (GK32) were solubilized with n-dodecyl-maltoside as described in Materials and Methods, and after ultracentrifugation, supernatants containing the solubilized membrane complexes were separated using BN-PAGE. (a) Detection of the active cbb3–Cox in BN-PAGE by staining with NADI (N 0 ,N 0 -dimethyl-p-phenyldiamine (DMPD)C O2/indophenol blueCwater). A blue-stained band that appeared upon staining with NADI is indicated by an asterisk (*). (b) Detection of membrane-bound c-type cyts in BN-PAGE by TMBZ (3,3 0 ,5,5 0 -tetramethylbenzidine) staining. In the presence of H2O2, the intrinsic peroxidase-activity of c-type cyts is revealed by the appearance of light-blue bands (*). (c) Western blot analyses of membranes derived from the wild-type and the ccoP mutant M7G. Following BN-PAGE, proteins were electroblotted onto Immobilon-P membranes, and probed with polyclonal antibodies directed against CcoP (anti-CcoP) or CcoN (anti-CcoN). As control, membranes of the cbb3–Cox-overproducing strain were separated on BN-PAGE and stained with TMBZ to reveal c-type cyts.

to the smaller amounts of solubilized cbb3–Cox. In comparison to the NADI stain, the TMBZ stain appeared to be more diffuse, suggesting the presence of more than one c-type cyt complex in this region of the BN-PAGE. Although small membrane protein complexes are not well resolved by BN-PAGE, and their detection is difficult due to their proximity to the Coomassie brilliant blue front at the bottom of the BN- PAGE gels, we reproducibly observed additional TMBZ-stainable bands below the 69 kDa marker band (Figure 1(b)). They were most pronounced in membranes derived

from the cbb3–Cox over-expressing strain, but a weak TMBZ-stainable band was recognized in the cbb3–Cox knock-out strain GK32, suggesting the presence of other monomeric c-type cyt in these membranes. In order to confirm that the 230 kDa complex contained the subunits of the cbb3–Cox, we used immune-detection methods. Wild-type membranes were separated on BN-PAGE and, after transfer to a polyvinylidene difluoride (PVDF) membrane, decorated with antibodies directed against either CcoN (anti-CcoN) or CcoP (anti-CcoP).

992 The anti-CcoN antibodies recognized two bands, one at 230 kDa and a second band running at about 210 kDa (Figure 1(c)). The 230 kDa complex, but not the 210 kDa complex, was also recognized by antiCcoP antibodies. A band running in BN-PAGE below the 69 kDa marker band was recognized by anti-CcoP, but not by anti-CcoN, antibodies (Figure 1(c)). The 230 kDa complex was not detected by anti-CcoN antibodies in membranes of the ccoP mutant M7G, which assembles an inactive cbb3–Cox consisting of only CcoN and CcoO but lacking CcoP.5,14 Instead, the amount of the 210 kDa complex in M7G membranes was increased significantly compared to wild-type membranes (Figure 1(c)). In agreement with previous studies 5,14, antibodies directed against CcoP failed to detect any CcoP-containing complexes in M7G and could barely detect a very weak band running below the 69 kDa marker band (Figure 1(c)). Unfortunately, due to the lack of antibodies directed against the third subunit CcoO, we could not detect it immunologically. As mutants lacking CcoO do not assemble a stable cbb3–Cox and do not perform the NADI reaction,14 we surmised that the 230 kDa NADI-positive complex also contains CcoO, which was later confirmed directly (see Figures 3 and 7). The NADI-negative 210 kDa complex probably also contains CcoO, because, like the 230 kDa complex, it was TMBZ-stainable despite the absence of CcoP (Figure 1(c)). Again, the presence of CcoO in the 210 kDa sub-complex was later confirmed (see Figure 7). We conclude from these data that in DDM-solubilized R. capsulatus membranes, the subunits of cbb3–Cox are found in various pools: CcoP is either associated with CcoN to form the 230 kDa active cbb3–Cox complex, or it exists as a stable monomeric protein, while CcoN is present in

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both the 230 kDa active complex and the 210 kDa inactive sub-complex. Assembly of cbb3–Cox into the 230 kDa complex is independent of growth conditions In both eukaryotic and prokaryotic cells, the electron transfer chains appear to be organized in respiratory supercomplexes, referred to as respirasomes.22–24 These large macromolecular structures are believed to optimize electron transfer by minimizing the effect of diffusion on electron transport.25,26 However, how the composition of supercomplexes is modulated in response to various physiological conditions is not known. In R. capsulatus, multiple regulators 27–29, including RegB-RegA, FnrL and HvrA,30 modulate the expression of the cbb3–Cox under various growth conditions.31,32 We therefore determined whether complex formation of cbb3–Cox would be different in membranes from cells grown under aerobic, semiaerobic, or anaerobic-photosynthetic growth conditions. In BN-PAGE, a single NADI-stainable complex at 230 kDa was observed in all membranes. Use of different detergents and different protein/ detergent ratios also revealed the presence of only the 230 kDa active cbb3–Cox complex (data not shown). Moreover, a-CcoN antibodies revealed the presence of the 230 kDa and 210 kDa complexes, whereas a-CcoP antibodies recognized the 230 kDa complex and the monomeric CcoP (Figure 2). As observed earlier,31–33 steady-state levels of the 230 kDa cbb3–Cox complex were highest under semi-aerobic, and lowest under anerobic-photosynthetic, conditions (Figure 2). Thus, cbb3–Cox apparently does not assemble into different complexes in response to various growth conditions.

Figure 2. The 230 kDa cyt cbb3–Cox complex is assembled under various growth conditions. Membranes from aerobically, semi-aerobically and photosynthetically grown wild-type cells were solubilized as described for Figure 1, and solubilized membrane protein complexes were separated using BN-PAGE. Subsequently, proteins were electroblotted onto Immobilon-P membranes and decorated with polyclonal antibodies against CcoN (anti-CcoN, left) or CcoP (anti-CcoP, right).

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Subunit composition of the NADI-positive 230 kDa cbb3–Cox complex The subunit composition of the 230 kDa complex was analyzed by two-dimensional gel electrophoresis and subsequent mass spectrometry. Membranes from the cbb3–Cox over-expressing strain were separated by BN-PAGE, stained with the NADI reagent, and the stained band was cut out and separated by a second-dimension SDS-PAGE (Figure 3(a)). After staining with colloidal Coomassie brilliant blue, protein bands were cut out from the gel, subjected to in-gel digestion by trypsin and identified by mass spectrometry. The two prominent bands of about 50 kDa and 28 kDa (Figure 3(a)) gave rise to peptides homologous to CcoN and to CcoO, as well as to the membrane-bound c-type cyt cy, respectively. Similarly, a weaker band at about 32 kDa was identified as CcoP. In addition, peptides homologous to the predicted amino acid sequence of open reading frame (ORF)277 were identified also in the 28 kDa band. Although this ORF is located immediately upstream of the ccoNOQP operon in many cbb3–Cox-containing bacteria, it is not essential for cbb3–Cox assembly or activity in R. capsulatus,14 and its function remains to be determined. Nevertheless, the detection of

993 ORF277 peptides now establishes that it is translated into a protein. Several weaker bands, including light-harvesting proteins, which are most likely contaminants originating from the bacteriochlorophyll-containing complex running in BN-PAGE immediately above the 230 kDa complex, were also detectable (Figure 3(a)). Preliminary data identified these minor bands as ribosomal proteins (e.g. L19; EF-Tu), chaperones (e.g. Hsp60) and proteases (e.g. ClpB), which are expected to cross-contaminate membrane preparations, due to their cellular abundance. We were unable to detect the small subunit CcoQ by this procedure. As the predicted molecular mass of CcoQ is about 6.5 kDa, its detection by in-gel digestion and subsequent mass spectrometry is more difficult. Although CcoQ is not essential for cbb3–Cox activity in vivo,10 and a purified cbb3–Cox lacking it is still active,5 CcoQ is considered to be important for regulating the electron flow through cbb3–Cox.11 Thus, to determine whether CcoQ is part of the 230 kDa complex, a different approach was undertaken. We have shown recently that in vitro synthesized membrane proteins can be integrated into existing complexes without interfering with their biological activity,34,35 probably by exchanging with the endogenous subunits.

Figure 3. Analysis of the composition of the 230 kDa cbb3–Cox complex by 2D gel electrophoresis and mass spectrometry. (a) The first dimension BN-PAGE was performed using 400 mg of wild-type ICMs as described for Figure 1. The 230 kDa cbb3–Cox complex was visualized by NADI staining, cut out of the gel and separated on SDSPAGE. After staining with colloidal Coomassie brilliant blue, the detected protein bands were cut out and identified by nLC-MS/MS as described in Materials and Methods. (b) For in vitro labelling of the cbb3–Cox complex, R. capsulatus CcoQ was synthesized in vitro using an R. capsulatus cell-free transcription/translation system in the presence or in the absence of ICMs. Membranes carrying integrated radiolabelled CcoQ were pelleted by ultracentrifugation, solubilized and subjected to BN-PAGE as described for Figure 1. Following electrophoresis, radioactively labelled bands were visualized using a phosphorimager. The 230 kDa and 210 kDa cbb3–Cox complexes are indicated by asterisks (*). A band containing bacteriochlorophyll, which is highly abundant in R. capsulatus ICMs, and labelled by CcoQ is marked with a hash sign (#).

994 Accordingly, a R. capsulatus in vitro transcription/ translation system was used to synthesize radioactively labelled CcoQ and to determine its assembly into R. capsulatus membranes. In the absence of membranes, no protein complex was radioactively labelled with CcoQ (Figure 3(b)), but in the presence of wild-type membranes, CcoQ was found associated with both the 230 kDa and the 210 kDa complexes. Importantly, none of these complexes was labelled with CcoQ when membranes of the cbb3–Cox knock-out strain GK32 were used (Figure 3(b)), suggesting that CcoQ is an integral part of these two complexes. In addition, we noted that in wild-type membranes but not in membranes of the cbb3–Cox knock-out strain, a third band (Figure 3(b) (#)) running immediately above the 230 kDa complex was also labelled with CcoQ. This pigmented band was not recognized by anti-CcoN or anti-CcoP antibodies and was not stainable by TMBZ or NADI (Figure 1), suggesting that it most likely does not correspond to a third form of the cbb3–Cox complex (i.e. distinct from the 230 kDa and 210 kDa complexes). A likely possibility is that CcoQ labels unspecifically this abundant bacteriochlorophyllcontaining complex running immediately above the 230 kDa complex, although at present we cannot exclude the possibility of a functional interaction between CcoQ and this protein complex. It is noteworthy that in the absence of cbb3–Cox, the concentrations of bacteriochlorophyll-containing proteins are decreased (Figures 1(a) and 3(b)), which could explain why this band is not labelled by CcoQ in the cbb3–Cox knock-out strain. In any event, by combining a proteomics approach with in vitro labelling techniques, we were able to demonstrate that all four subunits of the cbb3–Cox

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assemble into an enzymatically active 230 kDa membrane complex. The 230 kDa cbb3–Cox complex assembles independently of its respiratory chain partners SDS-PAGE/mass spectrometry analyses (Figure 3(a)) indicated that the NADI-stained 230 kDa complex also contained the Rieske FeS protein subunit of the cyt bc1 complex and cyt cy, which is an electron carrier to the cbb3–Cox. Whether these proteins form a complex together with cbb3–Cox was analyzed by using membranes isolated from a cyt bc1 knock-out, a cyt c2 cyt cy double knock-out, and a cyt bc1 cyt c2 double knock-out strain. In all mutant membranes, the NADI-stainable 230 kDa complex was present (Figure 4(a)), indicating that neither the absence of the cyt bc1 complex, nor that of the cyts c2 and cy affected the presence of the cbb3–Cox complex in R. capsulatus membranes. Although the resolution of BN-PAGE does not easily reveal small variations in size, it appears unlikely that either the approximately 100 kDa monomeric, or 200 kDa dimeric, cyt bc1 complex36 is a part of the 230 kDa NADI stainable cbb3–Cox complex. Interestingly, immunoblot analyses using antibodies directed against the cyt c1 and Rieske FeS protein subunits of the cyt bc1 complex showed the existence of a cyt bc1 complex at about 230 kDa, which was not present in membranes of a cyt bc 1 knock-out strain (Figure 4(b)). The 230 kDa cyt bc1 complex was present even in a cbb3–Cox knock-out strain (data not shown), indicating that the 230 kDa region of BN-PAGE contains both the cbb3–Cox and the cyt bc1 complex, which apparently assemble as separate entities independently of each other. Antibodies

Figure 4. The 230 kDa cbb3–Cox complex assembles independently of the cyt bc1 complex and the cyts cy and c2. (a) ICMs derived from wild-type or the mutant strains MT-RBC1 (Dbc1), FJ2, (Dcyt c2 cyt cy) and MT-GS18 (Dbc1 Dcyt c2) were solubilized as described for Figure 1, separated on BN-PAGE and subsequently stained with NADI to reveal active cbb3–Cox. (b) Western blot analyses of wild-type and MT-RBC1 membranes using antibodies against the cbb3–Cox subunits CcoP and CcoN, and antibodies directed against the cyt bc1 complex subunits Rieske-FeS protein and c1.

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directed against cyt c1 recognized, in addition to the 230 kDa cyt bc1 complex, a band running below the 69 kDa marker band. Thus, a monomeric form of cyt c1 is present among the TMBZ-stainable low molecular mass bands, which are detected even in the absence of cbb3–Cox (Figure 1(b)). Furthermore, as both the cyt bc1 complex and the cbb 3–Cox interact with the membrane-bound electron carrier cyt cy,37 its detection in the second dimension SDS-PAGE suggested that it might be closely associated with either or both of these complexes. This was probed by separating the membrane-bound cyts present in the 230 kDa NADI-stainable band. First, we analyzed the cyt content in Rhodobacter membranes separated on SDS-Tris/Tricine polyacrylamide gels.38 TMBZstaining of wild-type membranes revealed the presence of four membrane-bound c-type cyts that correspond to the CcoP and CcoO subunits of the cbb3–Cox, the cyt c1 subunit of the cyt bc1 complex and cyt cy, along with that of the soluble cyts c2 and c 0 , which are trapped inside the membrane vesicles (Figure 5, lane 1). As expected, these bands were absent from the appropriate knock-out strains (Figure 5, lanes 2 and 3). In the cyt cy/c2 knockout strain, a strong TMBZ-stainable band was visible above CcoP, which corresponds to the soluble cyt c551 (S. Onder & F.D., unpublished results). Next, membranes of the cyt c2/cy doubleknock-out and the cyt bc1/c2 double knock-out strains were subjected to BN-PAGE and stained with the NADI reagent. The NADI-stainable 230 kDa band was cut out and separated on SDS-Tris/Tricine polyacrylamide gels followed by

995 staining with TMBZ. This analysis revealed the presence of CcoO, CcoP and cyt c1 in the cyt cy c2 double knock-out strain (Figure 5, lane 4), although CcoP and cyt c1 were not well separated. Importantly, even in membranes from the cyt bc1/c2 knock-out strain, cyt cy was still present in the 230 kDa complex (Figure 5, lane 5), which was confirmed by mass spectrometry (data not shown). These data indicate that cyt cy can be found together with cbb3–Cox even in the absence of the cyt bc1 complex. However, cyt cy appears to be present in sub-stochiometric amounts in the 230 kDa complex, as suggested by its weaker TMBZ-staining in comparison to the other mono-heme cyt CcoO. In addition, no apparent differences in molecular mass can be seen between the 230 kDa NADI-stainable complexes detected in a wild-type or in a cyt cy/c2 double knock-out strain (Figure 4). This suggests that the resolution of the BN-PAGE is insufficient to discriminate between cbb 3–Cox complexes associated with cyt cy and those lacking it. CcoH and CcoS are specific assembly factors for the 230 kDa cbb3–Cox complex In all organisms characterized so far, the ccoNOQP operon is followed immediately by the ccoGHIS gene cluster, which encodes four integral membrane proteins required for the production of an active cbb3–Cox.16,39,40 Although the exact functions of these gene products are unknown, mutagenesis studies have indicated that the individual gene products are involved in distinct steps during the assembly of an active cbb3–Cox.16

Figure 5. cbb3–Cox is found together with the membraneanchored cyt c y. Membranebound cyts in wild-type and mutant ICMs were revealed by staining with TMBZ. In lanes 1–3, ICMs were loaded directly onto an SDS-Tris/Tricine polyacrylamide gel and subsequently stained with TMBZ (1D SDS-PAGE), whereas in lanes 4 and 5, ICMs were first separated on BN-PAGE and stained with NADI. Subsequently, the blue 230 kDa complex was cut out and separated on a second dimension SDSTris/Tricine polyacrylamide gel, which was then stained with TMBZ (2D SDS-PAGE) to reveal the membrane-bound cyts present in this complex. CcoP and CcoO correspond to the cyt subunits of the cbb3–Cox, whereas cy and c1 correspond to the electron carrier cyt cy and the cyt c1 subunit of the cyt bc1 complex, respectively. C551, C2 and C 0 refer to the soluble cyts c that are trapped inside the ICMs.

996 In R. capsulatus, deletion of CcoG appears to have only minor effects on the activity of the cbb3–Cox, whereas in the absence of either CcoH or CcoI, the presence of an active cbb3–Cox is reduced drastically.16 A remarkable phenotype is observed upon deletion of CcoS. Although CcoN, CcoP and CcoO are fully detectable in Rhodobacter membranes using either appropriate antibodies or TMBZ staining,16 the cbb3–Cox is completely inactive due to the absence of a functional heme b3–CuB binuclear centre in CcoN. 16 While homology searches suggested that CcoI is a P-type ATPase, presumably involved in Cu trafficking,41 no function has been attributed so far to CcoH or CcoS by such in silico analyses. We therefore examined the function of the ccoGHIS gene products in the maturation and assembly of the cbb3–Cox by BN-PAGE of membranes derived from various ccoGHIS mutants. In agreement with published data,16 no NADI-stainable band was detected in membranes of mutants lacking either the entire ccoGHIS gene cluster, or only the single ccoI, or ccoH or ccoS products (data not shown). Interestingly, immunoblot analyses using anti-CcoP antibodies revealed significant differences in the amount of the monomeric CcoP present in these mutant membranes (Figure 6). As in wild-type membranes, both the 230 kDa complex and the monomeric CcoP were present in membranes of a ccoS knock-out mutant, except that the 230 kDa in the latter strain was not NADI-stainable. On the other hand, in membranes derived from the ccoGHIS knock-out strain, anti-CcoP antibodies barely detected any 230 kDa cbb3–Cox complex or monomeric CcoP. A somewhat similar situation was observed in the non-polar ccoH mutant, indicating that this small membrane protein is important for the presence of both the 230 kDa complex and the monomeric CcoP. Moreover, in membranes of the ccoH and ccoS deletion strains, a weak band of about 160 kDa was recognized by

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anti-CcoP antibodies (Figure 6). Finally, a very different situation was observed in the non-polar ccoI mutant. In agreement with its NADI-negative phenotype, no 230 kDa complex was detected using anti-CcoP antibodies, but large amounts of monomeric CcoP were found in these membranes. These data indicate that the lack of CcoH exhibits the strongest effect on cbb3–Cox, as DccoH mutants lack both the active 230 kDa cbb3–Cox complex and the monomeric CcoP. The lack of CcoI only impairs formation of the active 230 kDa cbb3–Cox complex, with no major effect on the stability of monomeric CcoP. Finally, only the activity of the 230 kDa cbb3–Cox complex, but not its assembly or the stability of monomeric CcoP, is impaired in the absence of CcoS. CcoH and CcoS are associated directly with the cbb3–Cox during its assembly The roles of CcoH and CcoS during the assembly of cbb3–Cox were analyzed further by monitoring the ability of in vitro synthesized CcoH and CcoS to associate with either the 230 kDa or the 210 kDa complexes. As an internal control, we first in vitro synthesized CcoO in the presence of wild-type membranes. After the isolation of CcoO-containing membranes by centrifugation, they were solubilized by DDM and separated on BN-PAGE. The data indicate that both the active 230 kDa complex and the inactive 210 kDa complex were labelled by in vitro synthesized CcoO (Figure 7). It should be noted that, although in vitro synthesized CcoO associates specifically with both complexes, it is unknown whether it contains its heme group. The in vitro system used in this study contains all factors required for targeting and insertion of membrane proteins and has been used to study the translocation of cyts.42,43 However, it has not been determined whether this system contains all the

Figure 6. Roles of the ccoGHIS products for the assembly and stability of the 230 kDa cbb3–Cox complex. Wild-type and mutant ICMs were solubilized and separated on BN-PAGE as described for Figure 1, and antibodies against CcoP (anti-CcoP) were used to detect the 230 kDa complex and the monomeric form of CcoP. The 230 kDa cbb3–Cox complex and the monomeric CcoP are indicated; in addition, a band at 160 kDa (#) is weakly recognized by anti-CcoP antibodies.

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Figure 7. The putative assembly proteins CcoS and CcoH bind to the 210 kDa cbb3–Cox sub-complex. CcoO, CcoS and CcoH were synthesized in vitro as described for Figure 3 in the presence of wildtype or M7G (ccoP) membranes. The amount of radioactively labelled CcoS and CcoH used in these experiments is indicated at the bottom of the gel. Radioactively labelled bands (*) were visualized using a phosphorimager.

components required for heme insertion into an apo-cyt. Strikingly, when CcoS or CcoH were synthesized in vitro in the presence of wild-type membranes, we found that both proteins associated only with the 210 kDa sub-complex but not the 230 kDa active complex (Figure 7). This could suggest that CcoS and CcoH interact with the inactive cbb3–Cox before CcoP is incorporated to yield a functional enzyme complex. Consistent with this observation, a much stronger labelling of the 210 kDa sub-complex by CcoS and CcoH was observed if both proteins were synthesized in vitro in the presence of M7G membranes (Figure 7), which lack CcoP.5,14 It should be emphasized that the same amount of radioactively labelled proteins were used with wild-type and M7G membranes. The observation that only the 210 kDa complex but not the 230 kDa active complex was labelled with CcoH and CcoS is consistent with the observation that they were not detected by mass spectrometry analyses of the 230 kDa complex (Figure 3(a)). In addition to the 210 kDa complex, a band at about 160 kDa was labelled weakly with CcoH in wild-type membranes. Whether this band is identical with the 160 kDa band observed in ccoH and ccoS knock-out mutants (Figure 6) remains to be determined. In any event, the data obtained using radiolabelled CcoH and CcoS clearly indicate for the first time that both proteins interact specifically with an inactive cyt cbb3–Cox sub-complex and strongly support the notion that CcoH and CcoS are indeed bona fide assembly factors for the biogenesis of the cyt cbb3–Cox.

Discussion The cbb3–Cox complex in native R. capsulatus membranes Multimeric membrane protein complexes are involved in many important regulatory and catalytic processes, and their assembly is an inherently complex process, involving accessory proteins required for membrane targeting and insertion, cofactor incorporation and post-translational maturation.44 In this work, we have analyzed the assembly of cbb3–Cox in R. capsulatus, a wellcharacterized model organism in which cbb3–Cox is the only cyt c oxidase.14 In comparison to the better studied aa3-type cyt c oxidases,2 very little is known about the assembly process of cbb3–Cox, despite its general role in cellular energy transduction and its suspected importance for the pathogenicity of microaerophilic pathogens like H. pylori and N. menigitidis.3 First, we have examined the steady-state presence of cbb3–Cox in native membranes using BN-PAGE. In-gel activity measurements employing the cyt c oxidase, specific NADI reaction revealed the presence of a 230 kDa active complex, which by Western blotting, mass spectrometry, TMBZ staining, and in vitro labelling approaches was shown to contain CcoN, CcoO, CcoQ and CcoP (Figures 1 and 3). Whether the 230 kDa complex represents a monomeric or dimeric state of cbb3–Cox in membranes is unknown. The electrophoretic mobilities of CcoN, CcoO, CcoQ and CcoP in SDS-PAGE are in

998 agreement with their predicted molecular masses of 58 kDa, 27 kDa, 6.5 kDa and 32 kDa, respectively, suggesting a molecular mass of about 120 kDa for the monomeric cbb 3–Cox. Thus, the 230 kDa complex could indicate a dimeric state of the cbb3–Cox in R. capsulatus. However, molecular mass estimations by BN-PAGE are apparently highly influenced by the amount of Coomassie brilliant blue retained by membrane protein complexes. Examination of known monomeric and homo-dimeric membrane proteins led to an empirical conversion formula, MBNPZMAA!1.8, with MBNP corresponding to the molecular mass on BN-PAGE and MAA corresponding to that predicted from the amino acid sequences.45 Although it is not clear if this conversion is generally applicable to all membrane protein complexes, if it is valid for the cbb3–Cox complex, then a 230 kDa complex would rather reflect a monomeric state of cbb3–Cox in R. capsulatus membranes. In Pseudomonas stutzeri, analytical ultracentrifugation experiments suggested the presence of a monomeric cbb3–Cox complex in membranes,46 and known bacterial three-dimensional structures of cyt c oxidases,47 unlike the cyt bc1 complexes,48 are also monomeric. In energy transducing membranes of both prokaryotic and eukaryotic cells, respiratory complexes are often organized in supercomplexes or respirasomes,26 which presumably increase their functional efficiencies as well as their steady-state stability. Under the different solubilization conditions with different detergents used here, no change in the presence of the 230 kDa active cbb3–Cox complex was seen in R. capsulatus membranes derived from cells grown under different conditions (Figure 2). In addition to enhancing the catalytic activity, the structural stabilization of labile membrane protein complexes is considered to be a major reason for supercomplex formation.26 Accordingly, highly stable membrane protein complexes, like complex I from the yeast Yarrowia lipolytica,49 often do not organize themselves into larger macromolecular structures. Indeed, a stable and highly active cbb3–Cox complex from R. capsulatus membranes has been purified to homogeneity.5,14 It is noteworthy that the membrane-anchored cyt cy and the Rieske-FeS protein of the cyt bc1 complex were found reproducibly when the composition of the 230 kDa complex was analyzed by mass spectrometry. The cyt bc1 complex appears to form a separate complex running also at about 230 kDa in BN-PAGE, which is detectable immunologically in membranes containing or lacking the cbb3–Cox complex. As cyt cy acts as an electron acceptor from the cyt bc1 complex and a donor to the cbb3–Cox complex in Rhodobacter sp.,37,50 it is expected that it interacts physically with its physiological partners. However, the presence of the 230 kDa, NADI-stainable active cbb 3–Cox complex does not seem to depend on either of these partners, because its presence is not impaired in mutants lacking either the cyts cy and c2 or the cyt bc1 complex (Figures 4 and 5). Membrane-attached

cyt cbb3 Oxidase Biogenesis

electron carrier cyts, such as the Paracoccus denitrificans cyt c552,51 have initially been considered to act as linker proteins in respiratory supercomplexes. However, more recent studies indicate that cyt c552 is not essential for supercomplex formation in P. denitrificans.52 In any case, the formation of the 230 kDa cbb3–Cox complex does not seem to depend strictly on the presence or absence of other monomeric or multimeric respiratory chain proteins. A multi-step assembly pathway for the R. capsulatus cbb3–Cox complex A major challenge in understanding the formation of multi-subunit complexes is the spatial and temporal dissection of their assembly pathways. In this regard, one important finding of our study is the detection of a 210 kDa NADI-negative complex, containing CcoN, CcoO and CcoQ, but lacking CcoP and a pool of monomeric CcoP in Rhodobacter membranes. In principle, this 210 kDa sub-complex could be an artefact generated by the dissociation of CcoP from intact cbb3–Cox under the solubilization conditions used here. However, we rather propose that it represents a cbb 3–Cox assembly intermediate devoid of CcoP, similar to that suggested to be present in B. japonicum ccoP mutants.10 This is deduced from our observation that in membranes of the ccoP mutant M7G, which has been shown to contain CcoN and CcoO but not CcoP,14,19 the 230 kDa complex is absent, while the concentration of the 210 kDa cbb3–Cox subcomplex is increased (Figure 1(c)). We additionally employed in vitro labelling techniques to demonstrate that CcoS and CcoH, which are required for the formation of an active cbb3–Cox complex, associate specifically with the 210 kDa sub-complex in wild-type membranes and even more in M7G membranes lacking CcoP (Figure 7). These findings are consistent with the 210 kDa sub-complex being an assembly intermediate, and they indicate for the first time that the postulated assembly factors CcoH and CcoS16 indeed physically associate with the cbb3–Cox complex during its biogenesis. The Rhodobacter in vitro system used in this study has been shown to contain all the accessory proteins required for membrane transport,42,43 and similar systems have been used to monitor the interaction between membrane proteins in Escherichia coli.34,35 Thus, the combination of BN-Page with in vitro labelling techniques provides a powerful tool to reveal the association of a putative assembly factor with its substrates. In the absence of CcoH, neither the complexes nor the monomeric CcoP were detectable, pointing out that CcoH might be required for their stability. Considering that we did not observe binding of in vitro labelled CcoH to the 230 kDa complex, it is tempting to speculate that recruitment of CcoP into the 210 kDa sub-complex to form the active 230 kDa cbb3–Cox complex concomitantly induces

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dissociation of CcoH. Our observation that in a mutant like M7G, which lacks CcoP, a much stronger labelling by CcoH is observed as compared to wild-type membranes, supports this idea (Figure 7). Alternatively, because the concentration of the 210 kDa complex is increased in M7G (Figure 1(c)), the enhanced labelling could just reflect an increased number of binding sites for CcoH. Production of an active cbb3–Cox requires that both the b-type and c-type hemes and the Cu atom have to be inserted into properly localized apoproteins via specific post-translational maturation processes. Available data indicate that incorporation of the c-type hemes into CcoO and CcoP is mediated by the type I cyt c biogenesis pathway.53,54 Our data suggest that maturation of CcoO and CcoP most likely precedes the formation of the 210 kDa sub-complex. This is deduced from the observation that both the 210 kDa sub-complex and the monomeric CcoP exhibit peroxidase activity, indicative of a covalently attached heme group (Figure 1(b)). On the other hand, maturation of CcoN is apparently more complex, as it involves incorporation of both a bis-His axially coordinated low-spin b-type heme and an all-His coordinated high-spin heme b3–CuB binuclear centre. Currently, how the b-type hemes are incorporated into their cognate proteins, and how this process is coordinated with cellular Cu trafficking, and Cu insertion into CcoN is unknown.55,56 In the case of the cbb3–Cox, Cu delivery to CcoN is predicted to involve CcoI, which shows a high level of sequence homology to Enteroccocus hirae CopA and other Cu-uptake P-type ATPases. 15,16,41 Membranes of a CcoI knock-out mutant are devoid of the 230 kDa complex, which suggests that Cu incorporation is probably a prerequisite for the formation or the stability of the 230 kDa cbb3–Cox complex. Furthermore, the presence of monomeric CcoP in CcoI knock-out membranes (Figure 6) indicates that Cu-incorporation and c-type cyt maturation are independent processes. Previous studies have demonstrated that membranes lacking CcoS contain mature CcoO and CcoP as well as CcoNapoprotein, lacking the heme b and the functional heme b3–CuB binuclear centre.14,16 These findings suggest also that the maturation of the c-type cyts and of CcoN are not tightly linked to each other. The data presented here show that in the absence of CcoS, monomeric CcoP can still associate with the 210 kDa sub-complex to yield a 230 kDa cbb3–Cox complex (Figure 6), which, however, is not active. This implies a role of CcoS in a very late step of cbb 3–Cox complex assembly, probably during cofactor insertion. We are unable to attribute unambiguously a specific role to CcoS in the insertion of the b-type hemes or Cu or both into CcoN. As in the absence of the predicted Cu-uptake ATPase CcoI, even the 230 kDa complex is not detectable, an involvement of CcoS in the insertion of the b-type hemes appears more likely. Although in vitro synthesized CcoS is found associated with

the 210 kDa sub-complex and not with the 230 kDa complex, it remains to be determined whether the CcoS-dependent step during the biogenesis of cbb3–Cox occurs prior or subsequent to the association of the 210 kDa sub-complex with CcoP. In summary, our overall data indicate that in DDM dispersed R. capsulatus membranes a 230 kDa complex corresponding to the active cbb3–Cox, and a 210 kDa inactive sub-complex along with a monomeric pool of CcoP are present. Furthermore, a multi-step biogenesis process (Figure 8) seems to assemble the independently matured CcoO and CcoP subunits (Figure 8, step 1) via direct interactions with the assembly factors CcoH and CcoS, initially into a CcoNOQ–CcoHS inactive subcomplex (Figure 8, step 2), which is then converted into the active enzyme upon association of CcoP and probably dissociation of the CcoH and CcoS (Figure 8, step 3). It should be noted that, although the concentration of the 230 kDa complex is reduced in membranes derived from anaerobicphotosythetically grown cells, the ratio between the 230 kDa CcoNOQP complex and the 210 kDa CcoNOQ–CcoHS complex is not influenced (Figure 2). This probably suggests that the expression of the ccoNOQP operon and that of the ccoGHIS cluster are coregulated. Future studies on the distinct steps depicted by the assembly process proposed here will clearly be rewarding in progressing our understanding of the assembly of multimeric cofactor containing membrane protein complexes such as the cbb3–Cox of R. capsulatus.

Materials and Methods Bacterial strains and growth conditions The strains and plasmid used are listed in Table 1. E. coli strains harbouring plasmids were grown in LB medium supplemented with appropriate antibiotics (100 mg/ml, 50 mg/ml and 12.5 mg/ml for ampicilin, kanamycin and tetracycline, respectively). R. capsulatus strains were grown in Sistrom’s minimal medium A (Med A)57 or in enriched medium MPYE58 at 35 8C in liquid cultures in the dark and were shaken at 100 rpm and filled up to half the container volume (semiaerobic growth) or 200 rpm and filled up to one-tenth the container volume (aerobic growth). For photosynthetic growth conditions 1 l Duran bottles were filled completely with MPYE medium and the culture was stirred on a magnetic stirrer, under continuous illumination using OSRAM light bulbs (230 V, 60 W). Molecular genetic techniques Standard molecular genetic techniques were performed as described.59 For in vitro synthesis of CcoO, CcoS, CcoH and CcoQ, the corresponding genes were PCR amplified and cloned into appropriate pET expression vectors (Novagen, Bad Schwalbach, Germany). In the case of CcoO, the plasmid pCW24 was used as a template with the primers CcoO-NdeI-F 5 0 -GAT CCA TAT GTC AAT CAT GGA CAA ACA CC-3 0 and CcoO-HindIII-R 5 0 -ACG TAA GCT TGC GAC TTG CCA CGG GTT GGA-3 0 ,

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cyt cbb3 Oxidase Biogenesis

Figure 8. A multistep assembly model for the biogenesis of the cyt cbb3–Cox in R. capsulatus. Step 1: the available data suggest that after translation and membrane integration of the structural subunits CcoP, CcoO, CcoQ and CcoN of the cbb3–Cox, the c-type hemes are inserted via the type I cyt c biogenesis pathway into the di-heme c-type cyt CcoP and the mono-heme c-type cyt CcoO. Step 2: dodecyl maltoside-dispersed R. capsulatus membranes separated by BN-PAGE show the presence of both an inactive 210 kDa sub-complex containing the CcoN, CcoQ and CcoO subunits of the cyt cbb3–Cox associated with the assembly factors CcoH and CcoS, and a monomeric form of CcoP, probably also associated with CcoH, which is proposed to stabilize both the monomeric CcoP and the sub-complex CcoNQO. CcoS is required for the acquisition of the b-type hemes and formation of a functional binuclear centre,16 but in its absence an inactive cyt cbb3–Cox can still be assembled. Step 3: finally, the recruitment of CcoP is proposed to convert the 210 kDa sub-complex into the active cbb3–Cox. During this step, the assembly factors CcoH and CcoS probably dissociate. It should be emphasized that, although the maturation of the c-type cyts occurs early and independently of the maturation of CcoN, it is not known when the b-type hemes are inserted into CcoN, or how this event is coordinated with the CcoI-dependent Cu acquisition. the PRC product was digested with NdeI and HindIII restriction enzymes and cloned into similarly digested pET22b, yielding a CcoO-His6-tagged construct. CcoH was amplified using genomic DNA isolated from R. capsulatus 37b4 with CcoH-NdeI-F 5 0 -GTA CCA TAT GGC GAA ACC GCT GAC C-3 0 and CcoH-BamHI-R 5 0 -GAT CGG ATC CTC AGC TTT CGA CGA AGA AAT CG-3 0 , and cloned into NdeI/BamHI-digested pET22b. Similarly, CcoS was amplified using the primers ccoSNdeI-F 5 0 -CAT GCA TAT GTC GGT CCT GAC CTA TCT G-3 0 and ccoS-EcoRI-R 5 0 -GAT CGA ATT CTC AGC TTT TCG GGC GGT CGT C-3 0 using the plasmid CcoST7pET17b (CcoS cloned in pET17b via HindIII/EcoRI yielding a T7-tag–CcoS construct; unpublished results) as a template, and cloned into pET17b. CcoQ was amplified from the plasmid pCW25 with the primers CcoQ-NdeI-F 5 0 -GAT CCA TAT GGA CTA TCA TAT CTT GCG TG-3 0 and CcoQ-XhoI-R 5 0 -CTA GCT CGA GGC CCC GGC CAT GAT C-3 0 and cloned into pET22b via

NdeI/XhoI. To obtain an R. capsulatus CcoGHIS-NOQP over-expression strain, the plasmid pCW2516 was conjugated into the R. capsulatus wild-type strain MT1131 as described.60 Preparation of cell extracts and intracytoplasmic membranes High-speed supernatants (S-135 extract) of cell homogenates from R. capsulatus strain 37b4 and intracytoplasmic membranes (ICMs) from the strains MT1131, MT1131-pCW25, MT-RBC1, FJ2, GK32, M7G, CW1, CW2, CW6 and BK6 (Table 1) were prepared essentially as described,42 but with the following modifications. Cells were grown semi-aerobically at 35 8C. Membrane-free S-135 extracts were obtained by centrifuging 1 ml of S-30 supernatants in a Beckmann TLA-100 ultracentrifuge at 90,000 rpm for 9 min using the TLA-100.2 rotor, and only

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cyt cbb3 Oxidase Biogenesis

Table 1. Strains and plasmids used in this study Strain E. coli DH5a R. capsulatus MT1131 37b4 MT1131/pCW25 M7G CW1 CW2 CW6 BK6 GK32 FJ2 MT-RBC1 MT-GS18

Relevant phenotypes/properties

Genotype supE44 DlacU169(F80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 crtD121 RifR

ccoP269 DccoGHISTspe DccoITspe DccoHTkan DccoSTkan DccoNOTkan DcycATkan DcycYTspe D(petABCTspe) DcycATkan D(petBCTspe)

Plasmids CcoO-pET22b

ccoO cloned into pET22b via the NdeI/HindIII sites

CcoST7-pET17b

ccoS cloned into pET17b via the HindIII/EcoRI sites

CcoS-pET17b CcoH-pET22b

ccoS cloned into pET17b via the NdeI/EcoRI sites ccoH cloned into pET22b via the NdeI/BamHI sites

CcoQ-pET22b

ccoQ cloned into pET22b via the NdeI/XhoI sites

pCW25

Source or reference 59

Wild-type (NADIC) Wild-type (NADIC) cbb3–Cox overexpression NADIK NADIK NADIK NADIK NADIK NADIK K cyt cK 2 , cyt cy cyt bcK 1 K cyt cK 2 , cyt bc1

67 68 This study

In vitro expression of CcoO In vitro expression of CcoS As above In vitro expression of CcoH In vitro expression of CcoQ

This study

19 16 16 16 16 14 50 69 70

This study This study This study This study

ccoNOQP–ccoGHIS in pRK415

the top 750 ml of the supernatant was used as S-135 extract for cell-free protein synthesis. For pelleting ICMs, the S-30 supernatant was centrifuged for 2 h at 140,000g in a 50.2TI rotor in a Sorvall Discovery 90 ultracentrifuge. In vitro protein synthesis and integration into membrane vesicles, preparation of membranes for blue native-PAGE The proteins CcoS, CcoH, CcoQ and CcoO were expressed from the plasmids pET17b–CcoS, pET22b– CcoH, pET22b–CcoQ and pET19b–CcoO, respectively, under the control of the phage T7 RNA polymerase promoter. Cell-free protein synthesis with R. capsulatus S-135 extracts was carried out for 30 min at 35 8C as described.43 For co-translational integration of in vitro synthesized proteins into membranes, ICMs were added after 5 min of synthesis, and the reaction mix was incubated for 25 min at 35 8C. For blue native (BN)PAGE analyses, 100 ml of reaction mix containing 200 mg of ICMs was used per lane. Immediately after synthesis and integration, aggregates were removed from the reaction mix by centrifugation for 10 min at 14,000 rpm in a tabletop centrifuge at 4 8C. Membranes were then collected by ultracentrifugation at 100,000g for 30 min and resuspended in 25 ml of lysis buffer (50 mM NaCl, 5 mM 6-aminohexanoic acid, 50 mM imidazole–HCl, pH 7.0), solubilized with n-dodecylmaltoside (Roche Diagnostics, Mannheim, Germany) at a 1:1 ICM protein:detergent ratio from a 10% (w/v) dodecylmaltoside stock solution in lysis buffer, and incubated for 10 min at 25 8C. After ultracentrifugation at 100,000g for 30 min in a TLA100.2 rotor, the supernatant was collected and supplemented with 6 ml of loading buffer (5% (w/v) Coomassie brilliant blue in 500 mM 6-aminohexanoic acid) before loading onto the gel for BN-PAGE, as described.20

16

In-gel heme staining, in-gel NADI staining and immunoblotting BN-PAGE or SDS-PAGE gels were treated with 3,3 0 ,5,5 0 tetramethylbenzidine (TMBZ) to reveal the c-type cyts as described.21 In-gel activity staining for the cyt cbb3–Cox was performed by incubating BN-PAGE gels with a 1:1 (v/v) mixture of 35 mM a-naphthol dissolved in ethanol and 30 mM N,N-dimethyl-p-phenylenediamine in water,61 which produced a blue color in the presence of active enzyme. For immunoblot analyses, proteins were electroblotted onto Immobilon-P transfer membranes, and polyclonal antibodies against CcoP, CcoN, RieskeFeS protein and cyt c114 were used with horseradish peroxidase-conjugated goat anti-rabbit antibodies from Caltag Laboratories (Burlingham, CA, USA) as secondary antibodies. 2D-PAGE and liquid chromatography tandem mass spectrometry (nLC-MS/MS) analyses For determination of the composition of the 230 kDa complex, BN-PAGE was used in the first dimension stained with NADI reagent. The blue band corresponding to the active cbb3–Cox was cut out of the gel, and incubated in equilibration buffer (50 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 6 M urea, 30% (v/v) glycerol) for 30 min at room temperature. These gel fragments were analyzed using either SDS/10–20% polyacrylamide gradient gels62 for nano-LC-MS/MS analyses, or SDS, Tris/Tricine 16.5% polyacrylamide gels38 for TMBZ staining. For nano-LC-MS/MS analysis, gels were stained with colloidal Coomassie brilliant blue (34% (v/v) methanol, 2% (v/v) phosphoric acid, 17% (w/v) ammonium sulfate, 0.066% (w/v) Coomassie brilliant blue G250) for at least 24 h, and the stained bands were

1002 cut out, subjected to in-gel digestion with trypsin and subsequent lyophilization was as described.63 For nano-LC-MS/MS analyses, lyophilized peptide extracts were dissolved in 0.5% (v/v) trifluoroacetic acid (TFA) and loaded onto a nano-LC column (75 mm fused silica capillary with 8 mm tip opening (from New Objective, Cambridge, MA, USA) packed with C18 beads, 3 mm size (Dr A. Maisch, Ammerbuch, Germany)) using a high-pressure loading device for column preparation. Peptides were then eluted with an aqueous/organic gradient (solution A, 0.5% formic acid (SdS GmbH, Rastatt, Germany); solution B, 0.5% formic acid, 80% (v/v) acetonitrile (Sigma, Taufkirchen, Germany) in water (Merck, Bad Schwalbach, Germany), 65 min, 3–100% solution B) at a flow rate of approximately 200 nl/min using a nanoflow-HPLC system (Ultimate & Famos autosampler, Dionex, USA), and electrosprayed into a QStar Pulsar i (Applera, Darmstadt, Germany) tandem quadrupole time-of-flight (TOF) mass spectrometer. The system used corresponds to the previously described instrumentation and protocols.63–65 Each measurement cycle consisted of one MS spectrum followed by up to four MS–MS spectra (IDA mode). MS–MS data were extracted using the appropriate software modules provided by the manufacturer (BioAnalyst, Applera, Darmstadt, Germany) and searched by an in-house MASCOT-server (MatrixScience, UK) against appropriate databases.66

Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft/SFB388 (to H.G.K.), and NIH GM 38237 and DOE ER 9120053 (to F.D.). We thank Dr Martin Biniossek, Institut fu¨ r Molekulare Medizin und Zellforschung, Universita¨t Freiburg, for his help with nLC-MS/ MS analyses and Dr Matthias Mu¨ller for discussion.

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Edited by M. Moody (Received 11 August 2005; received in revised form 2 November 2005; accepted 12 November 2005) Available online 1 December 2005