the putative assembly factor ccoh is stably associated with the

JOURNAL OF BACTERIOLOGY, Dec. 2010, p. 6378–6389 Vol. 192, No. 240021-9193/10/$12.00 doi:10.1128/JB.00988-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Putative Assembly Factor CcoH Is Stably Associated with thecbb3-Type Cytochrome Oxidase�

Grzegorz Pawlik,1 Carmen Kulajta,1 Ilie Sachelaru,1 Sebastian Schroder,1 Barbara Waidner,2Petra Hellwig,3 Fevzi Daldal,4 and Hans-Georg Koch1*

Institut fur Biochemie und Molekularbiologie, Albert-Ludwigs-Universitat Freiburg, D-79104 Freiburg, Germany1; Institut furMedizinische Mikrobiologie und Hygiene, D-79104 Freiburg, Germany2; Laboratoire de Spectroscopie Vibrationelle et

Electrochimie des Biomolecules, Institut de Chimie, UMR 7177, Universite de Strasbourg, 67000 Strasbourg, France3;and 103B Carolyn Lynch Laboratory, Department of Biology, University of

Pennsylvania, Philadelphia, Pennsylvania 191044

Received 20 August 2010/Accepted 4 October 2010

Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexesbecause the need for cofactor incorporation adds an additional level of complexity to their assembly. cbb3-typecytochrome c oxidases (cbb3-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cyto-chrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb3-Cox stability. Biogenesisof cbb3-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb3-Cox. Mostbacteria expressing cbb3-Cox also contain the ccoGHIS genes, which encode putative cbb3-Cox assembly factors.Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb3-Cox assembly andshowed that CcoH is a single spanning-membrane protein with an N-terminus-out–C-terminus-in (Nout-Cin)topology. In its absence, neither the fully assembled cbb3-Cox nor the CcoQP or CcoNO subcomplex wasdetectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembranedomain to the CcoP subunit of cbb3-Cox. A second hydrophobic stretch, which is located at the C terminus ofCcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the activecbb3-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQsubcomplexes into the active cbb3-Cox. Surprisingly, CcoH does not seem to interact only transiently with thecbb3-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behavesmore like a bona fide subunit of the cbb3-Cox than an assembly factor per se.

The heme-copper oxidases comprise a family of functionallyrelated enzyme complexes, which terminate respiratory chainsin both prokaryotic and eukaryotic cells (38). In addition totheir physiological importance for all oxygen-dependent cells,these enzyme complexes have been used as model complexesfor studying the assembly of cofactor-containing multisubunitprotein complexes (18). All members of the heme-copper ox-idase family are characterized by the presence of a membraneintegral subunit I, which binds a low-spin heme and a charac-teristic high-spin heme-CuB binuclear center. There is, how-ever, a remarkable diversity in respect to subunit composition,electron donor, and oxygen affinity. Due to this diversity, mem-bers of the heme-copper oxidase family have been classifiedinto three major subfamilies (33). Subfamily A heme-copperoxidases are the most abundant and are present in mitochon-dria and in many bacterial species. They use two proton trans-fer pathways (21), while subfamily B heme-copper oxidasesappear to use only one proton channel (5, 17). The bacterialcbb3-type cytochrome (cyt) c oxidases (cbb3-Cox) constitutethe prototype for the C subfamily and are the second-most-abundant oxidases (8, 17). They are encoded by the ccoNOQP

operon (fixNOQP in rhizobia) and have so far been found onlyin eubacterial species. ccoN encodes the catalytic subunit I,which harbors the conserved histidines required for ligating theheme b and the heme b3/CuB binuclear centers (11, 12, 37).Different from the aa3-type cytochrome c oxidases (aa3-Cox),cbb3-Cox lack a second Cu center (CuA) in subunit II andinstead contain two membrane-bound c-type cytochrome sub-units, the monoheme subunit CcoO and the diheme subunitCcoP. As in many bacterial cytochrome oxidases, a fourthsubunit (called CcoQ) is present in most cbb3-Cox. This sub-unit does not seem to contain any cofactor and is not essentialfor function. Nevertheless, in the absence of CcoQ the amountof the functional cbb3-Cox complex is reduced in Rhodobactercapsulatus (34).

The first X-ray structure of a cbb3-Cox from Pseudomonasstutzeri has recently been solved at 3.2 Å (3). The architectureof the catalytic CcoN subunit displays a remarkable similarityto the known structures of cytochrome oxidases of the A and Bfamilies (21, 36, 47, 54) despite the rather low level of sequenceconservation. Differences are detectable in the heme b and hemeb/CuB environments, which probably contribute to the high levelof oxygen affinity of the cbb3-Cox. Based on the orientation of theCcoP and CcoO subunits relative to the CcoN subunit, electronsare most likely transferred from a donor cytochrome to thepartly solvent exposed outer heme group of CcoP and subse-quently via the inner heme group of CcoP to CcoO. FromCcoO, the electron is then shuttled via heme b to the hemeb3/CuB binuclear center, where oxygen reduction takes place.

* Corresponding author. Mailing address: Institut fur Biochemieund Molekularbiologie, Albert-Ludwigs-Universitat Freiburg, ZBMZ,Stefan-Meier-Str. 17, D-79104 Freiburg, Germany. Phone: 0049-761-203-5250. Fax: 0049-761-203-5289. E-mail: [email protected].

� Published ahead of print on 15 October 2010.

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The structural diversity of the heme-copper oxidase family isalso reflected by the requirement for different assembly fac-tors. For aa3-Cox, more than 30 proteins are required for theirassembly in eukaryotes (4, 23, 46). Of those, only a few are alsorequired for the assembly of the simpler bacterial aa3-Cox.Examples are Surf1, which is required for heme a insertion intosubunit I (2, 45), and CoxG, a Cox11 homologue required forCu delivery to the CuB site (13, 19). Neither Surf1 nor CoxGappears to be required for cbb3-Cox assembly (1, 2).There arealso indications, however, for a dual function of assembly fac-tors in both aa3-Cox and cbb3-Cox assembly. SenC, a homo-logue of the mitochondrial ScoI, is involved in CuA centerassembly in aa3-type cytochrome oxidases (20) but is also re-quired for cbb3-Cox activity in R. capsulatus (49) and Pseudo-monas aeruginosa (9) but not in Bradyrhizobium japonicum (1).Since cbb3-Cox lack a CuA center, the exact function of SenC/ScoI for cbb3-Cox activity remains to be determined.

The active R. capsulatus cbb3-Cox migrates on blue native(BN) gels as a 230-kDa complex that is assembled via a 210-kDa CcoNO subcomplex to which an approximately 50-kDaCcoQP complex is recruited (26, 34). This multistep assemblydepends on the proteins encoded by the ccoGHIS gene cluster(25, 26, 35), which is located immediately downstream of theccoNOQP operon in many bacteria (8, 22, 24). The ccoG geneencodes a putative polyferrodoxin and is the most conserved ofthe four genes (8). Nevertheless, its deletion in R. capsulatusdoes not abolish the cbb3-Cox activity (25). A different pheno-type is observed with a ccoI deletion; here no cbb3-Cox activityis detectable, and the cbb3-Cox subunits fail to form a stablecomplex in the membrane (26). The ccoI gene product showsall features of a P-type ATPase, and it has been suggested thatit is involved in copper delivery to cbb3-Cox (25, 35), althoughthis suggestion still awaits experimental verification. WhetherCcoI is a dedicated Cu delivery system for cbb3-Cox is alsounknown; it has recently been suggested that the CcoI homo-logue in Rubrivivax gelatinosus might also be involved in pro-viding Cu to other Cu-containing enzymes like NosZ (14). TheccoS gene encodes a small, putative membrane protein, whichappears to be specifically involved in cofactor insertion. In theabsence of CcoS, cbb3-Cox assembles without the heme b andthe heme b3/CuB binuclear centers (25, 26). Whether CcoS isable to bind heme or Cu directly is currently unknown, but itsprimary sequence does not show any motifs that would point inthis direction. Finally, CcoH also encodes a small putativemembrane protein without any defined motif or any homologyto proteins of known function. Deleting ccoH prevents thestable assembly of cbb3-Cox (26), but the exact function ofCcoH has remained unclear.

The available data indicate that distinct cellular machineriesare involved in catalyzing similar, or even identical, steps dur-ing the assembly of aa3-Cox and cbb3-Cox The frequent occur-rence of cbb3-Cox in many pathogenic bacteria and the obser-vation that this enzyme is the only terminal oxidase recognizedin the genomes of Helicobacter pylori and Neisseria meningitides(30) render the assembly process and the involved assemblyfactors attractive as targets for antibacterial treatments. In thecurrent study, we analyzed the function of CcoH in cbb3-Coxassembly and stability. Our data demonstrate that CcoH is asingle spanning-membrane protein with an N-terminus-out–C-terminus-in (Nout-Cin) topology that specifically contacts the

CcoP subunit of cbb3-Cox. Surprisingly, CcoH does not seemto interact only transiently with the cbb3-Cox, but under thetested conditions, it stayed tightly associated with the active,fully assembled complex, almost behaving like a bona fidesubunit of the cbb3-Cox rather than an assembly factor per se.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The strains and plasmids used arelisted in Table 1. Escherichia coli strains harboring plasmids were grown in LBmedium supplemented with the appropriate antibiotics (100, 50, and 12.5 �g/mlof ampicillin, kanamycin, and tetracycline, respectively). R. capsulatus strainswere grown in Sistrom’s minimal A medium or in enriched MPYE medium (6,44) at 35°C in liquid cultures in the dark with the appropriate antibiotics (10 and2.5 �g/ml of kanamycin and tetracycline, respectively). For semiaerobic growth,500-ml cultures were grown in the dark in 1,000-ml flasks and were shaken at110 rpm.

Molecular-genetics techniques. Standard molecular-genetics techniques wereperformed as described by Sambrook et al. (40). For in vitro synthesis, the ccoHgene was cloned into pET19b and pET22b (Novagen, Bad Schwalbach, Ger-many) by using BamHI and NdeI restriction sites to obtain N-terminally orC-terminally His-tagged CcoH derivatives under the control of the T7 promoter.Truncated CcoH versions for in vitro synthesis were generated by incorporatingstop codons into the ccoH sequence by using the following primers: 5�-CGCGGAATAAACCTAGTCGGTCCGGGTC-3� and 5�-CCCCTCAGCCTTGCCCCCGGGGCCTG-3� for deleting the second hydrophobic segment and 5�-CCCCGGCTAGGTCTTCACC-3� and 5�-CTTGAAGTGGCGAATTCCTATGTCG-3� for deleting the entire cytoplasmic domain. For in vivo truncation of CcoH,an additional stop codon and an XbaI restriction side were introduced into CcoHin pET17b-CcoGHI by using primers 5�-GTCCGT CCG GGTCATCTGC-3�and 5�-TGATCTAGATCCGCGCCCCTC-3�, and the modified ccoH gene wassubsequently subcloned into pRK415 using BamHI and XbaI.

Preparation of ICMs and high-speed supernatants. High-speed supernatants(S-135 extracts) of cell homogenate from wild-type R. capsulatus strain MT1131and intracytoplasmic membranes (ICMs) from MT1131 and mutant strains wereprepared essentially as described previously (55), with the following modifica-tion: cells were grown semiaerobically at 35°C. Membrane-free S-135 extractswere obtained from strain MT1131 by French pressing cell suspensions at 8,000lb/in2 followed by a first centrifugation at 30,000 � g for 30 min. The resultingsupernatant (S-30) was centrifuged in 1-ml aliquots in a Beckmann TLA-100ultracentrifuge at 90,000 rpm for 9 min using a TLA-100.2 rotor. The top 750-�lof the supernatant was used as an S-135 extract for cell-free protein synthesis.Preparation of Helicobacter pylori membranes was done according to the protocoldesigned for E. coli membranes (7). In brief, after breaking the cells using aFrench-pressing cell and a first centrifugation as described above, the pellet wasresuspended and centrifuged for 2 h at 40,000 rpm in a Beckmann Ti50 rotor.The crude membrane pellet was then resuspended and subjected to three-stepsucrose gradient centrifugation as described previously (7). Membranes werefinally resuspended in INV buffer (50 mM triethanolamine acetate [pH 8.0]–250mM sucrose).

In vitro protein synthesis, protease protection assay, and carbonate resistance.T7 RNA polymerase-dependent in vitro expression of ccoH was achieved usingplasmid pET19b-ccoH. Cell-free protein synthesis using [35S]methionine andcysteine with R. capsulatus S-135 extracts was carried out for 30 min at 35°C asdescribed previously (15, 26, 55). For cotranslational integration of in vitro-synthesized proteins into membranes, ICMs were added after 5 min of synthesis,and the reaction mixture was incubated for 25 min at 35°C. For protease treat-ment, samples were incubated with 0.5 mg of proteinase K/ml for 20 min at 25°Cfollowed by trichloroacetic acid (TCA) precipitation (5% final concentration).The TCA pellet was resuspended in sodium dodecyl sulfate (SDS) loading bufferand loaded onto an 18% SDS-polyacrylamide gel. For analyzing carbonate re-sistance, freshly prepared Na2CO3 (pH 11) was added to the in vitro reactionmixture (final concentration, 0.18 M), and the samples were incubated on ice for30 min. After the mixture was centrifuged in a Beckmann TLA-100.3 rotor for 15min at 70,000 rpm, the pellet thus obtained was directly dissolved in SDS loadingbuffer. The supernatant was neutralized with glacial acetic acid, TCA precipi-tated, and centrifuged for 10 min at 20,000 � g, and the pellet obtained wasdissolved in SDS loading buffer. Radiolabeled proteins were visualized by phos-phorimaging using a Molecular Dynamics phosphorimager and quantified byusing ImageQuant software from Molecular Dynamics.

BN-PAGE. For analyses by BN-polyacrylamide gel electrophoresis (BN-PAGE), ICMs (50 �g of total protein) were resuspended in 10 �l of 2� lysis

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buffer (50 mM NaCl, 5 mM 6-aminohexanoic acid, and 50 mM imidazole/HCl[pH 7.0]), adjusted to 20 �l with water, and solubilized with n-dodecylmaltoside(DDM; Roche Diagnostics, Mannheim, Germany) at a 1:1 (wt/wt) ICM protein-to-detergent ratio from a 10% dodecylmaltoside stock solution in lysis buffer(final DDM concentration, 1%). After being incubated for 10 min at 25°C, thesamples were centrifuged for 15 min at 70,000 rpm in a Beckmann TLA-100.3rotor. After centrifugation, 15 �l of the supernatant was supplemented with 2 �lof loading buffer (5% Coomassie blue in 500 mM 6-aminohexanoic acid) and 5�l of 50% glycerol and then loaded onto a 5 to 20% BN-polyacrylamide gel.

Activity assays. (i) Oxygen uptake. N,N,N,N-tetramethyl-p-phenylenediamine(TMPD) oxidase activity was measured at 28°C in a closed reaction chamber(1-ml volume) with a fiber optic oxygen meter (Fibox 3; PreSens GmbH, Re-gensburg, Germany). R. capsulatus membranes were dissolved in ICM buffer (50mM triethanolamine, 1 mM EDTA, 1 mM dithiothreitol [DTT], and 0.5 mMphenylmethylsulfonyl fluoride [PMSF]) to a final concentration of approximately0.1 mg/ml. Oxygen consumption was initiated by the addition of 10 �l of 1 Msodium ascorbate (final concentration, 10 mM) and 5 �l of 24 mM TMPD (finalconcentration, 0.2 mM). Oxygen consumption was recorded at 28°C using Oxy-View 3.5.1 software (PreSens GmbH) and was terminated after several minutesof recording by the addition of 0.1 mM NaCN (final concentration). Net TMPDoxidase activity was determined by subtracting the endogenous respiration ratefrom that induced by ascorbate.

(ii) In-gel heme staining and on-plate or in-gel NADI staining. To reveal thec-type cytochromes, SDS-Tris-tricine-polyacrylamide gels were treated with3,3,5,5-tetramethylbenzidine (TMBZ) according to the method of Thomas et al.(51). For activity staining of cbb3-Cox in growing cells via �-naphthol plusN,N-dimethyl-p-phenylenediamine (DMPD) (NADI) reaction, cell cultures weregrown on MPYE plates with the appropriate antibiotics at 35°C. Staining wasperformed by incubating the plates with a 1:1 (vol/vol) mixture of 35 mM�-naphthol dissolved in ethanol and 30 mM NADI in water, which turned bluein the presence of active enzyme. NADI staining of BN-polyacrylamide gels wasperformed as described previously (26).

Immunodetection methods. For immunoblot analyses, proteins were electro-blotted onto Immobilon-P transfer membranes. Polyclonal antibodies againstCcoP, CcoN, and CcoH were used with horseradish peroxidase-conjugated goatanti-rabbit antibodies from Caltag Laboratories (Burlingame, CA) as secondary

antibodies, and enhanced chemiluminescence (ECL) reagent (GE Healthcare,Munich, Germany) was used as the detection substrate. Antibodies against theHis6 tag were obtained from Roche (Mannheim, Germany). Immunoprecipita-tions were performed as described previously (26).

RNA isolation and RT-PCR. Semiaerobic cultures of R. capsulatus in MPYEmedium were grown to mid-log phase (optical density at 652 nm [OD652], 0.8 to1.0). Approximately 1� 109 cells were centrifuged, incubated in TE buffer (10mM Tris–0.5 mM EDTA [pH 7]) containing 10 mg/ml of lysozyme for 15 min in37°C and passed 5 times through an 0.8-�m-diameter needle into RNase-freetubes. Isolation of total RNA was achieved by using a GE Healthcare minispinkit according to the suggested protocol. Two micrograms of total RNA wastreated with DNase I for 30 min at 25°C in the presence of the RNase inhibitorRNasin. Fifty nanograms of treated RNA was used in reverse transcriptase(RT)-PCRs with a OneStep RT-PCR kit (Qiagen, Hilden, Germany). The prim-ers used for detection of ccoH mRNA were 5�-CGACCAGAACCTCGAGCTGG-3� and 5�-ACCGCTGACCGGCCGCAAG-3�; the primers used for detec-tion of ccoN mRNA were 5�-CATGTTGCACATCGTCAACAACC-3� and 5�-CGAAGGTGATCATGCCGTTCC-3�. As a control for detecting genomic DNAcontamination, PCRs were performed with the same primer without the reversetranscriptase step using Phusion DNA polymerase (Finnzymes USA). Sampleswere separated on a 1.2% agarose gel.

RESULTS

CcoH is required for the activity and stability of cbb3-typecytochrome oxidase. The crucial importance of CcoH for cbb3-Cox assembly and activity has been demonstrated in previousstudies (25, 26); however, the exact function of CcoH hasremained unclear. In R. capsulatus, the active cbb3-Cox is de-tectable as a 230-kDa complex in BN-PAGE and is assembledvia a 210-kDa intermediate containing at least CcoN and CcoOand an approximately 50-kDa intermediate containing at leastCcoP and CcoQ (26, 34). In the ccoH deletion mutant CW6,

TABLE 1. Strains and plasmids used in this studya

Strain or plasmid Genotype Relevant phenotype or applicationb Source orreference

StrainEscherichia coli

DH5� supE44 �lacU169 (�80lacZ�M15) hsdR17 recA1 endA1gyrA96 thi-1 relA1

52

HB101 F� proA2 hsdS20(rB� mB

�) recA13 ara-14 lacY1 galK2rpsL20 supE44 xyl-5 mtl-1

52

Rhodobacter capsulatusMT1131 crtD121 rifR Wild-type, NADI� 41MT1131-pCW25 pRK415-ccoNOQP-ccoGHIS cbb3-Cox overexpression 29GK32 �ccoNO::kan NADI� 30M7G ccoP269 (stopc) NADI� 28CW6 �ccoH::kan NADI� 30CW1 �ccoGHIS::spe NADI� 30

PlasmidpRK415-ccoH ccoH cloned into pRK415 via BamHI/XbaI In vivo expression of CcoH 27pRK415-ccoH-M1 ccoH cloned into pRK415 without second HD via

BamHI/XbaIIn vivo expression of ccoH lacking the

second HDThis work

pET19b/pET22b Ampr NovagenpET19b-ccoH ccoH cloned into pET19b via NdeI/BamHI In vitro synthesis of N-His-CcoH 27pET19bccoH-M0 ccoH cloned into pET19b via NdeI/BamHI

(105)TTC3AUGIn vitro synthesis of N-His-CcoH lacking the

cytoplasmic loopThis work

pET19bccoH-M1 ccoH cloned into pET19b via NdeI/BamHI(378)TGG3AUG

In vitro synthesis of N-His-CcoH lacking thesecond HD

This work

a HD, hydrophobic domain; N-His-CcoH, N-terminally His-tagged CcoH.b NADI� indicates that the strain is positive in the NADI reaction (see Materials and Methods), while NADI� indicates that the strain does not respond within 30

min to the NADI reaction.c R. capsulatus M7G carries a premature stop codon at position 269.

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neither the fully assembled 230-kDa complex nor the 210-kDaand 50-kDa intermediates were detectable (Fig. 1A); thus,CW6 displayed the same phenotype as the cbb3-Cox knockoutstrain GK32. In SDS-PAGE, the catalytic subunit CcoN ofcbb3-Cox was undetectable in membranes of CW6 by immu-nodetection. The c-type cyt subunits CcoO and CcoP were alsoundetectable by heme staining, while the absence of CcoH didnot impair the steady-state stability of cyt c1 of the cyt bc1

complex or the stability of the membrane-bound electron car-rier cyt cy (Fig. 1B). In agreement with the lack of individualsubunits of cbb3-Cox, we also failed to detect cbb3-Cox-depen-dent oxygen uptake activity in membranes of the CcoH dele-tion strain (Fig. 1C). All phenotypes of the CcoH mutant werefully restored by expressing CcoH in trans (Fig. 1), indicatingthat deleting CcoH does not induce polar effects on the down-

stream ccoI and ccoS genes, which are also essential for theassembly of a functional cbb3-Cox (25). In comparison to thatin wild-type membranes, we observed a slightly increased ox-ygen uptake activity in membranes from cells of the CW6mutant expressing plasmid-borne ccoH, suggesting that underthe conditions tested, CcoH might be a limiting factor forsteady-state levels of cbb3-Cox activity.

CcoH is a type I integral membrane protein. ccoH is pre-dicted to encode a small protein of 151 amino acids, whichpresumably spans the membrane at least once with an N-terminally located transmembrane domain. However, a secondhydrophobic stretch at the C terminus could constitute anadditional transmembrane domain, as predicted by many sec-ondary-structure prediction programs. For determining thetopology of CcoH, we employed an R. capsulatus in vitro

FIG. 1. CcoH is essential for cbb3-Cox stability and activity in R. capsulatus. (A) Intracytoplasmic membranes (ICMs; 50 �g of protein) of theindicated R. capsulatus strains were solubilized with dodecylmaltoside and separated by BN-PAGE. CW6/CcoH corresponds to a genomic ccoHdeletion mutant carrying a copy of ccoH on a plasmid. After being transferred onto a polyvinylidene difluoride (PVDF) membrane, complexes weredecorated with antibodies against the CcoN and CcoP subunits (�-CcoN and �-CcoP). (B) The ICMs shown in panel A were separated onTris-tricine gels and either transferred onto PVDF membranes to detect the steady-state amount of CcoN (upper panel) or analyzed by hemestaining, which revealed the membrane-bound c-type cytochromes of R. capsulatus (lower panel). Indicated are the subunits CcoP and CcoO ofcbb3-Cox, cyt c1 of the bc1 complex, and cyt cy. (C) cbb3-Cox activities in different ICMs were measured as oxygen uptake activities in at least threeindependent experiments.



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transcription-translation system together with inverted intracy-toplasmic membrane vesicles (26, 34). CcoH was synthesized invitro in the presence or absence of ICMs and subsequentlysubjected to alkaline carbonate extraction, which allows differ-entiation between integral membrane proteins and proteinsthat are soluble or only peripherally attached to the membrane(10). In vitro synthesis of CcoH resulted in a radioactivelylabeled protein of 17 kDa (Fig. 2A), which is in line with thepredicted molecular mass of CcoH. In the absence of ICMs,CcoH was found predominantly in the supernatant (S) aftercarbonate extraction and ultracentrifugation. The smallamount of CcoH found in the pellet fraction (P) even in theabsence of ICMs is most likely the result of aggregation, whichis frequently observed if small hydrophobic proteins are syn-thesized in vitro (7, 35). In the presence of wild-type ICMs,however, the majority of CcoH was carbonate resistant, i.e.,recovered from the pellet fraction after centrifugation. Thisresult demonstrates that CcoH is an integral membrane pro-tein. Carbonate resistance of CcoH was also observed in mem-branes of the cbb3-Cox knockout strain GK32, suggesting thateven in the absence of cbb3-Cox, in vitro-synthesized CcoH canbe inserted stably into ICMs.

To analyze the topology of CcoH in the membrane, weemployed proteinase K protection assays of in vitro-synthesizedCcoH. CcoH was in vitro synthesized in the absence of ICMsand subjected first to carbonate extraction. After ultracentrif-ugation, the supernatant and the pellet fractions were treatedwith proteinase K, which completely digested CcoH (Fig. 2B,left panel). In the presence of ICMs, proteinase K treatment ofthe carbonate-resistant material resulted in a proteinase K-protected fragment of about 5 kDa, which was not observedafter proteinase K treatment of the supernatant, i.e., of thecarbonate-sensitive material. The occurrence of a 5-kDa pro-tease-protected fragment supports a topology of CcoH inwhich the N-terminal hydrophobic domain (amino acids 10 to33) serves as a transmembrane domain, while amino acids 34to 151 are accessible to proteinase K and thus reside on thecytoplasmic side of the inverted-membrane vesicles. In orderto verify this topology, we made use of the N-terminal His tagpresent in the in vitro-synthesized CcoH. Anti-His6 antibodiesimmunoprecipitated both the full-size CcoH and the protease-protected 5-kDa band, demonstrating that the N-terminal Histag was protected against proteinase K digestion (Fig. 2B, rightpanel).

Because these data cannot completely rule out that the sec-ond hydrophobic domain is also involved in membrane bindingof CcoH, we analyzed the proteinase K protection of two invitro-synthesized CcoH truncations (Fig. 2C). In vitro synthesisof CcoH-M1, which lacks the second hydrophobic domain (aminoacids 120 to 150), resulted in a slightly smaller in vitro-synthesizedproduct (Fig. 2D), but after proteinase K treatment we observedthe 5-kDa protected fragment, also seen for full-size CcoH.Proteinase K treatment of a CcoH derivative, which consistedalmost exclusively of the first transmembrane domain (CcoH-M0, amino acids 1 to 35), also resulted in the occurrence of the5-kDa protease-protected fragment (Fig. 2D). That the dele-tion of the second hydrophobic domain did not interfere withmembrane integration of CcoH or its topology in the mem-brane was further confirmed by carbonate extraction, whichdemonstrated that the two C-terminally truncated CcoH

derivatives were, like full-size CcoH, carbonate resistant(Fig. 2E).

In summary, these data demonstrate that the first hydropho-bic domain of CcoH serves as a transmembrane domain, whilethe second hydrophobic domain is not required for stablemembrane integration of CcoH in vitro. Thus, CcoH is a typicaltype I membrane protein with a single transmembrane domainand an Nout-Cin topology.

The second hydrophobic stretch of CcoH is required for itsstability in vivo. Although the second hydrophobic stretch ofCcoH does not seem to be required for membrane integration,its high level of sequence conservation among different speciessuggests an important role for the function of CcoH. Its rolewas analyzed by testing whether the CcoH-M1 construct, whichlacks the second hydrophobic stretch, could complement theCcoH deletion strain CW6. NADI staining allows the detectionof active cbb3-Cox in whole colonies. DMPD is oxidized bycbb3-Cox and, together with �-naphthol, forms the dye indo-phenol blue (24). Colonies of the R. capsulatus wild-type strainMT1131 turned blue within 30 s after treatment with the NADIreagent, and a similar response was observed for the CW6strain complemented with full-size CcoH (CW6/CcoH) (Fig.3A). In contrast, CW6 expressing CcoH-M1 (CW6/CcoH-M1)did not show any response even after 30 min, indicating thatthe second hydrophobic domain of CcoH is essential for func-tion. This was confirmed by biochemical analyses using BN-PAGE, immunodetection, and heme staining, which revealedthat neither the 230-kDa cbb3-Cox complex nor the individualsubunits of cbb3-Cox were detectable in membranes of CW6expressing CcoH-M1 (Fig. 3B).

Because CW6 and CW6/CcoH-M1 displayed exactly thesame phenotype, we analyzed whether the truncated CcoHderivative was stable in R. capsulatus membranes. We wereunable to detect the truncated CcoH version (Fig. 3C) byimmunodetection using anti-CcoH antibodies, but RT-PCRusing a specific primer pair for the central part of the ccoHgene revealed the presence of an identical PCR product usingRNA isolated from wild-type, CW6/CcoH, or CW6/CcoH-M1cells as a template (Fig. 3D). This indicates that ccoH-M1 istranscribed into mRNA but fails to yield a stable protein in themembrane in vivo. As the in vitro data (Fig. 2D and E) seemedto indicate that CcoH-M1 can be stably integrated into ICM,the apparent difference between the in vitro and in vivo data(Fig. 3) is probably due to the fact that the in vitro experimentswere performed in the presence of a protease inhibitor cocktailand for a short period of time (i.e., 30 min).

CcoH stability in the membrane depends on cbb3-Cox. Theinstability of the truncated CcoH derivative in vivo could indi-cate that the lack of the second hydrophobic domain precludesthe protein-protein interactions that are required for CcoHstability. Because CcoH is essential for cbb3-Cox assembly, wetested whether the stability of CcoH in the membrane wasinfluenced by the absence of cbb3-Cox. By Western blottingusing anti-CcoH antibodies, CcoH was detectable in wild-typeR. capsulatus membranes but not in membranes of the cbb3-Cox deletion strain GK32 (Fig. 4A). CcoH was also not de-tectable in GK32 expressing multiple ccoH copies from a plas-mid (Fig. 4B). RT-PCR revealed that the lack of the cbb3-Coxgenes in GK32 did not influence transcription of the ccoH gene(Fig. 4B). As a control, we used a primer pair specific for ccoN,



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FIG. 2. CcoH is an integral membrane protein with an Nout-Cin topology. (A) CcoH was in vitro synthesized in an R. capsulatus cell-freetranscription-translation system in the absence or presence of ICMs. Subsequently, the reaction mixtures were treated with alkaline carbonate, andcarbonate-resistant (pellet, P) and carbonate-sensitive (supernatant, S) materials were separated by ultracentrifugation. Membrane-associated orsoluble proteins were found in the supernatants after CO3 treatment and centrifugation. (B) CcoH was in vitro synthesized in the presence of ICMsand carbonate extracted. The carbonate-resistant material was then resuspended in ICM buffer and treated with proteinase K. Samples wereseparated on a Tris-tricine gel. Immunoprecipitations (IP) were performed with anti-His antibodies, which recognized the N-terminal His tag ofCcoH. Asterisks (*) indicate the proteinase K-protected fragments of CcoH, while a pound sign (#) indicates an unspecific band that wasimmunoprecipitated by anti-His antibodies. (C) Schematic representation of C-terminally truncated CcoH derivatives. wt, full-sized CcoH; CcoH-M0, aCcoH derivative that consists mainly of the transmembrane domain; CcoH-M1, a derivative that lacks the second hydrophobic segment. (D) ProteinaseK digestion of the in vitro-synthesized CcoH derivatives in the presence of wild-type ICMs. Arrows indicate the in vitro-synthesized derivatives withoutproteinase K treatment, and asterisks indicate the proteinase K-protected fragments. (E) Carbonate extraction of in vitro-synthesized constructsperformed in the presence of wild-type ICMs (MT1131) and ICMs lacking cbb3-Cox (GK32) and without ICMs (�).

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which encodes the catalytic subunit of cbb3-Cox. The ccoNmRNA was detectable by RT-PCR in wild-type cells, CW6,and CW6 expressing plasmid-borne ccoH (Fig. 4C). These datademonstrate a mutual dependence of cbb3-Cox and CcoH.In the absence of CcoH, cbb3-Cox is not stably assembled,and in the absence of cbb3-Cox, CcoH is not stable in mem-branes. The RT-PCR data indicate that this is not the resultof a transcriptional coupling, because the mRNA levels are notsignificantly influenced in the respective mutants. Gene expres-

FIG. 3. The second hydrophobic segment of CcoH is crucial for itsfunction and stability. (A) The truncated CcoH-M1 derivative or full-size CcoH were expressed from a plasmid in the ccoH deletion strainCW6, and cbb3-Cox activities were determined by NADI staining of R.capsulatus colonies. The dark color indicates active cbb3-Cox. MT1131corresponds to wild-type R. capsulatus. (B) Steady-state stabilities ofcbb3-Cox complexes and individual subunits in membranes from dif-ferent R. capsulatus strains. The upper panel shows the results ofBN-PAGE with subsequent immunodetection using antibodies againstthe CcoN subunit. The middle panel shows the presence of the CcoNsubunit after separation of membranes by SDS-Tris-tricine-PAGE andsubsequent immunodetection. The lower panel depicts a heme-stainedgel. (C) Detection of the truncated CcoH-M1 derivative expressed instrain CW6 by using anti-CcoH antibodies. (D) Results of RT-PCRusing total RNA from different R. capsulatus strains showing ccoHexpression.

FIG. 4. CcoH stability is dependent on cbb3-Cox. (A) The presenceof CcoH was analyzed by Western blotting in either wild-type strainMT1131 expressing a plasmid containing the complete ccoNOQP-ccoGHIS gene cluster or in the cbb3-Cox deletion strain GK32.(B) The same as panel A but with GK32 expressing a plasmid-bornecopy of ccoH. (C) Results of RT-PCR performed by using total RNAderived from wild-type strain MT1131, cbb3-Cox knockout strainGK32, CcoH knockout strain CW6, and CW6 complemented withthe ccoH gene. The reactions were performed with primer pairsbinding to ccoH (upper lane) or ccoN (middle lane). A control PCRfor detecting genomic DNA contaminations was performed with thesame RNA preparations but without the reverse transcriptase step(lower lane, �RT).



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sion studies using a lacZ-based translational reporter fusion toccoH showed comparable -galactosidase activities in wild-type and GK32 cells (data not shown). Therefore, the lack ofstability is posttranscriptional and is probably the result of aposttranslational degradation event.

The stabilizing effect of cbb3-Cox on CcoH could most easilybe explained by their direct physical contact. We thereforeemployed an in vitro cross-linking approach using the mem-brane-permeable chemical cross-linker DSS (disuccinimidylsuberate). CcoH was in vitro synthesized in the absence ofICMs or in the presence of either wild-type or GK32 ICMs.After the addition of DSS, the reaction mixture was immuno-precipitated using anti-CcoP antibodies. In the presence ofwild-type ICMs, anti-CcoP antibodies immunoprecipitated aradioactively labeled band at about 50 kDa of the expected sizeof a CcoH-CcoP cross-linking product (Fig. 5). No cross-link-ing product was observed in the absence of membranes nor inthe presence of cbb3-Cox deletion membranes. We also tried todetect a possible interaction between CcoN and CcoH by DSScross-linking and immunoprecipitation, but the cross-linkingproduct was too weak to make a definite statement about apossible CcoN-CcoH interaction. Nevertheless, the strongcross-link between CcoH and CcoP demonstrates that CcoH isin close proximity to the CcoP subunit of cbb3-Cox.

CcoH is stably associated with the cbb3-Cox complex. Theinteraction between CcoP and CcoH would be in agreementwith a function of CcoH during the assembly of the cbb3-Coxcomplex, which supports its proposed role as a dedicated cbb3-Cox assembly factor (25, 26). In addition, the observation thatCcoH is stable only in the presence of cbb3-Cox suggests thatCcoH might associate more permanently with this enzyme.

In order to address this question, we combined BN-PAGEwith in vitro labeling. CcoH was in vitro synthesized and inte-

grated into wild-type R. capsulatus membranes, which weresubsequently separated by BN-PAGE. If CcoH bound tothe cbb3-Cox complexes, it should radioactively label them andthe complexes should be detectable by autoradiography. In thepresence of wild-type ICMs, CcoH labeled the 230-kDa com-plex, which corresponds to the active, fully assembled cbb3-Coxcomplex, and weakly labeled the 210-kDa complex (Fig. 6A),which represents an intermediate containing at least the CcoNand CcoO subunits (26). As observed previously (26), thesmallest complex, which represents a CcoP-CcoQ complex(34), was not detectable by BN-PAGE with in vitro labeling.Whether this is related to a low level of labeling efficiency or tothe low level of abundance of the small complex is currentlyunknown. The specificity of in vitro labeling was controlled byusing ICMs of the cbb3-Cox deletion strain GK32, which werenot specifically labeled by CcoH. In contrast, in ICMs of theccoP deletion strain M7G, which assembles the 210-kDa inter-mediate but not the 230-kDa complex, only labeling of the210-kDa complex was observed. Since the 210-kDa intermedi-ate does not contain the CcoP subunit, these data also indicatethat CcoP is not the only contact partner of CcoH.

In vitro labeling also allowed us to determine whether theC-terminally truncated CcoH derivatives maintained their abil-ity to bind to the complexes. CcoH-M1 was in vitro synthesizedand integrated into wild-type, GK32, and M7G (�ccoP) mem-branes. For full-sized CcoH, we observed labeling of both the230-kDa and the 210-kDa complexes in wild-type membranesand labeling of only the 210-kDa complex in �ccoP mem-branes. The labeling in M7G ICMs appeared to be strongerthan the labeling in wild-type ICMs, which is probably relatedto the larger amount of the 210-kDa complex in M7G com-pared to that in wild-type strains. Nevertheless, these dataindicate that CcoH-M1 is still able to bind to the cbb3-Cox

FIG. 5. CcoH interacts with the CcoP subunit of cbb3-Cox. CcoH was in vitro synthesized in the absence of ICMs or in the presence of eitherwild-type strain MT1131 ICMs or ICMs derived from the cbb3-Cox knockout strain GK32. After in vitro synthesis and integration into themembrane, one part was treated with dimethyl sulfoxide (DMSO), while the other part was incubated with DSS dissolved in DMSO. TheDSS-treated sample was further split, and one part was directly TCA precipitated, while the other part was subjected to immunoprecipitation usingantibodies against the CcoP subunit of cbb3-Cox. The asterisk (*) indicates the CcoH-CcoP cross-linking product.



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complexes. Finally, we also tested the CcoH-M0 derivative,which lacks the complete cytoplasmic domain. However, herewe observed no binding to either the 230-kDa nor the 210-kDacomplex. The differences in labeling were not the result oflarge differences in the amounts of in vitro-synthesized pro-teins, as indicated in Fig. 6B. In summary, these data demon-strate that CcoH binds to both the fully assembled cbb3-Coxand to the 210-kDa cbb3-Cox intermediate. Because CcoP ismissing in the 210-kDa complex, CcoH apparently binds notonly to CcoP but also to another subunit of cbb3-Cox.

We could not determine by using the in vitro-labelingtechnique whether binding of in vitro-synthesized CcoH oc-curs via exchange with the endogenous CcoH or through anunoccupied binding site (26, 34).Therefore, we determinedwhether CcoH was detectable in the cbb3-Cox complexes byimmunodetection. Wild-type membranes were separated byBN-PAGE, and after Western transfer, were decorated withanti-CcoH antibodies. The antibodies recognized two bands at230-kDa and 210-kDa and a third band running below the66-kDa marker band with BN-PAGE (Fig. 7). Strikingly, thesewere exactly the bands that were also recognized by antibodiesagainst CcoN (Fig. 1 and 2) and CcoP (Fig. 6). All threecomplexes were undetectable in membranes of the cbb3-Coxdeletion strain GK32 (Fig. 7) and became significantly strongerin membranes from wild-type cells expressing the complete

FIG. 6. CcoH binds to a preassembled cbb3-Cox. (A) In vitro-synthesized wild-type CcoH (wt) or the truncated CcoH versions CcoH-M0 andCcoH-M1 were in vitro synthesized in the presence of the indicated membranes. MT1131 contains wild-type amounts of cbb3-Cox, while in GK32,cbb3-Cox is deleted. M7G corresponds to an R. capsulatus ccoP mutant that assembles the 210-kDa CcoNO subcomplex. After in vitro synthesis,samples were separated by BN-PAGE, and radioactively labeled bands were detected by phosphorimaging. Asterisks (*) indicate the fullyassembled 230-kDa cbb3-Cox complex, while plus signs (�) indicate the 210-kDa subcomplex lacking the CcoP subunit. (B) Synthesis control ofthe in vitro reactions described for panel A. Note that the three panels correspond to the same gel, which was split into three parts. Therefore, thesize differences among the three CcoH derivatives are not visible.

FIG. 7. CcoH is a component of the cbb3-Cox complexes. ICMsfrom the indicated strains were solubilized with DDM and separatedby BN-PAGE as described for Fig. 1. Complexes were subsequentlydetected by antibodies against CcoH or CcoP. MT1131/pCW25 carriesthe ccoNOQP-ccoGHIS gene cluster on a plasmid. CW1 correspondsto the ccoGHIS deletion strain.



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ccoNOQP-ccoGHIS gene cluster (pCW25). The three bandswere also undetectable in membranes of the ccoGHIS deletionmutant. Like the in vitro-labeling data (Fig. 6A), these datashow that CcoH contacts not only the CcoP subunit but alsoother subunits of the cbb3-Cox complex, because the 210-kDacomplex is recognized by anti-CcoH antibodies despite the factthat CcoP is not present in this subcomplex. In addition, thesedata demonstrate that CcoH contacts the cbb3-Cox complexesnot only transiently during assembly but that it stays attachedto the active 230-kDa cbb3-Cox complex.

DISCUSSION

The assembly of a cofactor containing multisubunit proteincomplexes is a highly ordered process that is orchestrated bymany auxiliary proteins. These proteins are referred to as as-sembly factors because they are usually not present in themature complex, but in their absence, enzyme complexes fail toassemble. Although a single assembly factor has been shown tocontrol the proper assembly of the heterotrimeric nitrate re-ductases (27), typically protein complexes assemble along anassembly line involving many of these dedicated assemblyfactors (18). Respiratory complexes like cytochrome oxi-dases have been used as model enzymes for analyzing theseprocesses because the need for cofactor incorporation addsan additional level of complexity to their assembly.

In a previous study we proposed two possible roles for theputative assembly factor CcoH in cbb3-Cox assembly. CcoHcould be required for stabilizing the CcoQ-CcoP and CcoN-CcoO subcomplexes. In addition, CcoH could facilitate theassociation of both subcomplexes in the functional 230-kDacbb3-Cox complex (26). Our data now demonstrate that CcoHis indeed present in both complexes and that CcoH can becross-linked to CcoP by the amine-specific cross-linker DSS.CcoH contains four lysine residues flanking the transmem-brane domain, and lysine residues are also clustered on the cisside of the transmembrane segment of CcoP. Thus, the CcoH-CcoP contact could occur primarily via their respective trans-membrane segments. The core complex of cbb3-Cox, consistingof CcoN, CcoO, and CcoP, has recently been crystallized fromP. stutzeri (3), and the X-ray structure shows that the trans-membrane region of CcoP is freely accessible and appears tohave no contact with the other subunits of the cbb3-Cox com-plex (3). In addition, the transmembrane domains of bothCcoP and CcoH show high levels of sequence conservation,which would favor a specific CcoH-CcoP interaction in addi-tion to their role as membrane anchors.

Our data indicate that CcoP cannot be the only contact sitefor CcoH because CcoP is not detectable in the 210-kDa com-plex, which still contains CcoH. Whether this additional con-tact site is provided by CcoO or CcoN is currently unknown.Like CcoP, CcoO consists of a single transmembrane domainconnected to the large periplasmic heme binding domain.Therefore, possible contacts between CcoH and CcoO wouldprobably occur primarily via their respective transmembranedomains. However, the transmembrane domain of CcoOseems to be largely occupied by its interaction with the catalyticsubunit, as initially suggested by molecular-modeling studies(16, 42) and now confirmed by the X-ray structure (3). Contactof CcoH with the cytosolic N terminus of CcoO is also unlikely,

because the N terminus is rather short and not highly exposedin the crystal structure (3). Thus, an interaction between CcoHand CcoN in the 210-kDa complex appears to be more likely.Originally, two-dimensional predictions had pointed to thepresence of 14 transmembrane segments, with the N and Ctermini facing the cytoplasm (52); however, there was alsoexperimental evidence that the first two segments were notmembrane integrated but rather located on top of the mem-brane surface (56). In P. stutzeri, CcoN consists of 12 trans-membrane domains (3), but CcoN in P. stutzeri is also shorterthan that in B. japonicum or R. capsulatus. In R. capsulatus,CcoN contains an N-terminal extension of about 50 aminoacids, which could provide a contact site for the essential sec-ond hydrophobic segment of CcoH.

The second hydrophobic segment displays the highest levelof sequence conservation within the CcoH family of proteins,highlighting its functional relevance. If this segment is deleted,cbb3-Cox is not assembled, and as a consequence, CcoH is notdetectable. On the other hand, in vitro-synthesized CcoHlacking the second hydrophobic domain is able to bind to apreassembled cbb3-Cox complex and also to the CcoNOsubcomplex. This finding suggests that the lack of the sec-ond hydrophobic domain does not completely block the bind-ing of CcoH to the CcoNO subcomplex but rather prevents theformation of the stable 230-kDa cbb3-Cox holocomplex and isalso in agreement with our assumption that it is primarily thetransmembrane segment of CcoH that is responsible for mak-ing contact with the cbb3-Cox subunits. An attractive model forthe role of CcoH during cbb3-Cox assembly is that one copy ofCcoH binds via its transmembrane domain to CcoP in the50-kDa CcoPQ complex (Fig. 8). A second CcoH also binds viaits transmembrane domain to the 210-kDa CcoNO complex.These interactions stabilize both subcomplexes, and CcoH

FIG. 8. Model for the role of CcoH during assembly of cbb3 oxidasein R. capsulatus. After integration of the individual subunits of cbb3-Cox into the membrane, two subcomplexes are formed, the CcoQPsubcomplex and the CcoNO subcomplex. Both subcomplexes also con-tain CcoH, which is essential for their formation or their stability. Inthe next step, both subcomplexes assemble to form the active 230-kDacbb3-Cox complex. The second hydrophobic domain (hatched bars) ofCcoH could facilitate this assembly and serve as a dimerization domainto allow the CcoHQP and the CcoHNO complexes to fuse. Please notethat the indicated molecular masses have been deduced from analysesby BN-PAGE and therefore represent only approximations.



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would then facilitate in the next step the assembly of bothsubcomplexes into the final 230-kDa cbb3-Cox complex, possi-bly via a dimerization of the C-terminal hydrophobic domains.

Our data reveal the crucial role of CcoH in cbb3-Cox assem-bly and also demonstrate that CcoH displays a rather unusualbehavior for an assembly factor, because it can be detected asa stable component of the active cbb3-Cox complex in R. cap-sulatus membranes. Furthermore, the in vivo steady-state sta-bilities of cbb3-Cox and CcoH are dependent on each other,which is also unusual for an assembly factor. The lack of CcoHin the absence of cbb3-Cox is not due to impaired ccoH tran-scription but is most likely due to enhanced proteolysis ofCcoH in the absence of cbb3-Cox in vivo. This assumption is inline with our observation that CcoH synthesized in the in vitrosystem is stable even in membranes lacking cbb3-Cox. Theshort incubation time and the presence of protease inhibitorsare likely to prevent proteolysis in this system. In contrast tothe rhizobia, in which ccoGHIS is suggested to form anoperon with a single transcript (22), in Rhodobacter speciesthe ccoGHIS genes contain internal promoters and can beexpressed independently (25, 39), which indicates that ccoHexpression is not necessarily linked to the expression of ccoG,ccoI, and ccoS. The stable association of CcoH with cbb3-Coxis surprising, because so far CcoH has never been found inpurified-protein preparations (11, 12, 50, 57). However, inthese preparations, cbb3-Cox was always purified as a three-subunit complex lacking the CcoQ subunit. CcoQ was also notdetected in the recent X-ray structure of cbb3-Cox (3), whichindicates that CcoQ is easily lost during purification. Thus,either CcoH is also lost during purification, or it is substoichio-metric and not detected by SDS-PAGE Coomassie staining. Itis interesting to note, however, that an additional transmem-brane domain is detectable in the crystal structure, whichobviously cannot be assigned to CcoQ by either side-chainrecognition or by mass-spectrometric analysis of dissolvedcrystals (3).

Because the stable association of CcoH with cbb3-Cox wasdetected mainly by BN-PAGE in our study, it is important toemphasize that the presence of complexes in BN-polyacryl-amide gels primarily reflects their stability. Therefore, we can-not entirely exclude that the 230-kDa cbb3-Cox complex rep-resents a stable assembly intermediate, despite the fact that itis active (Fig. 8) (26, 34). It is also possible that CcoH is presentin only a fraction of cbb3-Cox complexes in R. capsulatus andthat this fraction is more stable than the cbb3-Cox complexeslacking CcoH. That CcoH is a stable component of cbb3-Cox isin line with the observation that CcoH is conserved in mostbacterial species that contain a cbb3-Cox complex (8). How-ever, in some species, e.g., Sulfurimonas denitrificans (43) andHelicobacter pylori (53), CcoH is missing. In many of theseorganisms, ccoH appears to be replaced by two genes (cog5456and cog3198) (8), which show sequence homology to ccoH andwhich probably functionally replace ccoH. However, we couldnot find any homologous gene in the Helicobacter pylori ge-nome. Interestingly, although the molecular masses of the He-licobacter pylori cbb3-Cox subunits are greater than those of theR. capsulatus subunits (31, 32), the Helicobacter pylori cbb3-Coxsubunits run slightly below the R. capsulatus cbb3-Cox subunitsin BN-PAGE (data not shown), which is expected if CcoH isstably associated with cbb3-Cox in R. capsulatus.

In summary, in our study we determined the important con-tribution of the predicted assembly factor CcoH to cbb3-Coxassembly. Our results reveal that CcoH displays a rather un-usual behavior for an assembly factor in that it not only inter-acts with subunits of cbb3-Cox during intermediate stages oftheir assembly but that it stays associated with the active, fullyassembled enzyme complex as well.

ACKNOWLEDGMENTS

This work was supported by grants from the Deutsche Forschungs-gemeinschaft (DFG-GRK1478 to H.-G.K. and P.H. and DFG-FOR929 to H.-G.K. and B.W.), the German-French-University (DFH)Ph.D. College on Membranes and Membrane Proteins to H.-G.K. andP.H., and the NIH (GM 38237) and DOE (91ER 20052) to F.D. I.S.was supported by a fellowship from the Erasmus program of theEuropean Union.

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