Biogenesis of Cytochrome C Oxidase — in Vitro Approaches to Study Cofactor Insertion Into a Bacterial Subunit I

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Biochimica et Biophysica Acta 1777 (2008) 904–911

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Review Biogenesis of cytochrome c oxidase — in vitro approaches to study cofactor insertion into a bacterial subunit I

Peter Greiner ⁎, Achim Hannappel, Carolin Werner, Bernd Ludwig

Institute for Biochemistry, Molecular Genetics, Johann-Wolfgang-Goethe University, Max-von-Laue Straβe 9, 60438 Frankfurt am Main, Germany

ARTICLE INFO ABSTRACT

Article history: Biogenesis of cytochrome c oxidase is a complex process involving more than 30 known accessory proteins in Received 30 November 2007 yeast for the regulation of transcription and translation, membrane insertion and protein processing, cofactor Received in revised form 25 March 2008 insertion, and subunit assembly. Here, we focus on the process of cofactor insertion into subunit I of Accepted 2 April 2008 cytochrome c oxidase using the soil bacterium Paracoccus denitriﬁcans as a model organism. The use of Available online 10 April 2008 bacterial systems facilitates biogenesis studies, as the number of required assembly factors is reduced to a

Keywords: minimum. Both, co- and posttranslational cofactor insertion scenarios are discussed, and several approaches Assembly to shed light on this aspect of biogenesis are presented. CtaG, the Paracoccus homolog of yeast Cox11 which is fi Cox11 involved in copper delivery to the CuB center, has been puri ed and characterized spectroscopically. A Copper chaperone previously unreported signal at 358 nm allows monitoring copper transfer from copper-loaded CtaG to an Heme a acceptor. Both CtaG and apo-subunit I were purified after expression in Escherichia coli to develop an in vitro Cell-free expression copper transfer system, probing the posttranslational insertion hypothesis. To mimic a potential fifi Paracoccus denitri denitri canscans cotranslational insertion process, cell-free expression systems using E. coli and P. denitrificans extracts have been established. Expression of subunit I in the presence of the detergent Brij-35 produces high amounts of “solubilized” subunit I which can be purified in good yield. With this system it may be feasible to trap and purify assembly intermediates after adding free cofactors, purified assembly proteins, or P. denitrificans membranes. © 2008 Elsevier B.V. All rights reserved.

1. Introduction are transferred to the ﬁnal electron acceptor oxygen via four redox

centers. The bi-copper CuA-site which is located in the peripheral Cytochrome c oxidase (COX) is a highly conserved, ancient enzyme domain of subunit II in the intermembrane space (IMS) (see Fig. 1) of evolutionary origins going back to times before atmospheric oxygen receives electrons from cytochrome c and passes them on to subunit I [1]. With oxygen levels rising, cytochrome c oxidase gained its vital (SU I), which is the heart of the enzyme. This 65 kDa integral membrane role as the basis for oxygen-dependent life on earth. Even though a vast protein with its 12 transmembrane helices is almost completely amount of insight in this enzyme has been gathered since its discovery, hydrophobic and contains, next to the ﬁrst electron acceptor heme a,

many basic questions remain to be solved [2–5]. We are only beginning the binuclear heme a3:CuB center, which is the active site of the protein, to unravel the mechanism that couples electron transfer to proton where the reduction of O2 to H2O takes place (see Fig. 1). During a full pumping [6], and with the investigation of COX biogenesis an exciting catalytic cycle eight protons are taken up from the mitochondrial matrix ﬁeld has evolved that will give rise to new experimental methods and [5,10,11]. Four are translocated across the inner membrane, building up add to our understanding of this important enzyme [7–9]. the proton gradient, and the other four are consumed in the formation of As terminal oxidase in the mitochondrial respiratory chain, water, thereby also contributing to the proton gradient, which

cytochrome c oxidase catalyzes the final step in electron transport, eventually drives ATP production by the F1FO ATP synthase. the reduction of molecular oxygen to water [10,11]. In this process four In mammalian mitochondria, cytochrome c oxidase consists of 13 electrons are taken up from the one-electron donor cytochrome c, and subunits, ten of which are encoded by the nucleus and have to be imported to the mitochondrion via the TOM (translocase of the outer mitochondrial membrane) and TIM (translocase of the inner mitochondrial membrane) complexes [12]. The remaining three subunits Abbreviations: BCA, 2,2′-bicinchoninic acid; CMC, critical micelle concentration; are encoded and expressed by the mitochondrion itself, and are highly COX, cytochrome c oxidase; EXAFS, extended X-ray absorption fine structure; IMM, conserved throughout evolution. These three subunits (SU I, II and III) inner mitochondrial membrane; IMS, intermembrane space; SU, subunit; TEV, tobacco form the functional core of the enzyme, and their overall structure is etch virus; TXRF, total reflection X-ray fluorescence ⁎ Corresponding author. Tel.: +49 69 798 29243; fax: +49 69 798 29244. virtually identical in mitochondria and bacteria [13,14]. Bacterial E-mail address: [email protected] (P. Greiner). cytochrome c oxidases lack any accessory subunits resembling the

Fig. 1. The catalytic core of cytochrome c oxidase. Subunits I and II contain all essential cofactors for catalytic activity of cytochrome c oxidase. The ﬁrst electron acceptor, the bi- copper CuA-center is located in the peripheral domain of subunit II (red). From there electrons are passed on to the other three redox active cofactors which are located in subunit I

(blue), namely heme a and the binuclear heme a3:CuB center, where the ﬁnal step in electron transfer, the reduction of molecular oxygen to water takes place. In some bacteria, genes coding for subunits II and III are arranged in an operon. In P..denitriﬁcans this cta operon contains additional genes for nonstructural proteins: CtaB is involved in heme a biosynthesis,

CtaG is responsible for CuB insertion and Surf1c is implicated in heme insertion (corresponding yeast nomenclature in parenthesis below). nuclear subunits in mitochondria, and are most probably involved in servation, using their calculated E-values (see legend Table 1). Human the regulation of COX activity [15], which is by far more complex in and P. denitrificans hits are listed in Table 1. eukaryotes than in bacteria. The most highly conserved assembly factors are all involved in the Phylogenetically the α-proteobacteria (e.g. Paracoccus denitrificans, formation or insertion of cofactors in one way or another, except for Oxa1/ Rhodobacter sphaeroides or Bradyrhizobium japonicum) are the closest YidC, which is responsible for the insertion of a large number of integral relatives to mitochondria [1], and are generally regarded as suitable membrane proteins into the lipid bilayer [17].InP. denitrificans we find model systems for studying mitochondrial respiration. While all homologs with a high degree of sequence conservation for this very set of present information suggests highly similar or identical mechanisms assembly factors, emphasizing their importance and suggesting that little of operation for both the mitochondrial and bacterial oxidases, the has changed in the core assembly processes throughout evolution (see latter are far simpler to access experimentally due to their smaller Table 1). Biogenesis in eukaryotes is certainly more complex, but those number of subunits, their genes being manipulated with ease, and the basic mechanisms of oxidase biogenesis delineated from bacteria will fact that strains with otherwise lethal mutations in COX are kept most likely hold true for the human oxidase as well. viable by the presence of alternative terminal oxidases. Interestingly, no human homologs exist in the databases for ten, i.e. almost one third, of the known yeast assembly factors. Four of 1.1. Bacteria contain a distinct subset of COX assembly factors these (Cox14, Mba1, Pet100, Pnt1) are not found in any other eukaryotic organisms other than fungi, and the other six occur very A complex system is required for proper regulation and coordina- rarely, i.e. in less than ten higher eukaryotic organisms (eumetazoa). tion of subunit assembly and cofactor insertion to ensure that no So despite the fact that yeast is a genetically well-exploited eukaryotic reactive assembly intermediates or free redox cofactors cause damage model organism, care must be taken when comparing information to the cell during biogenesis. In fact over 30 proteins that do not end up between yeast and human mitochondria. in the final complex are known to be involved in COX assembly in yeast [for excellent reviews on mitochondrial oxidase assembly see 7,8,9,16]. 1.2. Copper insertion into subunit I may occur co- or posttranslationally A protein blast of the known yeast assembly factors on all non- redundant protein sequences available via NCBI was carried out to In P. denitrificans, three of the most highly conserved assembly obtain an overview on the distribution and the degree of conservation factors – CtaB, CtaG and Surf1c (known as Cox10, Cox11 and Shy1 in of each assembly factor among different organisms. To this end, the yeast) – are part of the cta operon, which also codes for the structural blast results were sorted taxonomically and by their degree of con- subunits II and III of cytochrome c oxidase (see Fig. 1). ΔctaG and 906 P. Greiner et al. / Biochimica et Biophysica Acta 1777 (2008) 904–911

Table 1 Oxidase assembly factors as initially identiﬁed in yeast and their distribution across eukaryotic and prokaryotic organisms

Yeast Humana Paracoccusa Eumetazoab Bacteriab Function Cox15 Cox15 (2·10− 60) CtaA (5·10− 51) ++ +++ Heme a synthase (heme a biosynthesis) − 51 − 32 Cox11 Cox11 (3·10 ) CtaG (7·10 ) ++ +++ Copper chaperone required for copper insertion into subunit I (CuB) − 33 − 24 Sco1/Sco2 Sco1/Sco2 (2·10 ) PrrC, 2× (4·10 ) ++ +++ Sco1: copper chaperone required for copper insertion into subunit II (CuA) Sco1/2: also involved in regulation of copper homeostasis Cox10 Cox10 (3·10− 49) CtaB (4·10− 18) ++ +++ Farnesyl transferase (heme a biosynthesis) Shy1 surfeit 1 (7·10− 20) Surf1, 2× (2·10−10) ++ +++ Part of early assembly intermediates (with Mss51 and Cox14 in yeast) involved in insertion of heme a into subunit I Yah1 ferredoxin 1 (1·10− 17) ferredoxin, 2× (3·10− 08) ++ +++ Ferredoxin (heme a biosynthesis) Oxa1 Oxa1-like (4·10− 35) YidC (3·10− 02) ++ +++ IMM translocase required for membrane insertion of mitochondrial subunits of COX Arh1 AdR (2·10− 57) ++ +++ Ferredoxin reductase (heme a biosynthesis) − 21 Mia40 CHCHD4 (2·10 ) +++ +++ IMS protein, involved in import and assembly of twin Cx9C motif IMS proteins Yfh1 frataxin (6·10− 12) ++ +++ Frataxin, involved in mitochondrial iron homeostasis and the biogenesis of Fe/S-clusters and heme-biosynthesis − 11 Cox19 Cox19 (6·10 ) ++ + IMS twin Cx9C protein, putative metalloprotein (may, like Cox17, function in metal transport to mitochondria) Cox18 Cox18 (2·10−04) ++ ++ IMM translocase (Oxa2) required for membrane insertion of the C-terminal domain of subunit II (interacts with Pnt1 and Mss2) Member of the YidC/Oxa1/Alb3 family − 04 Cox23 CHCHD7 (2·10 ) ++ + IMS twin Cx9C protein, putative metalloprotein (may, like Cox17 and Cox19, function in metal transport to mitochondria) Pet117 CSRP2BP (5·10− 04) ++ + Precise function unknown − 02 Pet191 MGC52110 (3·10 ) ++ + Involved in maintenance of twin Cx9C proteins Cox17, Cox19 and Cox23 in the IMS Cox16 Cox16-like (3·10− 02) ++ + Precise function unknown Pet54 RBM9 (2·10− 01) ++ ++ Translational activator of subunit III mRNA (with Pet122 and Pet494), also required for splicing of subunit I mRNA

Cox17 Cox17 (1.0) ++ − IMS twin Cx9C protein involved in copper delivery to Cox11 and Sco1 Pet309 LRPPRC (4.6) ++ + Stabilizes and activates translation of subunit I mRNA

Cmc1 MGC61571 (6.8) ++ + IMS twin Cx9C protein, precise function unknown Coa1 hCG2036697 (7.1) + + Early step in assembly (interacts with Mss51/Shy1/Cox14) Coa2 −−Precise function unknown Cox14 −−Binds new subunit I and regulates its translation together with Mss51 Cox20 + + Chaperones subunit II through proteolytic processing Mba1 −−Part of an Oxa1-independent translocation machinery (involved in insertion of mitochondrial and nuclear subunits) Mss2 + ++ Facilitates translocation of the C-terminal tail of subunit II Mss51 + + Activates translation and stabilizes subunit I after membrane insertion Pet100 − + Required in a late state of biogenesis, facilitating the assembly of Cox7p, Cox8p and Cox9p into the holoenzyme Pet111 + + Translational activator of subunit II mRNA Pet122 + + Translational activator of subunit III mRNA (with Pet54 and Pet494) Pet494 + ++ Translational activator of subunit III mRNA (with Pet54 and Pet122) Pnt1 − + IMM protein involved in export of proteins form mitochondrial matrix

a values in parenthesis are E-values from local alignments of yeast sequences with human or Paracoccus denitrificans sequences respectively. They were used as a measure for the degree of conservation to sort the blast hits. The lower the E-value, the more significant the score. The E-value or expectation value, is defined as the number of alignments with an equivalent or better score that are expected to occur in a database search by chance. b − no obvious homologs; + less than ten; ++ between 10 and 100; +++ over 100 different organisms identified in the corresponding taxonomic group in a blastp of the yeast sequence on all non-redundant protein sequences in the NCBI database.

Δsurf1c strains have been obtained for P. denitrificans previously, with tribute to proper folding. But at least for the copper ion, a cotransla- results pointing toward a function in cofactor insertion into subunit I tional insertion could also render the metal temporarily solvent for CtaG [18] and heme insertion specifically for Surf1c (Bundschuh, exposed, increasing the risk of reactive oxygen species-production. submitted). Our finding that Surf1c is involved in heme insertion To reduce the toxic effects of solvent-accessible metal ions, an elaborate nicely ties together with previous work in R. sphaeroides [19]. system of metal transporters and shuttle proteins has evolved, with the Concerning CtaG, other groups have shown that it is required for effect that virtually all metal ions in living cells are chelated by proteins

CuB insertion and that it is a copper(I) binding protein [20,21],soitis or peptides [22,23]. One may assume that the cell takes quite some generally considered as the assembly factor that inserts copper into efforts to protect the CuB from becoming solvent-accessible during its the CuB center. However, despite many efforts, a final proof for this insertion. Experimental evidence has been provided for an association immediate function of CtaG is still elusive. A copper transfer from CtaG between the potential copper chaperone for the CuB center, Cox11 to subunit I or a physical interaction between CtaG and an assembly (CtaG in bacteria), and ribosomes in yeast, pointing toward a intermediate remain to be shown. cotranslational copper insertion [24]. But taking a closer look at the The manner of cofactor incorporation into subunit I is one of the structure of cytochrome c oxidase, a channel which gives access to the major issues in research on oxidase biogenesis. Are they inserted co- binuclear heme a3:CuB center can be identified underneath subunit II or posttranslationally, and what is the order of their insertion? (see Fig. 2 and [23]). It is compelling to speculate that CuB is inserted The cofactors of subunit I are buried deep inside the membrane and posttranslationally into the folded, heme containing subunit I through well within the framework of its 12 transmembrane helices, which this channel, and after successful cofactor insertion subunit II docks makes a posttranslational insertion unlikely at first glance. A and occludes the channel, thereby bringing in the last redox center cotranslational insertion instead might stabilize the protein and con- (CuA) and rendering the oxidase active. P. Greiner et al. / Biochimica et Biophysica Acta 1777 (2008) 904–911 907

Fig. 2. A channel in subunit I may allow access to the binuclear center, and is sealed by subunit II in the native complex. Surface representation of the P. denitriﬁcans cytochrome c oxidase (pdb code 1QLE). Subunits I and II are colored in blue and red, respectively. When subunit II is omitted from the representation, a channel becomes visible, granting access to the binuclear heme a3:CuB center (heme a3 in orange).

In order to answer the question whether CtaG/Cox11 inserts copper cavity is also lined by highly conserved hydrophobic amino acids (not in a co- or posttranslational manner we take several approaches: If CuB shown). No experimental hints exist for the function of this conserved is inserted cotranslationally, we should be able to follow the copper cavity, so its function can only be speculated upon. Since the copper insertion in an in vitro assay containing copper-loaded CtaG (Cu-CtaG) binding CFCF motif is flexible such that the copper liganding sulfurs can and ribosomes expressing subunit I. With this intention we have face away from the protein or towards the inner cavity (see Cox11 established a cell-free expression system for subunit I of cytochrome c structure ensemble 1SO9), one may assume that the copper is stored oxidase. inside this hydrophobic cage during transport to shield it from If, on the other hand, copper is inserted in a posttranslational surrounding solvent, thereby protecting the cell from unfavorable manner, we should be able to mimic this reaction in vitro by mixing redox chemistry. This speculation, however, contradicts previously the copper-loaded chaperone (Cu-CtaG) and a cofactor-free or heme a reported EXAFS-data [20,30] that suggest two copper ions liganded at containing subunit I (apo-SU I, heme-SU I). For this purpose we the interface of a CtaG/Cox11 dimer. heterologously expressed and purified CtaG and subunit I, established Some of the highly conserved surface residues identified in this a copper reconstitution procedure for CtaG and successfully looked for work have already been mutated previously [20,29] without knowl- a copper-dependent spectroscopic signal in the copper chaperone. edge of the CtaG structure [30], based only on sequence information. Furthermore we carried out a computational analysis of CtaG and Interestingly almost all mutations involving the residues mentioned subunit I in an attempt to identify conserved residues on the protein above resulted in complete respiratory deficiency (Table 2), confirm- surfaces that might be responsible for a protein–protein interaction. ing the quality and strength of the MultiSeq analysis. We predict that mutations in the other amino acids identified here will have severe 2. Computational analysis of CtaG effects on the function of CtaG as well. A similar analysis was carried out for subunit I of cytochrome c If copper is inserted into subunit I via an access channel (Fig. 2)ina oxidase with a special focus on surface residues in proximity of the posttranslational manner a transient interaction between the protein channel. Even though a number of conserved surface residues were surfaces of CtaG and subunit I is required. In order to identify residues identified, comparison of both surfaces failed to reveal a complemen- that might be responsible for such an interaction we analyzed the tary pattern of charged residues that could mediate an interaction. In known structures of CtaG (1SP0) and cytochrome c oxidase (1QLE) using fact on both protein surfaces there is an excess of negatively charged MultiSeq [25], an extension to the visualization program VMD [26].This residues suggesting that the two proteins actually repel each other. program can be used to sort, group, and align large numbers of protein Surface electrostatics, calculated for subunit I and apo-CtaG using APBS sequences and to visualize the alignment results in the protein [31], confirmed that the two proteins are indeed almost completely structure, highlighting conserved residues. A total of 341 CtaG/Cox11 negatively charged. Some more elaborate calculations that take into sequences was retrieved from a blastp [27] on the entire NCBI database account the influence of copper on the potential interaction should be using default settings (Word Size 3; Expect 10; Blosum62; Gap Costs: applied to the proposed SU I: Cu-CtaG docking, as has been done for existence 11, extension 1), but increasing the number of allowed results other complexes postulated to be involved in copper delivery to cyto- to 1000. Doubles and sequences lacking taxonomic metadata were chrome c oxidase [32]. removed. The remaining 247 CtaG sequences were grouped taxonomi- With the MultiSeq analysis of CtaG we have identified some cally into bacteria and eukaryotes, and the two groups were first aligned interesting candidate residues for mutational analysis, which may well separately, then to each other using ClustalW [28]. Finally the alignment be involved in protein–protein interactions, and we can state that a direct was colored by sequence identity and the coloring was applied to the posttranslational interaction between subunit I and CtaG seems unlikely, structure (Fig. 3). From this protein representation the surface residues due to the number of negatively charged residues on either surface. with the highest degree of conservation could be identified (Table 2 and Fig. 3). In addition to the copper binding CFCF motif five charged (acidic: 3. CtaG shows a copper-dependent spectroscopic signal E91, E134, E141, D152; basic: K126), seven polar (G75, Q82, G109, T133, T167, S169, T171) and nine apolar amino acids (W76, F78, P80, A111, If CtaG inserts copper into subunit I posttranslationally, it should P117, F124, M145, P146, V147) are conserved and surface-exposed (Table be possible to mimic this process in vitro by mixing the copper-loaded 2 and Fig. 3). Most of them are located close to the copper binding chaperone with cofactor-free or heme containing subunit I. An elegant cysteines and may well be involved in dimerization or an interaction experimental setup for this would include the possibility to monitor with a binding partner like subunit I. Interestingly, the protein contains a the kinetics of copper transfer by a spectroscopic feature of CtaG or pocket behind the copper binding CFCF motif [29], and this central subunit I. The copper transfer to Cox11, the yeast homolog of CtaG, has 908 P. Greiner et al. / Biochimica et Biophysica Acta 1777 (2008) 904–911

Fig. 3. Computational structure and sequence analysis of CtaG surface residues. The hydrophilic domain of CtaG/Cox11 (pdb code 1SP0) in surface representations showing (A) the degree of amino acid conservation as determined by a MultiSeq analysis of all available CtaG/Cox11 amino acid sequences. Blue coloring indicates high conservation, whereas residues colored in red are least conserved. (B) The most conserved residues are highlighted by amino acid type; red: acidic, blue: basic, orange: polar, green: nonpolar, copper binding cysteines in yellow. Most of the conserved surface residues are located on the copper binding face of the protein (“front”). All but one of the conserved charged residues are acidic.

been followed by a similar assay, taking advantage of a copper- weak signal centered around 358 nm arises. The broadening of the dependent fluorescence signal of Cox17, another copper chaperone peak at 280 nm has been reported previously for the yeast protein and further upstream in the copper delivery pathway [33]. In order to is most probably due to an overlap of aromatic amino acid absorption establish such an assay for CtaG and subunit I, the purified com- with S-Cu charge transfer bands [20]. But the 358 nm signal, which ponents and a copper-dependent spectroscopic signal of one of the becomes more distinct in the difference spectrum shown in the inset two proteins are required. of Fig. 4a, seems to be a feature that distinguishes the Paracoccus CtaG We expressed and purified two versions of the P. denitrificans CtaG from yeast Cox11. As UV/Vis spectroscopic data of Cu(I)-metallopro- using E. coli as a heterologous host: (a) The full length protein, teins is scarce, no clear statement can be made about the transition comprising a single membrane spanning helix at the N-terminus and that might be responsible for this signal. Spectroscopic changes the C-terminal soluble domain, which is the copper binding domain of arising from additional Cu-S charge transfer bands, internal ligand the protein and (b) a soluble fragment of CtaG consisting only of the transitions (e.g. p → p⁎ transitions) or transitions in the metal orbitals copper binding domain. Both versions were fused to a C-terminal TEV- (e.g. d d-transitions) may be involved. cleavable His6-tag. Even though the transmembrane helix is most If this signal actually depends on copper, it should disappear when probably essential for the recruitment of CtaG to subunit I or a copper is removed from the protein. So to further investigate the metal potential assembly complex and therefore for successful CuB insertion dependence of the 358 nm signal, kinetics after the addition of the in vivo, the soluble fragment contains all residues required for copper metal chelator EDTA were recorded with a 50 μM solution of Cu-CtaG binding, and as it yields far higher protein amounts it was chosen for (Fig. 4b). The addition of EDTA results in an exponential decay of the protein characterization. We further established a copper(I) recon- 358 nm signal on a slow timescale. A stable absorption value, stitution procedure that yields a stoichiometric Cu: protein ratio of ~1 suggesting an equilibrium between Cu·CtaG and Cu·EDTA, is reached (0.95±0.2). UV/Vis spectra of 50 μM apo- and Cu-CtaG solutions were after 45–60 min for EDTA amounts in the stoichiometric range. The recorded and normalized to the absorption at 280 nm. The spectrum decay is stronger when the concentration of EDTA is raised, in line with of Cu-CtaG does in fact diverge from the apoprotein in several distinct a shift in the equilibrium towards Cu·EDTA. As EDTA chelates copper features (Fig. 4a): the main absorption peak around 280 nm is slightly ions in the cupric form whereas CtaG binds the cuprous form, the broadened and blue-shifted upon copper binding and an additional kinetic characterization of the traces in Fig. 4b is not as straightforward P. Greiner et al. / Biochimica et Biophysica Acta 1777 (2008) 904–911 909

Table 2 4. Subunit I is devoid of cofactors when expressed in E. coli Most conserved surface residues on CtaG/Cox11

1SP0a Paracoccusb Yeastc % Known Mutant Reference In addition to purified Cu-CtaG and the spectroscopic signal, we conservationd mutations phenotype also need the purified cofactor-free subunit I (apo-SU I) of cytochrome in yeast c oxidase for the in vitro copper transfer experiment introduced above. E62 E91 E170 80% No method has been described so far for recovering apo-SU I from G80 G109 G188 78% G188D/A235D no growth [29] fully assembled COX without denaturing the protein. In contrast to R. A82 A111 A190 82% Y94 F123 Y202 83% sphaeroides where intermediates of subunit I containing one or more K97 K126 K205 100% K205E no growth [29] cofactors accumulate in deletion strains [34], P. denitrificans subunit I C100 C129 C208 100% C208A no growth [20,29] is either downregulated or rapidly degraded in the absence of subunit C208A/C210A no growth, [20] II [35]. We therefore decided to heterologously express the Paracoccus 0.3 Cu/ subunit I in E. coli. proteine C111A/C208A/ 0.2 Cu/ [20] E. coli seems to be an ideal host for the expression of apo-SU I C210A proteine because it neither contains a cytochrome c oxidase, nor heme a F101 F130 F209 100% F209A no growth [29] synthase or a homolog of CtaG. Its most prominent terminal oxidase is C102 C131 C210 100% C210A no growth [20,29] the bo3-type quinol oxidase, which is, however, structurally homo- C208A/C210A no growth, [20] 0.3 Cu/ logous to the aa3-type cytochrome c oxidase, still raising the proteine possibility of incorrect heme insertion to subunit I by the bo3 C111A/C208A/ 0.2 Cu/ [20] assembly machinery [36]. e C210A protein We successfully determined conditions under which subunit I can F103 F132 F211 100% F211A wt [29] be expressed and purified from E. coli membranes via a TEV-cleavable T104 T133 E212 37% E105 E134 E213 52% G169D/ no growth [29] C-terminal His6-tag. Optimal expression of subunit I was reached E213G/ with the pET-system (pET24a) and over night induction at 16 °C. D245G/ We checked the purified subunit I for heme and copper contents by D265G optical spectroscopy and TXRF (total reflection X-ray fluorescence E112 E141 E220 76% M116 M145 M224 76% M224L wt [20] P117 P146 P225 93% P225R no growth [29] V118 V147 V226 77% V226W no growth [29] D123 D152 D231 91% P46 G75 P154 72% W47 W76 W155 88% F49 F78 F157 97% P51 P80 P159 89% Q53 Q82 Q161 53% P88 P117 P196 99% P196A wt [29] T139 T167 I247 85% S141 S169 H249 82% T143 T171 T251 87% residues in italics are located on the backside of the protein, facing away from the copper binding cysteines. a numbering according to the solution structure (1SP0) of a soluble Cox11 fragment originating from Sinorhizobium meliloti. b Paracoccus denitrificans numbering for CtaG. c Saccharomyces cerevisiae numbering for Cox11p. d percentage of identical amino acids (referring to the S. meliloti sequence, pdb code 1SP0) in all 247 CtaG/Cox11 sequences. e Paracoccus wildtype CtaG binds 0.95±0.2 Cu/protein when reconstituted in vitro.

as it could be using Cu(I) speciﬁc chelators like BCA, BCS or DTT. However, the latter chelators all interfere with the 358 nm signal of CtaG and could therefore not be used to replace EDTA in this assay. The reaction we observe instead should be the result of three connected equilibria:

ð1ÞCuþd CtaG ⇌ Cuþ þ CtaG

ð2ÞCuþ ⇌ Cu2þ þ e−

ð3ÞCu2þ þ EDTA ⇌ Cu2þd EDTA

As the oxygen concentration reaches ~260 μM in air-saturated water (25 °C), not in large excess over Cu(I) (~50 μM in our assay), the Fig. 4. Copper-dependent spectroscopic signature of CtaG. (A) UV/Vis spectra of a heterologously expressed soluble CtaG fragment (100 μM) in the apo- and Cu(I)-form oxidation of Cu(I) to Cu(II) may well be the rate limiting step, thus (black and red, respectively). The difference spectrum (Cu- minus apo-CtaG) is colored explaining why an equilibrium is reached only on such a slow in blue and drawn at a larger scale in the inset. In addition to a broadened and blue- timescale. shifted 280 nm peak, an additional signal centered around 358 nm appears upon copper From the effect of EDTA on the newly observed 358 nm signal we binding. (B) 50 μM Cu-CtaG solutions were incubated with EDTA at various concentrations. 100 μM, 1 mM and 10 mM correspond to a 2-fold, 20-fold and 200- conclude that it reports the copper loading status of CtaG. This fold stoichiometric excess of EDTA. An exponential decay that increases with rising spectroscopic tool may also allow to monitor the copper transfer to EDTA concentrations is recorded at 358 nm. Incubating the apoprotein with the highest subunit I of cytochrome c oxidase in future in vitro experiments. EDTA concentration has no inﬂuence on the 358 nm signal. 910 P. Greiner et al. / Biochimica et Biophysica Acta 1777 (2008) 904–911 spectroscopy) and found that the protein is devoid of cofactors (data not shown). This proves that Paracoccus subunit I is not recognized as a substrate by the E. coli bo3 assembly machinery.

5. Coexpression with CtaG does not yield a copper-containing subunit I in E. coli

As CtaG may act cotranslationally, therefore requiring nascent chains of subunit I for copper insertion, we decided to coexpress the two proteins in E. coli. We devised an E. coli expression plasmid coding for wild type Paracoccus CtaG, i.e. CtaG without a tag but containing its N-terminal transmembrane domain. This plasmid was coexpressed with a second expression plasmid coding for subunit I containing a

TEV-cleavable His6-tag. The isolation of membranes by differential centrifugation and subsequent Western blotting with antibodies against CtaG, subunit I, and the His-tag showed that both proteins were successfully inserted into E. coli membranes. Subunit I was Fig. 5. Cell-free expression of subunit I using E. coli extracts. Western blot after cell-free expressed in the presence or absence of CtaG, purified, and the copper expression of P. denitrificans cytochrome c oxidase subunit I in E. coli extracts. Precipitated content of the two versions determined by TXRF and by a Cu(I) specific protein from a 100 μl reaction was pelleted by centrifugation at 10 000 ×g for 30 min and 10% of the pellet applied to lanes labeled “P”.10% of the supernatant were applied to lanes BCA-assay [37]. The amount of copper bound to subunit I does not labeled “RM” (reaction mixture). In the absence of detergent subunit I is found only in increase when subunit I is expressed in the presence of CtaG in E. coli precipitates. The presence of Triton X-100 or dodecyl maltoside (not shown) during (not shown), indicating that the copper chaperone by itself is unable to expression did not yield subunit I in a “solubilized” form. The only detergents suitable for stably insert copper into subunit I in the heterologous host system. This “solubilizing” subunit I during cell-free expression were members of the Brij family, Brij- μ may have several reasons: (1) CtaG itself may not get copper-loaded in 35 being most effective. 500 g of the 12 transmembrane helix protein (65 kDa, apparent MW: ~50 kDa, see arrow) can be purified from 1 ml of reaction mixture. the heterologous host, making a copper insertion into subunit I impossible. This option can be ruled out though, because a membrane- from P. denitrificans deletion strains during translation, aiming at bound version of CtaG was purified and found to contain copper. (2) accumulating and analyzing assembly intermediates. Another possibility could be an interference of the His-tag on subunit I, In order to gain access to nascent chains of subunit I we adopted but this can also be ruled out because expressing a subunit I containing the E. coli method of cell-free protein synthesis (see below). Future a His -tag at the same position in the native host P. denitrificans results 6 experiments using P. denitrificans extracts should best mimic the in a fully assembled and functional cytochrome c oxidase. (3) The native situation for subunit I biogenesis, and cell-free expression with orientation and/or localization of CtaG and subunit I in the membrane P. denitrificans extracts has been carried out with good results for GFP may differ from the native situation and lead to a failure of CtaG in as a control protein (data not shown). binding nascent subunit I. (4) Alternatively the presence of additional Using E. coli extracts we are able to express the entire 65 kDa, 12- proteins may be required to coordinate the assembly process and to transmembrane helix protein with a C-terminal His -tag (Fig. 5). After recruit individual assembly factors or other subunits during biogen- 6 screening several detergents we have further established conditions esis. (5) Furthermore the presence of heme a may be a prerequisite for under which subunit I is “solubilized” during the translation process copper insertion, or the absence of heme a may render the Cu center B (Fig. 5). This brings about the possibility for subunit I to acquire the unstable such that copper is lost during the purification procedure. (6) heme cofactor present in solution while emerging from the ribosome. Finally CtaG might not be the immediate copper insertion factor for The use of Brij-35 (40×CMC) as solubilizing agent yields highest subunit I after all, a situation that cannot be fully ruled out yet. amounts of “soluble” subunit I: 500 μg of the apoprotein can be In order to address these questions heme a needs to be included in purified from 1 ml of reaction mixture. the model system. At present we are devising an operon coding for the The question of co- or posttranslational cofactor insertion to P. denitrificans heme a biosynthesis proteins CtaB and CtaA and the cytochrome c oxidase and the immediate function of CtaG in the copper copper chaperone CtaG which will allow the expression of subunit I in delivery to the Cu center remain to be clarified, but experimental setups the presence of heme a and the copper chaperone. B to address these questions have been established and promise to shed In an alternative approach we decided to express subunit I in a cell- light on this aspect of COX assembly in the near future. free expression system, allowing the addition of free hemes and purified assembly factors and raising the possibility to trap assembly Acknowledgements intermediates like a potential cotranslational complex between CtaG and subunit I. The Collaborative Research Center “Molecular Bioenergetics” (SFB 472), the Center for Membrane Proteomics (CMP) and the Cluster of 6. Cell-free expression provides access to nascent chains of subunit I Excellence Frankfurt (Macromolecular Complexes, DFG Project EXC 115) are acknowledged for financial support. We thank F. Bernhard, C. The expression of membrane proteins in cell-free extracts is an Klammt and D. Schwarz from the Institute of Biophysical Chemistry emerging technique preferentially used for specific labeling purposes. (V. Dötsch group) for providing strains and valuable information High protein amounts of up to 6 mg/ml for integral membrane proteins concerning the cell-free expression system. We would further like to can be expressed in small volumes (1 ml of reaction mixture) [38,39]. thank H.-W. Müller for excellent technical assistance. Toxic effects that often arise with the heterologous overexpression of membrane proteins are decreased. 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