How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation^,

Background: α-Proteobacteria, extant relatives of mitochondria, are model organisms for studying assembly of bacterial and mitochondrial metalloenzymes.

Results: Periplasmic thioredoxin TlpA is a specific reductant for copper chaperone ScoI and cytochrome oxidase subunit II (CoxB).

Conclusion: Cysteines in the copper-binding sites of ScoI and CoxB must be reduced prior to metallation.

Significance: Structures of TlpA-ScoI and TlpA-CoxB intermediates reveal mechanistic details of the reduction process.

Abstract

Two critical cysteine residues in the copper-A site (Cu_A) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu²⁺ transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E′₀ = −231 mV) as compared with that of TlpA (E′₀ = −256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10⁴ m⁻¹ s⁻¹ that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu_A-CoxB complex may be bypassed in vivo by high environmental Cu²⁺ concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.

Introduction

This study addresses a hitherto unresolved issue in the biogenesis of the aa₃-type cytochrome c oxidase (COX),² a multimeric integral membrane complex that performs the ultimate step in cellular respiration in many aerobic bacteria and in mitochondria (1–4). Being a heme- and copper-containing enzyme complex, the question of how its subunits plus heme and copper cofactors are assembled in the membrane has been answered only partially. Particularly intricate is the biogenesis of the membrane-anchored subunit II (CoxB) because it carries a binuclear Cu-Cu center (Cu_A) on a domain that faces the periplasmic side of the cytoplasmic membrane in Gram-negative bacteria or the intermembrane space in mitochondria (1, 2). Copper ions must therefore be delivered to these compartments for Cu_A assembly. The Cu_A center is the entry point of electrons coming from reduced cytochrome c, which are then guided to the membrane-integral subunit I via the low-spin heme A to the high-spin heme A₃-Cu_B center, the site of oxygen reduction (4, 5). The two copper ions in the oxidized form of the Cu_A center have an overall valence of +3 (+1.5/copper ion), and cytochrome c reduces them to +2 (+1/copper ion) (6). Subunit II possesses a highly conserved amino acid sequence motif for copper binding (HX₃₄CXEXCX₃HXXM) where the six ligands (underlined) are provided by the sulfur atoms of the methionine and the two cysteines, one imidazole nitrogen atom each of the two histidines, and the peptide bond carbonyl oxygen from the glutamate (1, 2).

When the Cu_A-binding domain of subunit II is secreted from the cytoplasm to the periplasm in the course of bacterial protein synthesis and membrane insertion, the two neighboring cysteine thiols (CX₃C) in the aforementioned motif are prone to oxidation because the periplasm is an oxidizing environment (7, 8). Regardless of whether intramolecular disulfide bond formation occurs by air oxidation or is catalyzed by a DsbA-like dithiol oxidase (9, 10), such a constellation precludes complexation with copper ions. Enzyme-catalyzed re-reduction of such a disulfide bond (an off-pathway intermediate to Cu_A assembly) to the dithiol form would thus appear to be necessary for copper ion binding to subunit II. A strong candidate for a periplasmic CoxB reductase is the thioredoxin-like protein TlpA (11, 12), which has been discovered first in Bradyrhizobium japonicum, a member of the α-proteobacteria and one of the model organisms for studying bacterial and mitochondrial hemo- and metalloproteins (13–15). TlpA has a negative redox potential (E′₀ = −256 mV), which predicts that it is a reductant in vivo (16, 17). The protein is anchored to the cytoplasmic membrane, with its thioredoxin domain facing the periplasm and, hence, ideally positioned to reduce membrane-associated substrates in this compartment. A tlpA knock-out mutant of B. japonicum is defective for cytochrome oxidase activity (11). In this work, we provide compelling functional and structural evidence that the oxidized CoxB protein is a substrate of the disulfide reductase TlpA and that CoxB reduction is an essential step in the biogenesis of active COX.

Incidentally, a CX₃C site such as in CoxB is also present in the B. japonicum ScoI protein (14), which is a homolog of the mitochondrial copper chaperone Sco1 (18, 19). The Cu²⁺-binding domain of the membrane-anchored ScoI and Sco1 proteins faces the periplasm and intermembrane space, respectively, akin to CoxB. We had shown recently that TlpA serves as a reductant for ScoI (20). With few exceptions, the biogenesis of the Cu_A center in eukaryotic mitochondria and in prokaryotes depends on the presence of Sco1/ScoI (18, 19). One could argue, therefore, that the cytochrome oxidase deficiency in B. japonicum tlpA mutant cells is simply the consequence of a defect in forming a sufficient amount of intracellular Cu-ScoI complex for the synthesis of Cu_A on CoxB. As shown here, however, this explanation alone does not hold true because not only ScoI but also CoxB are direct substrates of TlpA. Furthermore, the requirement of ScoI for cytochrome oxidase activity, but not that of TlpA, can be bypassed by supplying high environmental copper concentrations. This strongly suggests that Cu_A can be assembled on CoxB only after the latter had been reduced by TlpA.

EXPERIMENTAL PROCEDURES

Whole-cell Cytochrome Oxidase Test

B. japonicum 110spc4 (WT (21) and mutants ΔtlpA (Bj3556 (17)), ΔscoI (Bj2575 (14)), coxA::Tn5 (COX132 (22)), and ΔcoxB (Bj3563 (14)) were grown aerobically at 30 °C in PSY medium (21) containing either 100 μg/ml spectinomycin (for wild type) or 100 μg/ml spectinomycin plus 100 μg/ml kanamycin (all other strains). To test for possible complementation of cytochrome oxidase deficiency, the medium was supplemented with 50 μm CuCl₂. Cells from 2-ml culture volume were harvested by centrifugation and suspended in PSY to a final A₆₀₀ of 25. Samples (5 μl) of these suspensions were spotted onto a filter paper soaked with saturated TMPD solution. Formation of indophenol blue upon COX-catalyzed air oxidation of TMPD was recorded after 5-min incubation.

Expression Plasmids

The bacterial expression plasmids for production of CoxB_PD (residues 128–265) and TlpA_S (TlpA residues 38–221) and variants thereof were constructed using standard molecular cloning techniques. Detailed information on the individual plasmids including the coding nucleotide and the corresponding protein sequences is available from the authors on request. For production of ScoI_S^C74S, the previously described plasmid pRJ8336 (20) was used.

Protein Production and Purification

For production of CoxB_PD and its variants CoxB_PD^C229S and CoxB_PD^C233S in cytoplasmic inclusion bodies, Escherichia coli strain BL21(DE3) transformed with pET11a-coxB_PD, pET11a-coxB_PD^C229S, or pET11a-coxB_PD^C233S, respectively, was used. The purification procedure for CoxB_PD, CoxB_PD^C229S, and CoxB_PD^C233S was identical. E. coli BL21(DE3) transformed with pET11a-coxB_PD was grown at 37 °C in 2YT medium (Tryptone, 16 g/liter; yeast extract, 10 g/liter; NaCl, 5 g/liter) containing ampicillin (100 μg/ml) until an A_{600 nm} of 0.5 had been reached. CoxB_PD expression was induced by the addition of 0.1 mm isopropyl-β-d-1-thiogalactopyranoside, and cells were further grown at 30 °C for 4 h. Cells were harvested by centrifugation, suspended in 100 mm Tris-HCl, pH 8.0, 1 mm EDTA (3 ml/g wet cells), mixed with DNase I (50 μg/ml final concentration), and lysed with a Microfluidizer M-110L (Microfluidics, Westwood, MA). After the addition of 0.5 volumes of 60 mm EDTA-NaOH, pH 7.0, 1.5 m NaCl, 6% (v/v) Triton X-100, the lysate was stirred at 4 °C for 1 h. The inclusion bodies were harvested by centrifugation (30 min, 48,000 × g, 4 °C) and washed five times at 4 °C with 100 mm Tris-HCl, pH 8.0, 20 mm EDTA to remove the Triton X-100. The inclusion bodies were solubilized in 100 mm Tris-HCl, pH 8.0, 6 m guanidinium chloride, 1 mm EDTA, 100 mm DTT (20 ml/gram of inclusion body) at room temperature under stirring for 2 h. Insoluble material was removed by centrifugation, and CoxB_PD was refolded from the supernatant by rapid dilution with 50 volumes of 20 mm Tris-HCl, 0.5 m arginine-HCl, 1 mm EDTA, 10 mm DTT (pH 8.0) at room temperature. Refolded, reduced CoxB_PD was concentrated to ∼1.5 mg/ml and dialyzed against 20 mm Tris-HCl, 1 mm EDTA, 5 mm DTT, pH 8.5 (4 °C). Precipitated protein was removed by centrifugation, and the supernatant was applied at 4 °C to a Resource Q column (GE Healthcare, Glattbrugg, Switzerland) equilibrated with the same buffer. The flow-through, containing reduced CoxB_PD, was concentrated and subjected to gel filtration on Superdex 75 (GE Healthcare) in 20 mm Tris-HCl, pH 8.5, 0.3 m NaCl, 0.5 mm EDTA, 5 mm DTT at 4 °C. The fractions containing pure, reduced CoxB_PD (as judged after Coomassie Blue-stained SDS-PAGE) were stored at −20 °C until further use. The final yield of purified, reduced CoxB_PD was 60 mg/liter of bacterial culture. The identity of the protein (including the N-terminal methionine) was verified by ESI mass spectrometry (calculated mass: 15,578.0 Da; found: 15,578.0 Da).

For the use of CoxB_PD^C233S in crystallization trials, a different expression construct was made that carried an N-terminal His₆ tag. E. coli strain BL21(DE3) harboring pProExHTa-His₆-coxB_PD^C233S was grown and induced with isopropyl-β-d-1-thiogalactopyranoside as described above for CoxB_PD. In addition to the C233S replacement, CoxB_PD^C233S differs from CoxB_PD by the sequence GAFLELD at the N terminus (replacing the N-terminal Met¹²⁷ in CoxB_PD) and a 14-residue longer C-terminal end corresponding to the end of the natural CoxB reading frame. Harvested cells were suspended in 20 mm Tris-HCl, pH 8.0, 1 mm β-mercaptoethanol (1.5 ml/g of wet cells). One cOmplete^TM protease inhibitor tablet (Roche Applied Science, Basel, Switzerland) per 50-ml suspension and DNase I (50 μg/ml final concentration) were added, and cells were disrupted with a Microfluidizer. The lysate was centrifuged, and the pellet was mixed with 2 volumes of buffer A (20 mm Tris-HCl, 0.5 m NaCl, 6 m guanidinium chloride, 2 mm β-mercaptoethanol, pH 8.0) and stirred at 4 °C for 1.5 h. Insoluble material was removed by centrifugation, the supernatant was mixed with imidazole/HCl, pH 8 (final concentration: 10 mm) and loaded onto nickel-nitrilotriacetate-agarose (Qiagen, Switzerland) equilibrated with buffer A. The resin was washed extensively with buffer A containing 20 mm imidazole/HCl. His₆-tagged CoxB_PD^C233S was eluted with buffer A containing 200 mm imidazole/HCl, and protein fractions were refolded by dilution with 100 volumes of 20 mm Tris-HCl, 0.5 m NaCl, 0.5 m arginine-HCl, 5 mm β-mercaptoethanol, pH 8.0, and incubation for 1 h at room temperature. Refolded, His₆-tagged CoxB_PD^C233S was concentrated to 1.5 mg/ml (96 μm), and aggregates were removed by centrifugation. The N-terminal His₆ tag was then cleaved off by incubation (18 h, 4 °C) with TEV protease (7 μm) in 20 mm Tris-HCl, 0.3 m NaCl, 5 mm β-mercaptoethanol, pH 8.5. The uncleaved protein and the cleaved tag were removed by binding to a nickel-nitrilotriacetate column as described above. The cleaved protein in the flow-through was concentrated to ∼1.5 mg/ml and subjected to gel filtration on Superdex 75. The final yield of purified CoxB^C233Swas 4.5 mg/liter of bacterial culture, and its mass was verified by ESI mass spectrometry (calculated mass: 17,396.9 Da; found: 17,397.0 Da).

TlpA_S, used for the disulfide exchange equilibrium with CoxB_PD, and TlpA_S^C110S, used for crystallization, were produced as fusions to the C terminus of maltose-binding protein (MalE) and purified after factor Xa cleavage from MalE as described previously (16, 20, 23). Alternatively, an analogous expression plasmid (pMal-p/TEV-TlpA_S) encoding the same fusion with a TEV protease instead of a factor Xa cleavage site was used for production of TlpA_S for stopped-flow kinetics. The corresponding fusion protein was digested with TEV protease as described above for CoxB_PD^C233S, and TlpA_S was further purified by anion exchange chromatography as described (23). TlpA_S purified with this alternative method bears two additional glycines at its N terminus, which proved to be necessary for efficient cleavage (calculated mass: 19,634.7 Da; found: 19,632.5 Da). ScoI_S^C74S was produced and purified as reported recently (20). TEV protease was purified as described (24).

Protein Concentrations

Protein concentrations were determined via the specific protein absorbance at 280 nm (CoxB_PD, 28,100 m⁻¹ cm⁻¹; His₆-CoxB^C233S, 35,410 m⁻¹ cm⁻¹; CoxB_PD^C233S, 29,450 m⁻¹ cm⁻¹; reduced TlpA_S and TlpA_S^C110S, 17,300 m⁻¹ cm⁻¹; ScoI_S^C74S, 16,100 m⁻¹ cm⁻¹; TlpA_S^C110S-ScoI_S^C74S, 34,170 m⁻¹ cm⁻¹; TlpA_S^C110S-CoxB_PD^C233S, 47,700 m⁻¹ cm⁻¹; TEV protease, 32,200 m⁻¹ cm⁻¹).

Disulfide-exchange Equilibrium between TlpA and CoxB

CoxB_{PD ox} and TlpA_{S red} were mixed at three different molar ratios (initial concentrations in μm: 10:5, 5:5, and 5:10, respectively) and incubated for 18 h at 25 °C in 50 mm NaH₂PO₄-NaOH (sodium phosphate), 0.1 mm EDTA, pH 7.0. The equilibria were quenched with formic acid (10% v/v final concentration), and all four redox species (CoxB_{PD ox}, CoxB_{PD red}, TlpA_{S ox}, and TlpA_{S red}) were separated by reversed-phase HPLC on a Zorbax C8 SB-300 column (Agilent Technologies, Basel, Switzerland) at 60 °C using an acetonitrile gradient (35–45%) in 0.1% TFA. Eluted proteins were detected at 220 nm, and their concentration was quantified by peak integration. The equilibrium constant (K_eq) and the redox potential of CoxB_PD were calculated according to Equation 1 and the Nernst equation (Equation 2), respectively, using a value of −256 mV for the redox potential (E′₀) of TlpA_S (20).

TlpA_{S red} and CoxB_{PD red} were obtained after reduction with excess DTT (10 mm for CoxB_PD and 5 mm for TlpA_S) for ≥30 min at room temperature in 20 mm Tris-HCl, 0.3 m NaCl, 0.5 mm EDTA, pH 8 (CoxB_PD) or 10 mm Tris-HCl, pH 8 (TlpA_S) followed by centrifugation of CoxB_PD at 100,000 × g for 30 min at 4 °C and removal of excess DTT from both proteins by gel filtration on Hi-Prep 26/10 Sephadex G25 columns (GE Healthcare). CoxB_{PD ox} was obtained after incubation of CoxB_{PD red} (10–20 μm) with a 1.5-fold molar excess of DTNB in 50 mm sodium phosphate, 0.1 mm EDTA, pH 7, at room temperature for 20 min followed by gel filtration on a Hi-Prep 26/10 column as described above in 50 mm sodium phosphate, 0.1 mm EDTA, pH 7. Complete oxidation of CoxB_{PD red} by DTNB was verified by reversed-phase HPLC (see above).

Stopped-flow Fluorescence Kinetics of Disulfide Exchange between TlpA and CoxB

The kinetics of the reduction of CoxB_{PD ox} by TlpA_{S red} were recorded at 25 °C in 50 mm sodium phosphate, 0.1 mm EDTA, pH 7. The reactions were initiated with stopped-flow mixing (1:1) in a SX20 stopped-flow instrument (Applied Photophysics, Leatherhead, UK) and followed via the decrease in tryptophan fluorescence of TlpA_S at 320 nm upon oxidation (excitation at 280 nm) (20). A constant, initial TlpA_{S red} concentration of 1 μm was used, whereas CoxB_{PD ox} was used in excess, with initial concentrations of 5, 10, or 15 μm. The kinetic profiles were fitted globally with Berkeley Madonna^TM (version 8.3.18), with a fixed value of 7.1 for the ratio between the forward (k₂) and reverse (k₋₂) reactions based on the determined equilibrium constant (K_eq = k₂/k₋₂).

CD Spectra and Thermal Unfolding Transitions of CoxB_PD

CD spectra of CoxB_PD (0.2 mg/ml) in 10 mm sodium phosphate, pH 7.0, were recorded at 25 °C with a J-715 spectropolarimeter (JASCO, Easton MD; 0.1-cm path length). Thermal unfolding transitions of oxidized and reduced CoxB_PD in 10 mm sodium phosphate, pH 7.0 (supplemented with 5 mm DTT in the case of reduced CoxB_PD) were followed via the increase in the far-UV CD signal at 218 nm upon unfolding (heating rate: 1 K/min) and fitted and normalized according to a reversible thermal unfolding equilibrium (Equation 3)

where S represents the observed CD signal at 218 nm, y_f and y_u are the y axis intercepts at zero K, and m_f and m_u are the slopes of the pre- and post-transition baselines, respectively, T is the temperature, T_m is the melting temperature, and ΔH_m is the enthalpy change of unfolding at T_m.

In Vitro Reconstitution of the Cu_A Center in CoxB_PD

The Cu_A center in CoxB_PD was reconstituted via the reaction scheme deduced from the recently described Cu_A reconstitution in subunit II of the ba₃-type cytochrome oxidase from Thermus thermophilus (6).

Reduced CoxB_PD (50 μm) was incubated with CuCl₂ (500 μm) in 50 mm Bis-Tris-HCl, pH 7.0, overnight at room temperature. Excess CuCl₂ was removed by gel filtration in 50 mm Bis-Tris-HCl, pH 7.0, the solution was concentrated to ∼150 μm protein, and precipitated material was removed by ultracentrifugation. The measured absorbance spectrum with its maxima at 367, 479, and 813 nm confirmed the formation of the Cu_A center.

Preparation and Crystallization of the TlpA-ScoI Mixed Disulfide Complex

Purified TlpA_S^C110S and ScoI_S^C74S were incubated with excess DTT (2 mm) in 10 mm Tris-HCl, pH 8 (TlpA_S^C110S) or 50 mm succinic acid-NaOH, 0.2 mm EDTA, 0.1 m NaCl, 1 mm DTT, pH 5.3 (ScoI_S^C74S) at room temperature for 30 min followed by the removal of DTT by gel filtration on Sephadex G-25 (GE Healthcare) in 10 mm Tris-HCl, pH 8. TlpA_S^C110S (40 μm) was activated by incubation with a 20-fold molar excess of DTNB in 10 mm Tris-HCl, pH 8, for 20 min. The resulting TlpA_S^C110S-TNB derivative was again purified by gel filtration and incubated with an equimolar amount (50 μm each) of ScoI_S^C74S overnight at room temperature. After gel filtration on HiLoad Superdex 75 16/60 column (GE Healthcare) in 20 mm Tris-HCl, 0.3 m NaCl, pH 8.0 (at 4 °C), fractions containing the pure TlpA_S^C110S-ScoI_S^C74S mixed disulfide complex were buffer-exchanged to 10 mm Tris-HCl, pH 8, and concentrated to 10 mg/ml. Fragile crystals in the form of clustering platelets were grown with the sitting-drop vapor diffusion technique by mixing 0.75 μl of the TlpA-ScoI complex (10 mg/ml in 10 mm Tris-HCl, pH 8) with 0.75 μl of precipitant solution (final concentration: 3% glycerol, 0.2 m NaSCN, and 16% (w/v) PEG 3350) and equilibration over 100 μl of well solution (3% glycerol, 0.2 m NaSCN, 28% (w/v) PEG 3350) at 4 °C. Crystals were cryo-protected in precipitant solution containing 25% ethylene glycol and flash-frozen in liquid nitrogen.

Preparation and Crystallization of the TlpA-CoxB Mixed Disulfide Complex

The TlpA_S^C110S-CoxB_PD^C233S complex was generated as described above for TlpA_S^C110S-ScoI_S^C74S. The final reaction that led to the mixed disulfide complex was performed with equimolar concentrations (15 μm each) of CoxB_PD^C233S and TlpA_S^C110S-TNB in 10 mm Tris-HCl, pH 8 (1 h at room temperature). The TlpA_S^C110S-CoxB_PD^C233S complex was purified by gel filtration on HiLoad Superdex 75 16/60 as described above and finally concentrated to 12 mg/ml in 10 mm Tris-HCl, pH 8.0. ESI mass spectrometry and SDS-PAGE analysis revealed that seven C-terminal residues in CoxB_PD^C233S (residues 273–279) had been cleaved off by an unknown protease (calculated mass of the cleaved mixed disulfide complex: 36,284.5 Da; found: 36,287.5 Da). The TlpA_S^C110S-CoxB_PD^C233S stock solution (3 μl) was mixed with 1 μl of precipitant solution (25% (w/v) PEG 2000 monomethyl ether, 0.8 m formic acid-NaOH, 0.1 m sodium cacodylate, pH 6.5). Rod-like crystals grew at 4 °C within 13 weeks (sitting-drop vapor diffusion). Crystals were cryo-protected in precipitant solution containing 15% glycerol and flash-frozen in liquid nitrogen.

X-ray Crystallography and Structure Determination

The crystal structure of the TlpA_S^C110S-ScoI_S^C74S mixed disulfide was solved by molecular replacement with PHASER (25), using the crystal structure of the soluble domain of B. japonicum TlpA (Protein Data Bank (PDB) entry 1jfu) and the crystal structure of Bacillus subtilis Sco1 (PDB entry 1xzo) as search models. The asymmetric unit of the TlpA-ScoI crystal structure contains four complexes, where chains A and B form complex 1, chains C and D form complex 2, chains E and F form complex 3, and chains G and H form complex 4. For the analysis of the structure, mostly complex 2 (chains C+D) was employed. The structure of the TlpA_S^C110S-CoxB_PD^C233S complex was also solved by molecular replacement with PHASER (25), using the structures of B. japonicum TlpA (PDB entry 1jfu) and subunit II of cytochrome c oxidase from Rhodobacter sphaeroides (PDB entry 1m56), in which the N-terminal membrane helices (residues 1–130), residues 151–191, the C-terminal helix (residues 270–289), and the loop (residues 250–261) containing the two copper-binding cysteines are absent. PHENIX (26) and Refmac (27) were used for refinement and model validation. COOT (28) was used for manual model building. Secondary structure elements were assigned with DSSP (29). T-Coffee (30) was used for multiple sequence alignments. The Pdb2pqr web server (31) and APBS (32) were employed for analysis of electrostatics, PyMOL (The PyMOL Molecular Graphics System, Version 1.6.0.0, Schrödinger, LLC) was employed for generation of structural figures, and ALINE (33) was employed for producing figures of sequence alignments. Interface analysis of the structures was performed with EPPIC (34). PISA (35) was used to localize hydrogen bonds and salt bridges in the dimer interfaces.

RESULTS

Excess Environmental Cu²⁺ Rescues Cytochrome Oxidase Activity in a scoI but Not in a tlpA Deletion Mutant of B. japonicum

B. japonicum scoI and tlpA mutants share similar phenotypes including a defect in COX activity; nevertheless, respiration in these strains is possible because of the presence of quinol oxidases (14). We noticed previously that excess Cu²⁺ ions in growth media can compensate for the requirement of the Cu²⁺-specific copper chaperone ScoI in forming active COX (14). The precise mechanistic reason for this bypass is not known. Surprisingly, we now observed that a tlpA mutant cannot be compensated in a similar way by high external copper supplementation (Fig. 1). Therefore, TlpA in cells ought to have at least a second function apart from serving as a reductant to the CX₃C motif in ScoI (20). This result prompted us to inquire into the possibility that TlpA interacts with CoxB because this COX subunit also carries a CX₃C motif as part of its Cu_A-binding site.

FIGURE 1.

Whole-cell cytochrome oxidase activity is rescued in B. japonicum scoI but not tlpA mutant by excess Cu²⁺ in the growth medium. The respective B. japonicum mutants were grown aerobically in PSY medium with or without 50 μm CuCl₂ and spotted onto a filter paper soaked with the redox indicator TMPD. Active COX in cells such as the wild type (WT) converts TMPD to indophenol blue. Mutants of subunits I (coxA) and II (coxB) of COX served as negative controls. − Cu, without copper supplement; + Cu, with copper supplement.

TlpA Is a Specific Reductant of CoxB

The possible target for TlpA-catalyzed thiol-disulfide exchange is the cysteine pair Cys²²⁹/Cys²³³ in the periplasmic domain of CoxB. This domain (CoxB_PD) was expressed in Escherichia coli in the form of cytoplasmic inclusion bodies and purified after in vitro refolding under reducing conditions (Fig. 2). The dithiol of Cys²²⁹/Cys²³³ in CoxB_PD could be quantitatively converted to the disulfide form by oxidation with DTNB. Both reduced and oxidized CoxB_PD showed almost identical, cooperative thermal unfolding transitions, arguing for an intact tertiary structure (Fig. 2, B and C). This was further confirmed by the successful in vitro reconstitution of the Cu_A center in reduced CoxB_PD by the addition of Cu²⁺ ions (Fig. 2D) (6). Oxidized CoxB_PD failed to assemble the Cu_A center.

FIGURE 2.

Characterization of CoxB_PD, the recombinant, periplasmic domain of CoxB (residues 128–265) from B. japonicum. A, ESI mass spectrum of purified, reduced CoxB_PD (calculated mass: 15,578.0 Da; found: 15,578.0 Da). Inset: Coomassie Blue-stained SDS-polyacrylamide gel of purified CoxB_PD (B-PD); St.: molecular mass standard. a. u., arbitrary units. B, far-UV CD spectra of oxidized (blue) and reduced (red) CoxB_PD at pH 7.0 and 25 °C. C, both oxidized and reduced CoxB_PD show cooperative thermal unfolding transitions, demonstrating the presence of intact tertiary structure. Thermal unfolding transitions of oxidized (blue) and reduced (red) CoxB_PD in 10 mm NaH₂PO₄-NaOH, pH 7.0, at a heating rate of 1 K/min were followed via the increase in the far-UV CD signal at 218 nm upon unfolding, tentatively fitted according to a reversible thermal unfolding equilibrium (solid lines), and normalized. Apparent melting temperatures of 317.5 ± 0.2 and 318.5 ± 0.2 K were obtained for CoxB_{PD ox} and CoxB_{PD red}, respectively. D, UV/VIS spectrum of Cu_A-CoxB_PD after in vitro reconstitution of the Cu_A center, showing Cu_A-specific absorbance maxima at 367, 479, and 813 nm.

Assuming a function of TlpA as a CoxB reductase, we predicted (i) that the cysteine pair Cys²²⁹/Cys²³³ in CoxB_PD is less reducing than the active-site cysteine pair of TlpA (Cys¹⁰⁷/Cys¹¹⁰), and (ii) that reduction of CoxB by TlpA would have to proceed fast, i.e. with a rate constant above the threshold (∼10³ m⁻¹ s⁻¹) for physiological disulfide-exchange reactions between thioredoxin-like oxidoreductases and their substrates (36). We first determined the equilibrium constant (K_eq) of the disulfide-exchange reaction at pH 7.0 and 25 °C between CoxB_PD and the well characterized, soluble TlpA variant TlpA_S, which lacks the N-terminal membrane anchor (12). Reduced TlpA_S was mixed with CoxB_{PD ox} at different ratios, and the reaction products were separated and quantified by reversed-phase HPLC after acid quenching (Fig. 3A). The equilibrium constants, deduced from the individual HPLC runs after peak integration, proved to be independent of the initial CoxB_PD:TlpA_S ratio within experimental error, demonstrating that equilibria had been attained. The deduced K_eq value of 7.1 ± 0.6 translates into a redox potential (E′₀) of −231 ± 1 mV for CoxB_PD. This shows that reduction of CoxB_PD by TlpA_S (E′₀ = −256 mV) is thermodynamically favorable. The equilibrium is, however, far less on the side of oxidized TlpA_S as compared with the reduction of ScoI_S (residues 30–196) by TlpA_S, which has an equilibrium constant of 1740 (20).

FIGURE 3.

Thermodynamics and kinetics of the disulfide-exchange equilibrium between TlpA and CoxB at pH 7.0 and 25 °C. A, determination of the equilibrium constant (K_eq) of the reduction of CoxB by TlpA. Reduced TlpA (TlpA_{S red}) and oxidized CoxB (CoxB_{PD ox}) were mixed at initial concentrations (in μm) of 5 and 10, 5 and 5, or 10 and 5, respectively, and incubated for 18 h. The reactions were quenched with formic acid. All four redox species were separated by reversed-phase HPLC, detected via absorbance at 220 nm, and quantified by peak integration. The equilibrium constants deduced from the individual HPLC runs proved to be identical within experimental error, demonstrating that the disulfide-exchange equilibrium had been attained. AU, absorbance unit. B, stopped-flow fluorescence kinetics of the reduction of CoxB by TlpA. TlpA_{S red} (1 μm) was mixed with a 5-, 10-, or 15-fold excess of CoxB_{PD ox}, and the reaction was followed via the decrease in tryptophan fluorescence upon oxidation of TlpA_S. Data were fitted globally (solid lines) with Berkeley Madonna^TM according to the indicated disulfide-exchange equilibrium, with a fixed ratio of 7.1 between the second-order rate constants of the forward and reverse reactions (k₂ and k₋₂, respectively), and normalized. The estimated error of the rate constants is about 20%.

Next, the kinetics of the attainment of the disulfide-exchange equilibrium between CoxB_PD and TlpA_S were followed after stopped-flow mixing of reduced TlpA_S with a 5-, 10-, or 15-fold excess of oxidized CoxB_PD, where we made use of the strong decrease in the tryptophan fluorescence of TlpA_S upon oxidation (16, 20). The obtained rate constants of 8.4 × 10⁴ and 1.2 × 10⁴ m⁻¹ s⁻¹ for the forward and reverse reaction, respectively, demonstrate rapid disulfide exchange between both proteins (Fig. 3B). The rate constant of CoxB_PD reduction is thus very similar to that of the reduction of ScoI_S by TlpA_S (9.4 × 10⁴ m⁻¹ s⁻¹) (20) and a strong hint that both CoxB and ScoI are physiological substrates of the periplasmic reductase TlpA.

Trapping and Crystallizing the TlpA-ScoI and TlpA-CoxB Heterodisulfides

Disulfide exchange between thioredoxins and their substrates occurs via mixed disulfide intermediates, in which the more N-terminal, active-site cysteine of a thioredoxin forms the intermolecular disulfide with the substrate protein (37). In accordance with this reaction scheme, the active-site thiol of Cys¹⁰⁷ in TlpA forms a transient mixed disulfide with Cys⁷⁸ of ScoI (20). A mixed disulfide is kinetically unstable because it is rapidly attacked by a neighboring cysteine thiol. Therefore, a stable mixed disulfide between TlpA_S and ScoI_S was prepared by using the single-cysteine variants TlpA_S^C110S and ScoI_S^C74S. Activation of TlpA_S^C110S with DTNB and subsequent reaction with ScoI_S^C74S yielded the TlpA_S^C110S-ScoI_S^C74S complex.

To determine the cysteine residue in CoxB_PD, which forms the mixed disulfide intermediate with TlpA, the single-cysteine variants CoxB_PD^C229S and CoxB_PD^C233S were tested for their reactivity with DTNB-activated TlpA_S^C110S in a similar way as described previously with ScoI (20). The result was that predominantly Cys²²⁹ of CoxB reacted with Cys¹⁰⁷ of TlpA. Based on this finding, a stable TlpA_S^C110S-CoxB_PD^C233S mixed disulfide complex was prepared as described above for the TlpA_S^C110S-ScoI_S^C74S complex. The two purified mixed disulfide complexes were crystallized, and the x-ray structures of TlpA_S^C110S-ScoI_S^C74S and TlpA_S^C110S-CoxB_PD^C233S were solved at 2.2 and 2.0 Å resolution, respectively (Fig. 4; Table 1).

FIGURE 4.

X-ray structures of TlpA in complex with ScoI or CoxB. A and B, the two mixed disulfides consisting of TlpA_S^C110S (gray) (A) and ScoI_S^C74S (orange) or TlpA_S^C110S and CoxB_PD^C233S (cyan) (B) are shown in the same orientation with respect to TlpA_S^C110S, highlighting the existence of two distinct binding interfaces in TlpA for the two substrates. The cysteines involved in formation of the intermolecular disulfides (Cys¹⁰⁷ of TlpA_S^C110S, Cys⁷⁸ of ScoI_S^C74S, and Cys²²⁹ of CoxB_PD^C233S) are shown as sticks. The intermolecular disulfide bond of the TlpA_S^C110S-CoxB_PD^C233S complex was lost due to radiation damage. The inserted boxes (see “Results”) present detailed views of specific, polar interactions. In some of the views, the structures were rotated by the indicated angle. Residues are shown as sticks, and hydrogen bonds are shown as red dotted lines. Yellow sticks: sulfur atoms; red sticks: oxygen atoms; blue sticks: nitrogen atoms; yellow broken line in lower right inset of B: distance between Cys²²⁹ of CoxB_PD^C233S and Cys¹⁰⁷ of TlpA_S^C110S, indicating the position of the intermolecular disulfide that was lost due to radiation damage; N: N terminus of the respective polypeptide chain.

TABLE 1.

Statistics of crystallographic data collection and refinement

a.u., asymmetric unit.

	TlpASC110S-CoxPDC233Sa	TlpASC110S-ScoISC74Sa
	PDB entry 4TXV	PDB entry 4TXO
No. of complexes per a.u.	2	4
No. of amino acids/complex	184 (TlpASC110S) + 159 (CoxBPDC233S)	184 (TlpASC110S) + 175 (ScoISC74S)
Processing
Space group	P21 21 21	P1 21 1
a, b, c (Å)	74.83, 81.63, 109.37	60.03, 173.22, 66.69
α, β, γ (°)	90.00, 90.00, 90.00	90.00, 89.97, 90.00
Resolution (Å)	65.4–2.0 (2.1–2.0)b	50.0–2.2 (2.3–2.2)b
Wilson B (Å2)	40.0	40.4
Rmeas (%)	7.5 (84.8)	15.8 (131.9)
CC1/2	99.9 (65.6)	99.6 (48.2)
I/σI	13.8 (1.8)	10.6 (1.3)
Completeness (%)	98.8 (97.7)	98.9 (94.7)
Redundancy	3.5 (3.5)	7.5 (4.5)
Refinement
Resolution (Å)	65.4–2.0	49.3–2.2
No. of reflections (test set)	301,996 (2304)	68,109 (1363)
Rwork/Rfree (%)	16.5/21.0	17.4/20.7
Twin law		h, -k, -l
Twin fraction		0.5
No. of atoms
Protein	4820	10,193
Ligand/ion		15
Water	444	590
B-factors (Å2)
Protein	38.9	44.2
Ligand/ion	44.2	35.0
Water	39.3	30.6
Ramachandran analysisc
Favored (%)	96.8	96.0
Allowed (%)	2.9	3.8
Outlier (%)d	0.3	0.2
Bond length (Å)	0.017	0.006
Bond angles (°)	1.832	1.080

^a Data were collected from a single crystal.

^b Highest resolution shell is shown in parentheses.

^c Determined with PHENIX.

^d Two Ramachandran outliers at the border of the allowed region (Pro⁷⁶ of ScoI_S^C74S (TlpA_S^C110S-ScoI_S^C74S, chains B and F), Pro¹⁴¹ of TlpA_S^C110S (TlpA_S^C110S-Cox_PD^C233S, chain A), and Ala²³⁶ of Cox_PD^C233S (TlpA_S^C110S-Cox_PD^C233S, chain B)). 2F_o − F_c density, however, mandates this conformation of the residues.

The Folds of TlpA, ScoI, and CoxB in the Context of Mixed Disulfide Complexes

In both mixed disulfide complexes, the structure of TlpA_S^C110S is identical to that determined previously for wild-type TlpA_S, which shows a characteristic thioredoxin-like fold with a central β-sheet flanked by five α-helices and the active-site cysteine pair at the N-terminal end of helix α2 (12) (Figs. 4 and 5). In the TlpA_S^C110S-ScoI_S^C74S complex, ScoI also displays a thioredoxin-like fold, composed of nine β-strands and six α-helices with a β-hairpin extension between α-helix 4 and β-strand 8, which is characteristic of Sco-like proteins (38). Unlike the active-site cysteines in bona fide thioredoxins, however, the Cu²⁺ ion-liganding cysteine pair Cys⁷⁴/Cys⁷⁸ is located at a different position in the thioredoxin fold, namely in the loop segment (residues 71–80) between strand β3 and helix α1 (Figs. 4A and 5). Given that physiological disulfide-exchange reactions between thiol-disulfide oxidoreductases with a thioredoxin fold have not been observed and are kinetically restricted (7, 36), the reaction between TlpA and ScoI is unusual. This interaction is probably due to the different location of the Cys⁷⁴/Cys⁷⁸ cysteine pair in ScoI, which might facilitate the accessibility of the Cys⁷⁴–Cys⁷⁸ disulfide bond in oxidized ScoI for rapid, nucleophilic attack by Cys¹⁰⁷ of TlpA.

FIGURE 5.

Primary structure, regular secondary structure elements, and residues forming specific interactions at the protein-protein interface in the mixed disulfide complexes of TlpA_S^C110S, CoxB_PD^C233S, and ScoI_S^C74S. The cysteines forming intermolecular disulfide bonds are highlighted in yellow. Interacting residues are highlighted in cyan-blue and orange for the TlpA-CoxB and the TlpA-ScoI complex, respectively. Magenta-shaded TlpA residues participate in interactions with both CoxB and ScoI.

Apart from two exceptions (see below), CoxB_PD^C233S in the mixed disulfide complex with TlpA_S^C110S adopts essentially the basic fold that had been observed in the structure of the periplasmic Cu_A center-containing domain of subunit Cox2 in the aa₃-type cytochrome oxidases from Paracoccus denitrificans (1, 39), bovine heart mitochondria (2, 40), and R. sphaeroides (41). Specifically, CoxB_PD^C233S adopts an extended Greek key-like five-stranded β-sheet (Greek key plus one additional, antiparallel strand), a three-stranded β-sheet, a β-hairpin, and a C-terminal α-helix (Figs. 4B and 5). The intermolecular disulfide bond in TlpA_S^C110S-CoxB_PD^C233S was photoreduced during x-ray data collection (Fig. 4B, lower right inset), with a final refined distance of 2.9 Å between the two S_γ atoms (which compares with an ideal distance of 2.05 Å). A proper disulfide between Cys¹⁰⁷ (TlpA) and Cys²²⁹ (CoxB_PD), however, can be modeled upon a slight conformational change of both cysteine side chains without changing the overall geometry of the interface.

TlpA Displays Non-overlapping Surface Areas for Recognition of Different Substrates

The individual monomers in both mixed disulfide complexes are oriented such that their N termini are located on the same side of the heterodimers (Fig. 6A). Due to the presence of an N-terminal transmembrane domain in full-length CoxB and the N-terminal membrane anchors in wild-type TlpA and ScoI, the structures are fully consistent with a topologically suitable positioning on the periplasmic side of the cytoplasmic membrane for disulfide exchange between TlpA and ScoI or CoxB in vivo.

FIGURE 6.

TlpA interacts with its substrates ScoI and CoxB via two distinct electrostatic surfaces. A, representation of the electrostatic surface of TlpA_S^C110S in complex with ScoI_S^C74S (panel 1) and CoxB_PD^C233S (panel 3). In panel 2, the structures of TlpA-ScoI from panel 1 and TlpA-CoxB from panel 3 were superimposed based on TlpA_S^C110S. ScoI_S^C74S (orange) and CoxB_PD^C233S (cyan) are shown as ribbon models. Cys¹⁰⁷ of TlpA, Cys⁷⁸ of ScoI_S^C74S, and Cys²²⁹ of CoxB_PD^C233S are highlighted in yellow. B, electrostatic surface representation contoured from −1 kT/e (red) to 1 kT/e (blue) of the dimerization interfaces of ScoI_S^C74S (panel 1), TlpA_S^C110S (panel 2), and CoxB_PD^C233S (panel 3), highlighting the surface charge complementarity in both complexes. Surface charge calculation was performed for the free, individual molecules. ScoI_S^C74S (panel 1) and CoxB_PD^C233S (panel 3) were rotated by 180° with respect to their orientation in panel A. Red: negatively charged surface; blue: positively charged surface; yellow: position of reactive cysteine; N, N terminus of the respective polypeptide chain.

Remarkably, the solved structures of the two mixed disulfide complexes uncovered two separate TlpA interfaces for interaction with either ScoI or CoxB (Figs. 4 and 6), i.e. both interfaces overlap only in a comparatively small area. The interface area of TlpA_S^C110S-CoxB_PD^C233S (997 Ų) is more spacious than that of TlpA_S^C110S-ScoI_S^C74S (775 Ų). An upper-bound estimate for the shared overlap area is about 350 Ų (measured on the TlpA surface with the program EPPIC (34)). Herein, the TlpA residues Cys¹⁰⁷, Lys¹⁷¹, and Met¹⁷⁹ are involved in similar types of interactions with both ScoI and CoxB (Table 2). Although Cys¹⁰⁷ forms the respective intermolecular disulfide bond, Lys¹⁷¹ forms a salt bridge with Glu⁸⁴ of ScoI (Fig. 4A, left inset) or with the Cu_A-coordinating Glu²³¹ of CoxB (Fig. 4B, lower right inset), and Met¹⁷⁹ forms a two-fold backbone-backbone interaction with Cys²²⁹ of CoxB (Fig. 4B, lower right inset) or with Cys⁷⁸ of ScoI (Fig. 4A, left inset) where the backbone amide and backbone carbonyl of Met¹⁷⁹ of TlpA are in close proximity to the backbone carbonyl and backbone amide of Cys²²⁹ of CoxB and Cys⁷⁸ of ScoI, respectively (Table 2).

TABLE 2.

Specific intermolecular interactions in the structures of the examined TlpA-ScoI and TlpA-CoxB mixed disulfides

sc, side chain; ab, amide backbone; cb, carbonyl backbone; residues in bold face are highly conserved. Chains A+B of TlpA_S^C110S-CoxB_PD^C233S, and chains C+D of TlpA_S^C110S-ScoI_s^C74S were analyzed.

TlpASC110S	TlpASC110S-CoxBPDC233S mixed disulfide		TlpASC110S-ScoISC74S mixed disulfide
	CoxBPDC233S	Interaction	ScoISC74S	Interaction
Ala59	Lys221	cb-sc
Gly61	Thr222	ab-cb
Gly61	Met224	cb-ab
Ala64	Lys221	cb-sc
Leu66	Lys221	cb-sc
Trp106			Phe123	π-Stacking
Trp106			Phe83	π-Stacking
Cys107	Cys229	Disulfide	Cys78	Disulfide
Pro115	Leu168	Hydrophobic
Arg139			Ser121	sc-cb
Lys163			Arg90	cb-sc
Phe167			Phe83	π-Stacking
Lys171	Glu231	Salt bridge (2×)	Glu84	Salt bridge
Lys171			Leu181	sc-cb
Met179	Cys229	ab-cb	Cys78	ab-cb
Met179	Cys229	cb-ab	Cys78	cb-ab
Pro180	Gln228	cb-sc
Pro198	Leu169	Hydrophobic
Pro198	Tyr226	Hydrophobic
Glu200	Leu168	sc-ab
Glu200	Leu169	sc-ab

All other interactions at the protein-protein interfaces are specific to the individual mixed disulfide complexes (Table 2; for example, see Fig. 4A, lower right inset). The interface between TlpA_S^C110S and ScoI_S^C74S is characterized by a unique cluster of π-stacking interactions between four aromatic residues (Trp¹⁰⁶ and Phe¹⁶⁷ of TlpA, and Phe⁸³ and Phe¹²³ of ScoI (Fig. 4A, upper right inset)). Trp¹⁰⁶ of TlpA and Phe¹²³ of ScoI are conserved in TlpA homologs and ScoI-like proteins, respectively (alignments are available from the authors on request). Additional minor interactions are listed in Table 2.

The specific interface between TlpA_S^C110S and CoxB_PD^C233S features eight unique hydrogen bonds (for example, see Fig. 4B, left inset) and additional hydrophobic interactions of Pro¹¹⁵ and Pro¹⁹⁸ of TlpA with the side chains of the leucine pair Leu¹⁶⁸/Leu¹⁶⁹ of CoxB (Fig. 4B, upper right inset), in which Leu¹⁶⁹ is fully conserved (Table 2). The backbone amides of these leucines are hydrogen-bonded with the carboxylate of Glu²⁰⁰ in TlpA (Fig. 4B, upper right inset).

A striking difference between the heterodimer interfaces of TlpA-ScoI and TlpA-CoxB is revealed by the surface electrostatics of the individual molecules (Fig. 6A). The possible implications of this observation will be discussed below.

DISCUSSION

Because of the unique subunit and cofactor composition and the peculiar topology of cytochrome oxidase in the bacterial cytoplasmic membrane or mitochondrial inner membrane, the biogenesis of this enzyme is a very complex, multifactorial process. Although a substantial number of assembly factors for heme and copper insertion have so far been identified, our mechanistic understanding about how they assemble and act in concert is still incomplete. Results reported here add an important piece to the puzzle by showing how TlpA prepares ScoI and cytochrome oxidase subunit II (CoxB) for copper ion insertion and formation of the Cu_A center. The epistatic nature of TlpA function became evident from the fact that the cytochrome oxidase defect in a B. japonicum scoI mutant, but not in a tlpA mutant, could be corrected phenotypically by supplying excess Cu²⁺ to the growth medium. Such a rescue of COX activity had been observed previously in Sco-deficient organisms including human cell lines (14, 42–44). How this rescue works remains enigmatic in view of the fact that intracellular metal levels are usually regulated tightly, and chelators adjust them to appropriate concentrations in vivo (18). It is often typical for chaperones to be important especially under conditions of stress or nutrient limitation. In fact, ScoI requirement for cytochrome oxidase activity in B. japonicum seems to be most distinct under copper-limiting growth conditions (14, 15). Moreover, the bypass of ScoI by copper-replete conditions finds a possible explanation in the ease with which we could reconstitute the Cu_A center in vitro simply by incubating reduced CoxB in the presence of Cu²⁺ (Fig. 2D), as it had been worked out by Chacon and Blackburn (6). Assuming that such a self-assembly at high external Cu²⁺ concentrations also works in vivo, this would explain why ScoI may become dispensable. By contrast, it is absolutely essential for the success of the in vitro reconstitution of the Cu_A center that CoxB is provided in reduced form. Extrapolating this sine qua non to the in vivo situation means that CoxB must always be kept reduced for copper ion insertion, which is precisely the function we ascribe to TlpA. Obviously, the TlpA requirement for the biogenesis of functional COX cannot be bypassed by copper-replete conditions.

Our in vitro experiments performed both with oxidized ScoI (20) and with oxidized CoxB as substrates (this work) clearly support the function of TlpA as a reductase. TlpA has a more negative redox potential than ScoI and CoxB, and the rates of reduction of both substrate proteins are fast and lie in a physiologically relevant range. This was already a strong hint for a specific protein-protein interaction between TlpA and ScoI as well as TlpA and CoxB, a postulate that was fully confirmed by the high-resolution crystal structures of the mixed disulfide TlpA-ScoI and TlpA-CoxB complexes. Apart from the detailed contact areas already described in Fig. 4 (see above), TlpA_S^C110S possesses a distinct “dipolar” interface in the region where ScoI_S^C74S and CoxB_PD^C233S bind. The TlpA interface shared with ScoI is of basic nature, whereas the interface shared with CoxB is mostly acidic. ScoI and CoxB complement this dipole as each protein provides the opposite charge to the interface (Fig. 6B). In conclusion, we suggest a model in which electrostatic interactions between TlpA and its substrates are also governed by an overall charge complementarity. Such a reciprocal electrostatic complementarity might contribute to the fast rates of ScoI and CoxB reduction by TlpA (45). Interestingly, although the CX₃C motif is the common target in both substrate proteins, TlpA attacks the more N-terminal cysteine in CoxB (i.e. Cys²²⁹ in the ²²⁹CX₃C²³³ sequence) as opposed to the more C-terminal cysteine in ScoI (i.e. Cys⁷⁸ in the ⁷⁴CX₃C⁷⁸ sequence). This might also be a reflection of the bipolar asymmetry on TlpA, which positions each of two protein substrates from different angles on top of its active site.

A bioinformatics analysis (available from the authors on request) has shown that TlpA is widespread in aerobic bacteria that respire with cytochrome oxidases. The peculiar, exposed location of the Cu_A center on the outer side of the membrane, there being confronted with an oxidizing environment, appears to make a TlpA-like reductase function mandatory for the biogenesis of cytochrome oxidase. Whether or not this prerequisite also applies to mitochondria is less obvious. Recent work has shown, however, that dithiol-disulfide oxidoreductase relay systems do exist in mitochondria, both in the oxidizing as well as in the reducing direction (8, 46), and thioredoxin-like proteins were found in the mitochondrial intermembrane space (47). The existence of a ScoI- and CoxB-specific reductase in the periplasm of Gram-negative bacteria inevitably leads to the question of where the reducing power is ultimately derived from. Although E. coli does not possess a cytochrome oxidase for aerobic respiration, a system similar to that of E. coli DsbD, which relays reducing power from the cytoplasm over the membrane into the periplasm (9, 48), is also a valid option for B. japonicum.

Another unsolved problem for future research is where the copper ions for the biogenesis of the Cu_A center are derived from, and how Cu_A assembly proceeds. Although the Sco1/ScoI protein is widely accepted to be the Cu²⁺ carrier to Cu_A (18, 19), the direct transfer of Cu²⁺ to apo-CoxB has not yet been demonstrated with in vitro experiments. Furthermore, formation of the Cu_A center as an electron-delocalized Cu^1.5+–Cu^1.5+ system implicates not only Cu²⁺ but also Cu⁺ in the metallation process (6). A good candidate for a Cu⁺ carrier to Cu_A is the PCu_AC protein (called PcuC in B. japonicum) (15, 49). How the ScoI- and PcuC-like copper chaperones cooperate in the assembly of Cu_A on the TlpA-reduced CoxB subunit of cytochrome oxidase remains to be elucidated.

In this context, it is noteworthy that our work allowed for the first time a description of the structural differences between apo- and holo-CoxB. A superposition of the B. japonicum CoxB_PD^C233S structure onto the structure of P. denitrificans subunit Cox2 of cytochrome oxidase, which carries an intact Cu_A center (PDB entry 3hb3), is shown in Fig. 7. The largest difference concerns the segment 229–240, which contains four of the six Cu_A-coordinating residues, namely Cys²²⁹, Glu²³¹, Cys²³³, and His²³⁷ (Fig. 7A). The positions at the C_α atoms deviate substantially from those of the Cu_A-coordinating residues of P. denitrificans Cox2, ranging from 6.5 to 12.1 Å. By contrast, the positions of Met²⁴⁰ and His¹⁹⁴ do not change with respect to Cox2. This finding suggests that the Cu_A loop 229–240 in CoxB undergoes a major conformational change in the course of metallation. Furthermore, before metallation, the two cysteines Cys²²⁹ and Cys²³³ in the segment 229–240 are surface-exposed such that a disulfide bond between them is accessible for reduction by TlpA. After reduction, the two cysteine thiols may bind copper ions, and the entire loop may undergo a structural rearrangement to form the mature Cu_A center in holo-CoxB. Besides the Cu_A loop, we noticed one other structural difference, which concerns the segments Leu¹⁵⁹–Asp¹⁷² of B. japonicum CoxB and Leu¹³⁷–Asp¹⁵⁹ of P. denitrificans Cox2. In Cox2, that region contains two short α-helices that contribute to the interface with the neighboring subunit I, whereas these helices are absent in B. japonicum CoxB (Fig. 7B).

FIGURE 7.

The polypeptide segment containing the Cu_A-coordinating residues Cys²²⁹, Glu²³¹, Cys²³³, and His²³⁷ of reduced B. japonicum CoxB is predicted to undergo major rearrangements during formation of the Cu_A center. A, right panel: superimposition of CoxB_PD^C233S (cyan) in the TlpA_S^C110S-CoxB_PD^C233S mixed disulfide with holo-Cox2 of P. denitrificans (deep purple, taken from PDB entry 3hb3). The two copper ions in the Cu_A domain of Cox2 are shown as orange spheres. Left panel: close-up view of the superimposed periplasmic domains of B. japonicum CoxB_PD^C233S and P. denitrificans Cox2. A segment of TlpA_S^C110S from the TlpA_S^C110S-CoxB_PD^C233S complex is also shown in the background (gray). Dashed lines: distances (in Å) between the C_α atoms of the Cu_A-coordinating residues of CoxB_PD^C233S and those of Cox2; yellow sticks: sulfur atoms; red sticks: oxygen atoms; dark blue sticks: nitrogen atoms. B, structure-based sequence alignment, obtained with the DaliLite workbench (50) and manually edited, of B. japonicum CoxB_PD^C233S with the corresponding periplasmic domain of Cox2 from P. denitrificans. Sequence identity is 44%. The cysteines liganding the copper ions in the Cu_A center are highlighted in yellow and the other copper-coordinating residues undergoing a large conformational rearrangement upon formation of the Cu_A center are highlighted in purple, whereas the copper-coordinating residues that undergo no conformational rearrangement upon Cu_A formation are highlighted in cobalt blue. Secondary structure elements (β-strands: blue arrows; α-helices: red cylinders) were assigned as indicated by DaliLite (50).