Mutational analysis of the mitochondrial copper metallochaperone Cox17.

The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochrome c oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys --> Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys --> Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.

Functional cytochrome c oxidase requires the assembly of a macromolecular complex involving nuclearly and mitochondrially encoded subunits (1)(2)(3). The assembly is dependent on the insertion of cofactors, including two heme A groups, three copper ions, one zinc ion, and one magnesium ion (2). A series of nuclearly encoded accessory proteins mediate formation of the oxidase complex, although the function of only a few is understood (3)(4)(5)(6)(7). Two accessory proteins are enzymes that modify protoheme to heme A (8). Delivery and insertion of Cu ions into the oxidase appear to require at least three proteins, Cox17, Sco1, and Cox11 (9 -12). Two Cu ions are inserted into cyto-chrome oxidase subunit II forming the binuclear Cu A site, and a single Cu ion into subunit I forming the binuclear Cu-heme a 3 reaction center (2). Because subunits I and II are mitochondrially synthesized, Cu ions must be delivered from the cytoplasm and inserted within the mitochondrion.
Cells harboring a mutant COX17 gene are respiratory-deficient but synthesize both mitochondrially and nuclearly encoded cytochrome oxidase subunits (9). Cox17 was implicated in copper ion delivery to mitochondria by the observation that the respiratory defect of cox17⌬ cells was suppressed by high exogenous Cu(II) levels (9). Exogenous Cu(II) did not rescue a subset of other cytochrome oxidase mutants (9). Two observations are consistent with the predicted role of Cox17 as a Cu shuttle protein. First, Cox17 is an 8057-dalton polypeptide localized in the cytosol and intermitochondrial membrane space (13). Second, Cox17 is a Cu(I)-binding protein with at least two Cu(I) ions bound in a polycopper cluster (14).
Cox17 has been proposed to shuttle Cu ions to the mitochondria and transfer Cu to Sco1 as an intermediate step in Cu donation to cytochrome oxidase (10). Sco1 was implicated in copper ion delivery by the observation that the respiratorydeficient phenotype of cox17-1 cells harboring a Cys 3 Tyr substitution in Cox17 was suppressed by high copy SCO1 or the homologous SCO2 gene (10). Sco1 is an inner mitochondrial membrane protein important specifically in cytochrome oxidase assembly (15). Cells lacking a functional Sco1 are defective in cytochrome oxidase activity; this phenotype is not reversed by high exogenous copper (10). In contrast, cells lacking a functional Sco2 are respiratory competent (10,16). The proposed transfer of Cu(I) ions from Cox17 to Sco1 is supported by the mutational analysis of Sco1 in which substitutions in the potential metal binding motif, CXXXC, abolish Sco1 function (11). Human Sco1 homologs are known, and mutations in human SCO2 result in cytochrome oxidase deficiency and a fatal cardioencephalomyopathy (17,18).
Cu metallation of the Cu B site may require the Cox11 protein. Cox11 was recently shown to be important for Cu B site formation in Rhodobacter sphaeroides (12). Saccharomyces cerevisiae Cox11 is a 28-kDa mitochondrial membrane polypeptide essential for the accumulation of oxidase subunit I (8). If yeast Cox11 is a metallochaperone for the Cu B site of cytochrome oxidase, the possibility exists that CuCox17 delivers Cu(I) to both Sco1 and Cox11 for subsequent donation to the Cu A and Cu B sites, respectively.
Previously, we demonstrated that Cox17 bound Cu(I) within a polycopper cluster with predominantly thiolate ligands (14). If Cox17 donates Cu ions, the prediction is that mutations of the Cox17 Cu-binding thiolates would abrogate function. Here, we test this prediction by mutagenesis of the seven cysteinyl residues in Cox17. We show that only three of the seven cysteinyl residues are essential for Cox17 function and that sub-stitution of multiple essential cysteines with serines abolishes copper ion binding.
Vectors-The SacI/KpnI fragment of YEp-pRS426 containing the MET25 promoter and CYC1 terminator was excised and cloned into the YCp-based vector pRS316 with URA3 selection (19). COX17 and mutants were cloned into the modified pRS316, designated YCpCOX17, to yield low copy expression plasmids. Methionine levels were altered in the growth medium to modulate expression levels. Cells were cultured in medium containing 134 M methionine to partially repress expression of the MET25/COX17 fusions. Cells cultured in this medium exhibit approximately 60% of the maximal expression observed in cells cultured in medium without methionine (19). For Escherichia coli expression, COX17 or mutant genes were PCR 1 -amplified to include 5Ј NdeI and 3Ј BamHI sites. The first five codons of each PCR product were optimized for E. coli expression (20). The resulting DNA products were subcloned into the E. coli (BL21) expression vector pAED4, which is a derivative of the T7-based expression vector, pET-3a (21).
Mutagenesis-COX17 point mutations were constructed using PCR primers containing mismatches at the appropriate codon. Because of the small size of the gene, each of the mutants could be constructed by varying the 5Ј or the 3Ј oligonucleotide PCR primer. Each of the COX17 gene variants was verified by sequencing.
Mitochondrial Isolation and Oxidase Activity-Mitochondria were isolated from S. cerevisiae as described previously (22). Mitochondrial protein concentration was determined by the Bradford assay (23). Five g of total mitochondrial protein were added to 100 l of cytochrome c reactions (45 M cytochrome c, 50 mM phosphate, 0.1% deoxycholate). Oxidation of reduced cytochrome c (prereduced with sodium dithionite) was measured by monitoring the absorbance at 550 nm for 3 min. Reaction rates were normalized to total mitochondrial protein. Similar data were obtained if cytochrome oxidase activity was normalized to mitochondrial DNA or succinate dehydrogenase activity. Potassium ferricyanide was added to each reaction at the end of 3 min to measure the background absorbance of the oxidized cytochrome c.
Antibody Production-Two New Zealand White rabbits (Western Oregon Rabbitry) were injected subcutaneously with 365 g of purified Cox17 protein emulsified in Freund's complete adjuvant (Difco Laboratories). After 4 weeks, the rabbits were boosted with 365 g of protein mixed with Freund's incomplete adjuvant. A second booster shot was administered 2 weeks later with 182 g of protein. After 12 days, serum was obtained by final bleeding.
Western Analysis-Mitochondrial and post-mitochondrial supernatant proteins (10 g, as determined by the Bradford assay) were separated on 16% acrylamide gels in the Laemmli buffer system. Proteins were transferred to nitrocellulose (Bio-Rad Laboratories) and the blots probed with polyclonal antibodies against Cox17 or monoclonal antibodies to mitochondrial porin and phosphoglycerate kinase (Molecular Probes) at a 1:5000 dilution. As secondary antibodies, horseradish peroxidase-conjugated donkey anti-rabbit antibody (for Cox17) or a goat anti-mouse antibody (Amersham Pharmacia Biotech) was used at a 1:3000 dilution. Cox17 protein was visualized with ECL reagents (Amersham Pharmacia Biotech) followed by autoradiography. Porin and phosphoglycerate kinase proteins were detected either with the ECL reagents or after the addition of 50 ml of phosphate-buffered saline, 10 ml of methanol, 50 l of 30% hydrogen peroxide, and 1 ml of 30 mg/ml 4-chloro-1-naphthol (dissolved in absolute ethanol).
Purification of Cox17-Cox17 was expressed as a soluble protein in E. coli. BL 21 cells harboring the pAED-COX17 plasmid were grown to an A 600 nm of 0.4 -0.6 prior to incubation with 0.3 mM isopropyl-1-thio-␤-D-galactopyranoside for an additional 3 h. Copper sulfate was added to the growth medium to a final concentration of 1.4 mM at 1 h prior to cell harvest. The cell pellet was washed with 0.25 M sucrose to remove any spuriously bound Cu(II). The cell paste was resuspended in lysis buffer (20 mM phosphate, pH 7.0, 1 mM dithiothreitol) and stored at Ϫ70°C. Cells were lysed by freeze-thawing and repeated sonication. The lysate was clarified by centrifugation at 100,000 ϫ g for 40 min at 4°C. The supernatant was filtered through a low protein-binding 0.45 m filter and loaded onto a HiPrep 16/10 DEAE column (Amersham Pharmacia Biotech) equilibrated with lysis buffer. Chromatography was done using a Pharmacia fast protein liquid chromatography unit. The protein was eluted by a 600-ml 0 -0.4 M KCl gradient. Fractions containing Cox17 were combined, diluted two-fold, and loaded onto a hydroxyapatite column pre-equilibrated with 20 mM phosphate buffer, pH 7, containing 1 mM dithiothreitol. The protein was then eluted with a 20 -200 mM phosphate gradient (pH 7.0). The fractions containing Cox17 were concentrated and subjected to size exclusion chromatography on a G-75 Superdex 26/60 column (Amersham Pharmacia Biotech) equilibrated in 50 mM phosphate (pH 7), 1 mM dithiothreitol, and 100 mM NaCl.
Analyses-The mass of Cox17 was assessed by electrospray mass spectrometry done at The Scripps Research Institute mass spectrometry facility. Protein was quantified by amino acid analysis after hydrolysis in 5.7 N HCl containing 0.1% phenol in vacuo at 100°C. The analysis was performed on a Beckman 6300 analyzer. The copper concentration of the protein samples was measured using a PerkinElmer (AAnalyst 100) atomic absorption spectrophotometer. Dialysis was performed in size exclusion buffer using Slide-A-Lyzer cassettes with a 3,350-dalton cutoff (Pierce).

RESULTS
Yeast Cox17 contains seven cysteinyl residues as shown in Fig. 1, yet only six of these are conserved between species. Cys 16 is the one cysteinyl residue in yeast Cox17 not conserved in other species. Yeast COX17 was mutated to generate variants with single Cys 3 Ser substitutions. The substitution at Cys 57 was made to a Tyr as that was the original mutation in the cox17-1 mutant that led to the cloning of the gene (9). Mutant COX17 genes were reintroduced into cox17⌬ cells on a YCp plasmid under the inducible MET25 promoter and terminator. The MET25 promoter was used to enable evaluation of Cox17 function under both low expression (high methionine levels) and high expression (no added methionine) conditions. Cells lacking a functional Cox17 are respiratory-deficient because of the lack of cytochrome c oxidase function but grow well on fermentable carbon sources (9). The respiratory defect of cox17⌬ cells was reversed in cells harboring episomal wild-type COX17 cultured in methionine-containing medium to limit expression of the MET25 promoter (Fig. 2). In addition, cells harboring mutant COX17 with Cys 3 Ser substitutions at codons 16, 36, and 47 were also respiratory competent, suggesting that these three cysteinyl residues are not critical for in vivo Cox17 function. Although the C36S Cox17 mutant grew on glycerol, the growth was reproducibly impaired relative to cells containing the wild-type protein. In contrast, cells harboring COX17 with Cys 3 Ser substitutions at codons 23, 24, and 26 failed to grow with glycerol as the sole carbon source. It was reported previously that cells with the C57Y substitution fail to grow on glycerol (9). Thus, Cys 23 , Cys 24 , Cys 26 , and Cys 57 appear essential for Cox17 function. Mutant cox17 cells with mutations in these four essential cysteines also failed to grow when the mutant genes were overexpressed (minus methionine), so failure to grow was not due to expression levels. The mutant proteins accumulated to approximately similar levels as determined by Western analysis (Fig. 3).
Wild-type cells were transformed with the mutant cox17 genes to ensure that mutations did not have a dominant negative effect on growth. All transformants of wild-type cells grew well on glycerol in either medium with high methionine or no added methionine (Fig. 4).
Cytochrome c oxidase activity was quantified in mitochondria isolated from each transformant (Fig. 5). As expected, cox17⌬ cells with wild-type, C16S, C36S, and C47S Cox17 exhibit near normal levels of cytochrome c oxidase activity. Consistent with the lack of growth on glycerol, cells with C23S, C24S, C26S, and C57Y show greatly reduced oxidase activity with respect to the wild-type control. The remaining cytochrome c oxidase activity seen with C57Y Cox17 cells was inhibited by KCN and was not augmented by overexpression of the mutant (data not shown).
Mutations resulting in the substitution of the two nonessential conserved cysteines (Cys 36 and Cys 47 ) or all three nonessential cysteines (Cys 16 , Cys 36 , and Cys 47 ) to serines were engineered to determine whether these cysteines had any synergistic effect in Cox17. Cells harboring the double or triple cox17 mutants grew well on glycerol (Fig. 1) and had appreciable cytochrome c oxidase activity (Fig. 5). The weak glycerol growth of the C36S Cox17 single mutant was not observed with the triple mutant.
The inability of C57Y mutant Cox17 to support growth of cells on glycerol may arise from the nonconservative substitution. A C57S substitution was engineered in Cox17, and cells harboring this mutant Cox17 were glycerol prototrophs, unlike the glycerol auxotrophy for the C57Y mutant (Fig. 6A). C57S Cox17 cells exhibited wild-type cytochrome oxidase activity in isolated mitochondria (Fig. 6B). Thus, Cox17 is functional with either a Cys or Ser at position 57. Only Cys 23 , Cys 24 , and Cys 26 are essential cysteinyl residues for Cox17 function.
Western analysis was carried out to verify that the mutant Cox17 proteins that failed to rescue nonfermentable growth of transformed cox17⌬ cells were expressed (Fig. 3). The analysis was carried out in cells cultured in methionine medium to limit COX17 expression. Cox17 was detected in the cytosol of cox17⌬ cells transformed with each of the mutants, although Cox17 protein levels were slightly attenuated for the three essential Cys mutants. Cox17 is co-localized in the cytosol and the intermitochondrial membrane space with 60% of the protein localized to the intermitochondrial membrane space (13). Overexpression of COX17 results in an increase in the total cellular content of Cox17 but a lesser elevation in the mitochondrial content (13). Thus, the mitochondrial content of Cox17 is limited. In the present experiments with COX17 expressed episomally, total Cox17 levels are elevated at least 4-fold relative to Cox17 levels in wild-type cells expressing COX17 chromosomally, and the percentage of total Cox17 within mitochondria is about 10%, a percentage similar to that seen previously with overexpressed Cox17 (13). Mutant Cox17 molecules with individual Cys 3 Ser substitutions at the three essential Cys positions (positions 23, 24, and 26) were also co-localized within the mitochondria, although the mitochondrial level of C26S Cox17 was partially attenuated relative to the wild-type protein. The only Cox17 mutant that failed to accumulate significantly within the mitochondria was the C57Y variant (Fig. 3). The C57Y mutant protein showed consistently attenuated mi- tochondrial levels, although the protein was abundant in the cytosol. The residual levels of C57Y Cox17 in the mitochondrial were at levels comparable with cytoplasmic contamination. Substitution of Cys 57 with Ser resulted in a mutant Cox17 that was functional, but protein levels were lower than wild-type Cox17 or the C57Y mutant protein in both the mitochondria and the cytosol (Fig. 7). The attenuated level of the C57S Cox17 was not because of poor reactivity with the antisera, as the antisera recognized purified C57S Cox17 (data not shown).
Mutant Cox17 proteins were purified to evaluate Cu(I) binding. Recombinant wild-type Cox17 purified without any purifi-cation tags bound 2.8 Cu ions/monomer (Table I). The Cu binding stoichiometry was not significantly altered in any of the single mutants (Table I). Mutant Cox17 molecules with one of the three essential Cys residues mutated to Ser bound the same quantity of Cu as the wild-type protein. However, Cu binding was abolished in Cox17 with a double C23S/C24S substitution.
Because Cu binding was abolished with the double C23S/ C24S mutant Cox17, the question arose whether the mitochondrial localization of Cox17 was related to the Cu-binding property of Cox17. Mitochondria were isolated from cells harboring the non-Cu-binding mutant Cox17 with the double C23S/C24S substitution. Western analysis of the mitochondrial extract and post-mitochondrial supernatant revealed the mutant protein was present in both compartments (Fig. 7). Thus, mitochondrial localization of Cox17 is not dependent on Cu loading of Cox17.

DISCUSSION
Yeast Cox17 contains seven cysteinyl residues, only six of which are conserved in fungi and animals. Three of the conserved cysteinyl residues (Cys 36 , Cys 47 , and Cys 57 ) are nonessential in that substitution of any one of these cysteines with serines results in a functional cytochrome oxidase complex. Likewise, replacement of the nonconserved Cys 16 with a Ser does not perturb cytochrome oxidase function. The nonessential Cys residues do not appear to have a synergistic effect in that cells containing a mutant Cox17 with a triple C16S/C36S/ C47S substitution have a functional cytochrome oxidase. Only three cysteinyl residues in Cox17 are essential, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines Mitochondria were isolated from W303⌬cox17 cells transformed with cox17 variants. Protein gels were blotted onto nitrocellulose membrane and probed with a polyclonal anti-Cox17 antibody or monoclonal antibodies to either porin, a mitochondrial outer membrane protein, or cytosolic phosphoglycerate kinase (PGK). Western analysis of the mitochondrial C57S mutant protein is not shown as the content of the C57S mutant protein was reproducibly less than that observed for the C57Y protein (Fig. 3), making visualization difficult. WT, wild type.

TABLE I
Cox17 and the mutant variants were purified as untagged molecules by conventional chromatography. The Cu binding stoichiometry was determined in the purified samples by a combination of atomic absorption spectrometry and quantitative amino acid analysis. The values shown represent the bound Cu content of each protein done in triplicate for one or more isolates. The variance is from all analyses of each isolate.

Protein
Cu:protein ratio Wild-type Cox17 2.6 Ϯ 0.5 (n ϭ 1) results in a nonfunctional cytochrome oxidase complex. Cells harboring these mutant Cox17 molecules exhibit no cytochrome c oxidase activity in purified mitochondria and fail to grow on medium with nonfermentable carbon sources. These mutants likely represent a loss of function, as wild-type cells containing one of the Cox17 mutants at varying expression levels failed to exhibit any dominant negative effects on cytochrome oxidase function. Although a Cys 3 Ser substitution at sequence position 57 is functional, a Cys 3 Tyr substitution in Cox17 was shown previously to result in non-respiratory cells (9). Cells harboring C57Y Cox17 exhibit residual cytochrome c oxidase activity, and therefore partial functionality exists in the mutant protein. As Tyr is less chemically related to Cys than Ser, the Tyr substitution likely perturbs some function of Cox17. The C57Y Cox17 mutant binds the same number of Cu ions, yet fails to accumulate within the mitochondria. The low mitochondrial content of the C57Y Cox17 may arise from impeded mitochondrial entry or reduced stability. The C-terminal segment of Cox17, including Cys 57 , may be part of an important mitochondrial targeting sequence. Most mitochondrial proteins are localized to the mitochondria through an N-terminal target sequence (24), although a series of molecules are known to contain internal or C-terminal targeting sequences (25,26). A second metabolite transporter, ATP/ADP translocase, which functions as an ADP/ ATP carrier, is targeted for mitochondrial uptake via the TOM (translocation of the outer membrane) translocation complex by an internal signal motif (27). However, it is unlikely that the nonfunctional status of the C57Y Cox17 relates primarily to low mitochondrial uptake or stability, as only minimal quantities of the functional C57S are found in mitochondria. Episomal expression of COX17 in these studies led to a 4-fold increase in total cellular Cox17, and therefore only a fraction of Cox17 in the mitochondria in the present studies is necessary for its function. An additional function of Cox17 is likely to be impaired in the C57Y Cox17 mutant. A second candidate function of Cox17 is the donation of Cu(I) ions in the mitochondrial intermitochondrial membrane space, presumably resulting from docking to the Sco1 accessory protein. The C-terminal portion of Cox17 containing Cys 57 may be important in docking. Two observations are consistent with this prediction. First, overexpression of the SCO1 homolog, SCO2, efficiently suppresses the respiratory defects of cox17-1 cells harboring the C57Y Cox17 mutation but not cox17⌬ cells (10). The residual cytochrome oxidase activity in cox17-1 cells may relate to limited functional interactions with Sco1/Sco2. It is conceivable that the C57S mutant Cox17 interacts better with Sco1 than the C57Y protein. Weak interactions of Cox17 with Sco1 may result in Cox17 instability and degradation. Second, a hemagglutinin epitope tag at the C-terminal end of Cox17 impairs Cox17 function, although mitochondrial uptake and Cu(I) binding are normal (data not shown). In contrast, an N-terminal HA epitope-tagged Cox17 is functional and accumulates normally within the mitochondria.
The mitochondrial localization of Cox17 is consistent with the postulate that Cox17 functions in copper ion insertion into cytochrome oxidase. Curiously, only minimal quantities of the functional C57S mutant Cox17 are found in mitochondria. The interpretation of this result is unclear. Cox17 may be functional without mitochondrial targeting. Alternatively, Cox17 levels in the intermitochondrial membrane space may be in excess of what is required for function; therefore, only a fraction of the protein in this compartment is critical for function. Nothing is known of the fate of mitochondrially localized Cox17. If Cox17 shuttles Cu(I) ions to the mitochondria, it is unclear whether the apo-protein shuttles out of mitochondria or is degraded. Low mitochondrial accumulation of C57S Cox17 may relate to reduced stability of the protein in the intermitochondrial membrane space.
The prediction that Cox17 functions as the mitochondrial copper metallochaperone rests largely on the observed suppression of the glycerol auxotrophy of cox17 cells by exogenous copper salts (9). The proposed model for Cox17 function involves Cu ion shuttling to the mitochondrial intermembrane space and subsequent transfer of bound Cu ions to subunit II of cytochrome oxidase, perhaps via Sco1 (13). The mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17, as the non-copper binding mutant Cox17 with the double C23S/C24S substitution localizes normally to mitochondria. This important result negates a model that Cu(I) binding induces a conformer in Cox17 competent for mitochondrial entry.
The copper content of Cox17 purified as an untagged molecule (3 mol eq) is higher than the 2 mol eq stoichiometry observed previously for GST-Cox17 fusion (14). The biophysical properties of CuCox17 purified as a GST fusion differ from those of the untagged CuCox17 complex even when the GST moiety is removed. 2 We had proposed that the two Cu(I) ions bound to Cox17 purified as a GST fusion exist within a binuclear thiolate center with either four or five thiolate ligands (14). It is now clear that the copper thiolate center in Cox17 is more complex than that proposed originally for the GST fusion protein. 2 Evidence will be presented elsewhere that the three Cu(I) ions in CuCox17 are bound within a polycopper cluster. The present mutagenesis data are consistent with only three cysteinyl residues being important for ligation of three Cu(I) ions. Additional studies are necessary to determine the Cu(I) binding geometry and whether other non-thiolate ligands are involved in Cu coordination. Because the copper content is not altered with single Cys substitutions in the three critical residues, Cys 23 , Cys 24 , and Cys 26 , the prediction is that Cu(I) binding in the single mutants occurs in an altered center with coordination by one or more of the nonessential Cys residues for ligation. The nonessential Cys residues may be spatially close to the essential three residues and may facilitate Cu release through ligand exchange reactions, as proposed for the transfer of Cu(I) from the Atx1 metallochaperone to Ccc2 (28). Biophysical characterization of the Cu(I) complexes of the single Cox17 mutants with changes at Cys 23 , Cys 24 , or Cys 26 reveals a distinct change in the polycopper center. 2 It is likely that Cys 23 , Cys 24 , and Cys 26 are essential because of Cu(I) ligation, as the C23S/C24S double mutant fails to bind Cu(I). However, the possibility remains that the three cysteine residues are important in other aspects of Cox17 function, such as protein docking within the intermembrane space. As details emerge on the interactions of Cox17 within the intermembrane space, additional studies with the mutants may be insightful in elucidating the physiological significance of the three important cysteine residues.