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Review

Function and redox state of mitochondrial localized cysteine-rich proteinsimportant in the assembly of cytochrome c oxidase

Oleh Khalimonchuk, Dennis R. Winge ⁎

University of Utah Health Sciences Center, Department of Medicine, Salt Lake City, Utah 84132, USAUniversity of Utah Health Sciences Center, Department of Biochemistry, Salt Lake City, Utah 84132, USA

Received 18 September 2007; received in revised form 22 October 2007; accepted 30 October 2007Available online 4 March 2008

Abstract

The cytochrome c oxidase (CcO) complex of the mitochondrial respiratory chain exists within the mitochondrial inner membrane (IM). Thebiogenesis of the complex is a multi-faceted process requiring multiple assembly factors that function on both faces of the IM. Formation of thetwo copper centers of CcO occurs within the intermembrane space (IMS) and is dependent on assembly factors with critical cysteinyl thiolates.Two classes of assembly factors exist, one group being soluble IMS proteins and the second class being proteins tethered to the IM. A commonmotif in the soluble assembly factors is a duplicated Cx9C sequence motif. Since mitochondrial respiration is a major source of reactive oxygenspecies, control of the redox state of mitochondrial proteins is an important process. This review documents the role of these cysteinyl CcOassembly factors within the IMS and the necessity of redox control in their function.© 2007 Elsevier B.V. All rights reserved.

Keywords: Cytochrome oxidase; Copper; Mitochondria; Intermembrane space

1. Mitochondrial organization

The mitochondrion consists of a continuous reticulum thatmakes up nearly 10% of the cell volume in respiring yeast cells.The tubular network is highly dynamic and changes size andshape through fission and fusion events [1]. A double mem-brane forming two internal spaces encloses the mitochondria.The space between the two membranes is called the intermem-brane space (IMS) and the volume enclosed within the innermembrane (IM) is designated as the matrix compartment. Thetwo membranes differ significantly in their composition withthe IM more highly enriched in protein content. The outermembrane (OM) envelope contains 25 Å nm pores that allowdiffusion of small ions such as glutathione. The IM is a barrierto diffusion, so the passage of metabolites requires transporters.As such, the import of glutathione across the IM is believed tooccur through dicarboxylate and 2-oxoglutarate transporters [2].

⁎ Corresponding author. University of Utah Health Sciences Center, Salt LakeCity, Utah 84132, USA. Tel.: +1 801 585 5103; fax: +1 801 585 5469.

E-mail address: dennis.winge@hsc.utah.edu (D.R. Winge).

0167-4889/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamcr.2007.10.016

Mitochondria maintain their genome within the matrix com-partment. This genome codes for a limited number of polypep-tides, 13 and 8 in humans and yeast, respectively. All, but one,of these proteins are components of the oxidative phosphoryla-tion system.

The double membrane of mitochondria is interrupted byjunction points of contact between the IM and OM [3]. Thejunction points are likely formed by protein assemblies involvedin protein import [4,5]. The mean distance across the OM andIM is 20 nm, although the distance narrows to only about 14 nmat junction points. In cells with high respiration rates, the IM isinvaginated, folding into tubular structures designated cristae.Cristae are enriched in the enzyme complexes involved in oxi-dative phosphorylation. Electron microscopy tomography re-vealed that the cristae tubules are 30–40 nm in diameter butnarrow to about 28 nm at junction points with the boundary IM,designated inner boundary membrane (IBM) [3,5]. The cons-triction of the cristae junctions at the IBM resolves the solubleIMS into separate volumes that appear to be in equilibrium onlyfor small molecules [6]. Cristae can exist as tubular structures ormerged to form flattened lamellar compartments. Stacked la-mellar cristae remain connected to the IBM by tubular cristae

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junctions. The cristae junctions are believed to be dynamic andmodulated by the energetics of the organelle as well as by thefusion/fission process.

In this review, the lumen of the cristae will be referred to asthe cristae lumen and the space between the boundary IM andOM as the boundary IMS. The generic term IMS will specifythe generalized volume between the OM and IM without sub-compartmentization specification.

Oxidative phosphorylation occurs predominantly, but notexclusively, on the cristae membrane (CM). The enrichment ofcytochrome c oxidase (CcO) in cristae is well established [7],but recent studies confirm the abundance of complexes III (cy-tochrome bc1 complex) and V (ATP synthase) within the CM[5]. Respiratory complexes I, III and IV are largely present assupercomplexes within the CM [8–11]. The yeast III/IV super-complex consists of a dimeric complex III species at the corewith one or two CcO complexes at opposite ends [11]. Synthesisand assembly of the respiratory complexes occurs preferentiallyon the CM as mitochondrial ribosomal proteins are associatedwith the CM [5].

2. Generation of reactive oxygen species in mitochondria

Mitochondrial respiration is a major source of reactive oxy-gen species. Respiring mitochondria convert 1–2% of theoxygen consumed to superoxide anion [12,13]. The bulk ofsuperoxide anion produced on a daily basis in most organismscomes from ubisemiquinone of coenzyme Q within the res-piratory chain [14]. CoQ shuttles electrons from complexes Iand II to complex III. The CoQ semiquinone generated either atcomplex I or during the Q-cycle in complex III can react withoxygen generating superoxide anions. Based on the sidednessof the Q-cycle in complex III, superoxide is generated in boththe matrix and IMS, although the bulk of the superoxide isgenerated within the matrix [15,16]. Superoxide may itselfcause damage or may react further to yield other reactive speciessuch as hydrogen peroxide or the hydroxyl radical. The pre-sence of reactive oxygen species can induce oxidation eventssuch as modification of protein thiols on both sides of the IM[17]. Endogenous reactive oxygen species generated at complexI and III were shown to modify nine distinct mitochondrialproteins [17]. Several of the modified proteins function in fattyacid oxidation.

The normal production of superoxide anion during respira-tion and subsequent generation of hydrogen peroxide and otheroxidants in both the IMS and matrix necessitates control oftransition metal ion availability to minimize Fenton chemistry togenerate more potent oxidants. Copper ions used in metallationreactions are protein bound to minimize the deleterious effects ofunbound Cu(I) ions. Copper metallation of CcO and superoxidedismutase (Sod1) within the IMS occurs by metallochaperoneproteins Cox17 and Ccs1, respectively. Only a small fraction ofthe cellular Sod1 exists within the IMS [18]. The transferreactions are protein-mediated. However, the Cu(I) sites on Ccs1are partially solvent accessible and this is likely also true forCox17. Although the level of copper complexes of these twoproteins isn't known within the IMS, it is possible that the level

of Cu(I) within the IMSmay be regulated tominimize chances ofcopper-induced oxidation reactions. The Cu(I) ions used in theIMSmetallation reactions derives from a storage pool within thematrix [19,20]. It is conceivable that the Cu(I) transporter withinthe IM that translocates Cu(I) to the IMS is regulated such thatthe Cu(I) transported is coupled to the biogenesis of CcO andSod1.

Another defense against deleterious oxidative processes isthe availability of redox systems to maintain redox homeostasis.The effectiveness of the matrix redox system is highlighted bythe observation that the apparent redox potential of the mito-chondrial matrix is more negative (−360 mV) relative to thecytoplasm (−320 mV) in HeLa cells using redox-sensitive fluo-rescent GFP variants [21,22]. No information is available on theredox potential of the IMS compartment.

The mitochondria matrix contains well-defined redox com-ponents including glutathione, thioredoxin and glutaredoxin sys-tems. A fraction of the cellular glutathione reductase Glr1 existswithin the matrix in yeast. The thioredoxin system involves themitochondrial-specific Trx3 thioredoxin and the Trr2 thioredoxinreductase. Both monothiol (Grx5) and dithiol (Grx2) glutaredox-ins exist within the matrix. Initiation at an upstream ATG in thetranscripts for Grx2 and Glr1 generate the mitochondrially-targeted variants. The presence of multiple protein reductants andthe overlapping functions of Glr1 and Trr2 reductases illustratesthat a robust redox pathway exists within the matrix. Apart froman initial report of mammalian thioredoxin reductase-I existingwithin the IMS [23], it is not clear whether other redox com-ponents are present within the IMS compartments. The porousOM suggests that GSHmay equilibrate across the membrane, butno evidence exists for the IMS presence of Glr1 tomaintainGSH/GSSG redox homeostasis.

3. Role of cysteines in IMS proteins

The mitochondrial proteome is expected to contain nearly850 proteins in yeast [24] but closer to 1000 distinct proteins inhumans [25]. About 14% of the proteins are involved in oxi-dative phosphorylation, whereas 25% are predicted to be in-volved in maintaining and expressing the mitochondrial genome[26]. A subset of the proteome resides within the IMS either assoluble proteins or as molecules tethered to the IM. Many ofthese proteins are either cysteine-rich or have functionally im-portant cysteine residues that can exist as within disulfide bondsor as reduced thiolates. The abundance of disulfide-containingmolecules within the IMS suggests that redox control within thiscompartment differs from that within the cytoplasm where di-sulfide bond formation is rare due to the high reducing potentialof the cytoplasm [27]. The constriction of the cristae junctionscreating both cristae lumen and the boundary IMS opens thepossibility that redox pathways may be distinct within the twosubcompartments.

A common structural motif of cysteine-rich proteins withinthe IMS is a helical hairpin conformer. The structural paradigmfor the helical hairpin motif in IMS proteins is small Timproteins (Tim9/Tim10) characterized by a conserved twin Cx3Csequence motif. Tim9 and Tim10 each form a helix-loop-helix

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conformer held together by paired disulfide bonds in the twinCx3C motif [28] (Fig. 1A). Tim9 and Tim10 form a heter-ohexameric complex that functions as a chaperone for incomingproteins as they are delivered to the TIM22 complex for IMinsertion and SAM complex for OM insertion [29]. A secondheterohexameric complex Tim8/Tim13 exists within the IMSthat also mediates import and insertion of polytopic IM proteins

Fig. 1. Structural organization of the Tim9–Tim10 complex. (A). Structure ofthe oxidized form of Tim9. Conserved cysteine residues forming twin Cx3Cmotif are shown in red. The two pairs are designated as the proximal pair (closestto chain termini and distal pair (furthest from chain termini). (B). Structure of theTim9–Tim10 hexameric complex. Each subunit is shown in different color. Thefour conserved cysteines in disulfide linkages within the twin Cx3C motif areshown in red in one subunit colored yellow.

Fig. 2. Disulfide bond in subunit VIB (yeast Cox12) of cytochrome c oxidase.The structure of bovine CcO is shown with the disulfide bonds in subunit VIBshown in red. The four cysteines are arranged in a Cx9C…Cx10C motif. Thissubunit projects to the IMS side of the inner membrane. Subunit VIB is shown ingray.

[30]. Within the twin Cx3C motif, one disulfide consists of themost N-terminal and C-terminal Cys residues (designated pro-ximal pair), whereas the second disulfide consists of the twointernal Cys residues (distal pair). The hexameric complex(Fig. 1B) is dependent on the disulfides within each subunit[31]. Reduction of the disulfides disassembles the complex.

Yeast contains two IMS cytochrome c heme lyases, Cyc3 andCyc7, for the assembly of cytochrome c and cytochrome c1,respectively [32]. The two lyases attach heme to their respectivecytochromeswithin the IMS.Human cells use only a single hemelyase to assemble cytochrome c and cytochrome c1. Whereas thetwo lyases are not cysteine-rich molecules, their substratescytochrome c and cytochrome c1 have essential cysteinyl resi-dues that become covalently attached to the bound heme. Thus,the cysteines in cytochrome c and cytochrome c1 must be main-tained in their reduced state for covalent attachment of hemes.The prevalence of respiratory complexes within the cristae sug-gests that cytochrome maturation occurs predominantly withincristae lumen. Nothing is known how the cytochrome cysteinesare maintained in their reduced state.

Three subunits in the cytochrome bc1 complex and CcO thatproject into the IMS, primarily the cristae lumen, containdisulfide bonds. The Rieske iron–sulfur protein Rip1 and Qcr2of the bc1 complex contains disulfide linkages [33]. The CcOsubunit VIB (yeast Cox12) contains two disulfide bonds in thebovine CcO structure [34] (Fig. 2).

Sod1 contains a disulfide bond that is essential for its en-zymatic function. The copper metallochaperone Ccs1 activatesSod1 during its folding by inserting the catalytic Cu(I) ion andin addition catalyzes formation of the essential disulfide in Sod1

Fig. 3. Role of the MIA machinery in the import of IMS proteins. (A). Mia40captures cysteinyl proteins imported by the TOM complex in a transientintermolecular disulfide. The Erv1 sulfhydryl oxidase generates the activedisulfide in Mia40 used to capture the imported protein. Disulfide exchangereactions resulting in intramolecular disulfide bond formation within theimported protein releases it from Mia40. (B). Import and maintenance of twinCx9C motif proteins require the same MIA import machinery and the ill-definedrole of downstream Pet191.

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[35]. Ccs1 itself has CxC and Cx2C motifs that may be redox-active. Ccs1 co-exists with Sod1 within the IMS. The distri-bution of Sod1 between the boundary IMS and cristae lumen isnot known, but its presence in the cristae lumen is expected asthe respiratory complexes within the cristae IM are the majorsource of superoxide anions. Recent studies on the IMS Sod1 inrat mitochondria suggest that the redox status of Sod1 is morecomplex [23,36]. Sod1 appears to exist within the IMS as aninactive, reduced enzyme. It is transiently activated either byexogenous peroxide or superoxide anions or disruption of theOM [23]. Pretreatment of mitochondria with alkylating agentsquenches activation of the enzyme. Thioredoxin reductase-I isshown to exist within the IMS and to be competent to reduceand inactivate Sod1. Inarrea et al. [23] postulate that the redoxstate, and therefore the enzymatic activity, of Sod1 is highlyregulated within the IMS.

A redox proteomic study attempted to identify oxidized pro-teins in yeast using an affinity capture protocol [27]. Of the 64proteins identified, two IMS proteins Ccs1 and Sod1 were id-entified, although as mentioned, both proteins also exist withinthe cytoplasm. The other mitochondrial proteins identified, Prx1,Hsp60, Yhb1, Ilv5 and Aco1, are matrix proteins.

4. Role of cysteines in the MIA import pathway

Cysteinyl residues in Tim proteins are additionally importantfor their import into the IMS. The import of Tim proteins isdependent on a disulfide relay system involving the MIA ma-chinery consisting of at least Mia40 and Erv1 [37–41]. Transitof the Tim proteins through the TOM complex in the outermembrane results in transient capture of the imported moleculesby Mia40 through disulfide bonding. Disulfide interchangebetween oxidized Mia40 and reduced thiolates on the importedprotein generate intermolecular disulfides that trap the mole-cules within the IMS. The most N-terminal Cys residue in theCx3C motif of Tim9 and Tim10 was recently shown to be im-portant for efficient capture by Mia40, whereas a mutant variantlacking all four Cys residues is not imported at all [42]. Theimportance of the most N-terminal Cys residue in both Tim9and Tim10 suggests that recognition by Mia40 is precise andspecific [42]. Replacement of the only distal Cys pair (Cys 2and 3 in Fig. 1A) enabled import, but the process was inef-ficient. The mechanism of Tim10 release from Mia40 mayinvolve intramolecular disulfide exchange with the fourth Cys(the Cys in the proximal pair) [43]. The third Cys in Tim9appears to be more important than the fourth Cys for Mia40release [37]. The distal Cys disulfide pair is important forassembly of the Tim hexameric complex but doesn't appearessential for the release of Tim from the Mia40 import complex[43].

Erv1 is a flavin-containing sulfhydryl oxidase that generatesdisulfides in Mia40 for IMS protein import [44] (Fig. 3). Erv1has a limited substrate specificity, and Mia40 is its only knownin vivo substrate [45]. Erv1 has a redox-active Cx2C motif closeto the FAD that forms an initial disulfide [46]. Through disulfideexchange, the disulfide is likely transferred to an N-terminalCx2Cmotif for transfer to its targetMia40 [47]. In addition, Erv1

appears to contain a structural disulfide in the flavin domain. Inthe absence of the N-terminal Cx2C motif, Erv1 can inducedisulfide bond formation in small molecules, but this truncate isnon-functional in vivo [47]. All three cysteine pairs are essentialfor normal function that also includes a role in cytosolic Fe/Scluster biogenesis [48]. Oxidants for Erv1 include cytochrome cand molecular oxygen [49]. Sulfhydryl oxidases that use oxygenas the electron acceptor generate one hydrogen peroxide forevery disulfide bond formed [50]. Thus, oxidative folding maycontribute to reactive oxygen species.

Protein substrates of the MIA pathway identified to date in-clude the Tim proteins with a conserved twin Cx3C motif, Erv1with a Cx2Cmotif and a series of proteins with twin Cx9Cmotifs[51]. The transient capture of Tim9/Tim10 proteins by Mia40was recently shown to be specific [42], suggesting that

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recognition may involve more than an exposed thiolate. Withinthe class of twin Cx9Cmotif protein are the IMS proteins Cox17,Cox19, Cox23, Mdm35, Mic14 and Mic17 (Fig. 4). Importof Sod1 may be MIA-dependent as it is absent in the IMS ofErv1-1ts cells cultured at the non-permissive temperature [52].Sod1 has only two conserved cysteinyl residues separated by 88residues. However, the import of Sod1 is dependent on itschaperone Ccs1 that itself has Cx2C and CxC motifs. Ccs1 maybe imported into the IMS through one or both of those conservedmotifs and the apparent MIA-dependency of Sod1 import mayarise from an indirect effect through Ccs1 [53].

The presence of a twin Cx9C sequence motif does not ensurethat import is MIA-mediated, since the CcO assembly proteinPet191 has a twin Cx9C motifs and is imported in a Mia40-independent manner [53]. Likewise, Mia40 has a twin Cx9Cmotif yet is imported through the classical mitochondrial pre-sequence pathway.

Proteins transiently trapped by Mia40 during import arereleased by disulfide exchange reactions resulting in disulfidesin the imported proteins. It is not clear whether the oxidativefolding of Tim proteins is a paradigm for the twin Cx9C motifclass of IMS proteins. For proteins with multiple disulfidebonds, Erv1 may have a role for disulfide bond formation ana-

Fig. 4. Mitochondrial IMS proteins with the twin Cx9C motifs. Schematic represeAll conserved cysteines and their relative spacing are shown in red. Positions of the

logous to its oxidative role on Mia40. Alternatively, an addi-tional mechanism may generate the other disulfide linkages.

5. Function of cysteinyl CcO assembly factors withinthe IMS

One class of CcO assembly factors within the IMS consistsof soluble proteins with a conserved twin Cx9C motif. Theseinclude Cox17, Cox19, Cox23 and Pet191 (Fig. 4). Three othermembers Mic14, Mic17 and Mdm35 have no defined functionin CcO biogenesis. In addition, one subunit of CcO, Cox12,projecting into the IMS contains a related twin Cx9C structuralmotif. The four cysteines in Cox12 are present in two disul-fides stabilized in a helical hairpin conformation (Fig. 2). Thestructure of Cox17, like Cox12, exist in a helical hairpin [54,55] (Fig. 5A). A second class of cysteinyl proteins important forCcO biogenesis includes the Sco protein family and Cox11.These proteins are tethered to the IM.

5.1. Cox17

Cox17 is a soluble copper metallochaperone within the IMSacting as a Cu(I) donor to two accessory proteins Sco1 and

ntation of the yeast IMS proteins possessing twin or quadruple Cx9C motif.Cx9C motifs are indicated.

Fig. 5. Structures of Cox17 and Sco1 (A). Solution structure of the oxidizedform of apo-Cox17. The four conserved Cys residues of the twin Cx9C motif areshown in disulfide linkage in red. Two additional Cys residues participating inCu(I) ligation are also shown. (B). Structure of the C-terminal globular domainof human Sco1 with a bound Cu(I) ion (colored in cyan). The two liganding Cysand His residues are shown in red and brown, respectively. Although the siteappears on the surface, the coordinated Cu(I) is solvent shielded. The arrowshown specifies the dynamic loop 8.

Fig. 6. Copper metallation of CcO subunits. Formation of the CuA and CuB sitesin Cox2 and Cox1, respectively is mediated by chaperone proteins. Cox17 is theCu(I) donor to both Sco1 and Cox11 for subsequent transfer to Cox2 and Cox1,respectively. Cox17 can exist in two Cu(I) binding states, a monomeric mono-copper configuration and a polycopper oligomeric conformer. The significanceof the two Cu(I) conformers of Cox17 is unknown.

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Cox11 implicated in the copper metallation of the CuA and CuBsites of CcO, respectively [56] (Fig. 6). The importance ofCox17 in CcO assembly is highlighted by the embryonic le-thality of embryos homozygous for COX17 disruption [57].Cox17 does not form a stable interaction with either Sco1 orCox11, yet appears to use distinct interfaces to transfer Cu(I) toeach target protein [56]. The C57Y Cox17 mutant is capable ofCu(I) transfer to Cox11, but not Sco1 [56]. One known con-former of Cu-Cox17 is a helical hairpin monomer stabilized bytwo disulfide bonds with Cys residues in the twin Cx9C motif[54,55]. Cox17 molecules have two additional conserved Cysresidues present just upstream of the first Cys of the twin Cx9Cmotif. Those three Cys residues in a C23CxC26 sequence motifare essential for in vivo function, only Cys26 is part of the twinCx9C motif [58]. The single copper is diagonally coordinatedby Cys23 and Cys26 or Cys23 and Cys24. The redox couple ofthe double disulfide configuration to the fully reduced state hasa midpoint potential of −340 mV consistent with the dual di-sulfide molecule being a likely species in vivo [59]. However, amutant form of Cox17 lacking the remaining three conserved

twin Cx9C motif cysteines is functional, suggesting that Cox17is functional without either of the two disulfides in the twinCx9C structural motif. Cu(I) coordination is, therefore, not de-pendent on the disulfide-bonded helical hairpin configuration.

The metal-free conformer is also a disulfide-bonded helicalhairpin. The apo-molecule is capable of forming three disul-fides, yet the midpoint redox potential of redox couple of thetriple disulfide form to the double disulfide form is −197 mVsuggesting that the triple disulfide form of Cox17 probablydoesn't stably exist in vivo [59].

A second Cu(I) conformer is an oligomeric protein com-plex containing reduced thiolates that is capable of binding apolycopper-thiolate cluster [60,61]. The polycopper protein is ina dimer/tetramer equilibrium, with the polycopper cluster likelyexisting at the dimer interface [60]. The tetracopper clusterconformer necessitates that multiple cysteine residues are in thereduced thiolate state. An unresolved major question is whetherthe physiological state of Cox17 is monomeric or oligomericand what the Cu(I) binding stoichiometry is within the IMS.Cox17 purified from the IMS is largely in the copper-free state(unpublished observation).

The mononuclear Cu(I) conformer was identified by in vitroCu(I) titration studies, whereas the polycopper conformer wasidentified from recombinant Cox17 purified from E. coli cul-tured in copper-supplemented medium. In the absence of coppersupplementation, the recombinant protein is devoid of bound Cu(I), but theE. coli cytoplasm has essentially no free Cu(I). TheKd

value for the Cu1Cox17 was reported to be between 10−6–10−7 M, as measured by isothermal titration calorimetry (ITC)[54]. In contrast, the dissociation constant for the tetracopperCox17 complex was determined to be 1×10−14 M [61]. Al-though the two complexes appear to differ markedly in Cu(I)affinity, it is likely that the ITC determination is an underestimate.

5.2. Cox19 and Cox23

Two additional twin Cx9C proteins exist in the IMS that arerelevant to CcO assembly. Cox19 and Cox23 have conserved

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twin Cx9C structural motifs and resemble Cox17 in beingsoluble proteins, although both Cox17 and Cox19 are functionalwhen tethered to the IM. Cells lacking either Cox19 or Cox23are respiratory deficient and have diminished CcO activity.Cox19 resembles Cox17 in its ability to coordinate Cu(I) [62].Cysteinyl residues in Cox19 are important for Cu(I) binding aswell for the in vivo function of Cox19 in CcO assembly. Cox19isolated from the IMS contains titratable thiolates, suggestingthe protein is largely in the reduced state. The correlation of itsability to bind Cu(I) and in vivo function suggests redox controlof cysteines in Cox19 is important for its function [62]. IfCox19 folds into a helical hairpin analogous to Cox17; themutational analysis of Cox19 reveals that, as with Cox17, theprotein is functional without disulfide bonding. Mutations ofcysteinyl codons that would be expected to make a disulfide in ahelical hairpin do not abrogate function. Little is known aboutthe function of Cox23 or its redox state.

5.3. Pet191

Another twin Cx9C motif protein localized within the IMS isPet191. The motif in Pet191 is a variant from the motif inCox17, Cox19 or Cox23 in that the twin motifs are separated by22–30 residues in Pet191 molecules from different species,unlike the short 9-11 linkers in Cox17, Cox19 and Cox23. Inaddition, two additional conserved Cys residues exist in thecandidate Pet191 linker, assuming it also folds in a helicalhairpin. Yeast cells lacking Pet191 are respiratory deficient andhave a specific defect in CcO assembly [63]. Steady state levelsof Cox1, Cox2 and Cox3 are markedly diminished. The res-piratory deficiency of pet191Δ cells correlates with the atten-uation in the IMS protein level of Cox17, Cox19 and Cox23[53]. Although the three Cox proteins are imported into themitochondrion by the MIA pathway, the MIA import pathwayremains functional in pet191Δ cells, as the import of other MIAtargets including Tim13, Sod1 and Ccs1 is normal [53]. Pet191does not have a specialized role in MIA-dependent import of theCox proteins, as in vitro import of radiolabeled Cox19 is normalin pet191Δ cells [53]. Pet191 is tightly associated with a mem-brane, presumably the IM, and is facing the IMS. We are cur-rently assessing the role of Pet191 in the maintenance of twinCx9C motif proteins within the IMS.

5.4. Sco1/Sco2

Sco1 is implicated in formation of the mixed valent CuA sitein Cox2. Yeast lacking Sco1 are devoid of CcO activity andshow greatly attenuated Cox2 protein levels [64,65]. Humancells have two functional Sco molecules that are required forviability [66]. Yeast have a second Sco, Sco2, but cells lackingSco2 are not respiratory deficient. Mutations in either hSCO1 orhSCO2 lead to decreased CcO activity and early death. Patientswith mutations in SCO2 have a clinical presentation distinctfrom that of SCO1 patients [67]. SCO2 patients present withneonatal encephalocardiomyopathy, whereas SCO1 patientsexhibit neonatal hepatic failure. The distinctive clinical presen-tation is not a result of tissue-specific expression of the two

genes, as SCO1 and SCO2 are ubiquitously expressed andexhibit a similar expression pattern in different human tissues.Studies with immortalized fibroblasts from SCO1 and SCO2patients suggest that Sco1 and Sco2 have non-overlapping butcooperative functions in CcO assembly [66].

Sco1 and Sco2 localize to the IM and are tethered by a singletransmembrane helix. A globular domain of each exhibitinga thioredoxin fold protrudes into the IMS [68,69,76]. A singleCu(I) binding site exists within the globular domain of Sco1and Sco2 consisting of two cysteinyl residues within a Cx3Cmotif and a conserved histidyl residue. Mutation of the Cys orHis residues abrogates Cu(I) binding and leads to a non-functional CcO complex [68,69]. The structure predicts that thesingle Cu(I) ion coordinated to Sco1 is solvent-exposed andpoised for a ligand exchange transfer reaction (Fig. 5B). Thestructures of the metal-free human Sco1 and Cu1Sco1 complexare similar with only one loop showing significant rearrange-ments [70] (loop marked by arrow in Fig. 5B). The movement ofthis loop orients the Cu(I) binding His residue in the properorientation for metal binding. Although the Cu binding site issomewhat disordered in the apo-conformation, the site is largelypreformed poised for Cu(I) binding [71–73]. The structuraldynamics of loop 8 suggests it may be important interface forinteractions with Cox17 and/or Cox2 [70]. In addition to bindingCu(I), Sco proteins bind Cu(II) [74]. The Cu(II) site has a highercoordination number than the three-coordinate Cu(I) site [75]. Itis not clear whether Sco1 transfers both Cu(I) and Cu(II) ions tobuild the mixed valent, binuclear CuA site in Cox2. The humanSco2 conformer resembles human Sco1, although Sco2 showsgreater conformational dynamics [76]. It remains unclearwhether human Sco2 participates in Cu(I) transfer reactionsduring CcO assembly.

Cu(I) binding to Cox17 and Sco1 necessitates that the Cu(I)-binding cysteines be maintained in the reduced state during theCu(I) transfer reactions. Mutations that alter the redox state orCu(I) binding capacity of either protein are expected to atten-uate CcO assembly. Although no respiratory deficient mutationsin human COX17 have been described, missense mutations id-entified in SCO1 (P174L) and SCO2 patients (E140K andS240F) are located near the conserved Cx3C and essential Hisresidues, suggesting that the loss of function in both proteinsmay relate to aberrant copper binding. However, the P174Lsubstitution in hSco1 does not affect the ability of the protein tobind and retain Cu(I) or Cu(II) [77], yet its function in vivo isseverely compromised as evidenced by the pronounced CcOassembly defect in SCO1 patient tissues and fibroblasts [66,78].The molecular defect in the P174L mutant Sco1 is an impairedability to be copper metallated by Cox17 using both in vitroand in vivo assays [77,79]. The defect is attributed to eitheran attenuated interaction with Cox17 [77], or to an attenuatedCu(I) binding affinity due to a structural defect [79]. DefectiveCox17-mediated copper metallation of Sco1, and subsequentfailure of CuA site maturation, is the basis for the inefficientassembly of the CcO complex in SCO1 patient fibroblasts.

Cu ions associated to Sco1 are postulated to be delivered toCox2 thereby forming the binuclear CuA (Fig. 6). The CuA sitein Cox2 is formed within a ten-stranded β-barrel and two

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cysteine residues within a Cx3C motif bridging the two Cu ions[80]. The domain of Cox2 containing the CuA site protrudes intothe IMS with the CuA site 8 Å above the membrane surface [34].CuA site formation requires the cysteines to be reduced thio-lates. If the two copper sites are filled sequentially, it is con-ceivable that the Cys residues within the Cx3C motif in Cox2may be involved in ligand exchange reactions to move Cu(I)from Sco1 to Cox2. An initial Cu(I) coordination in Cox2 maybe a distorted two-coordinate complex involved the two Cysresidues prior to rearrangements to form the final coordinationsites.

The thioredoxin-like fold of Sco1 suggested that Sco1 mayhave a redox function, perhaps as a thiol:disulfide oxidor-eductase to maintain the CuA site cysteines in the reduced stateready for metallation [71,72]. Alternatively, Sco1 may functionas a redox switch, in which oxidation of the two Cys residues inthe Cu(I) binding Cx3C motif may facilitate Cu(I) transfer toCox2 [73]. In support of a redox role for Sco1, sco1Δ yeastcells were observed to be sensitive to hydrogen peroxide [71].Subsequently, we showed that the hydrogen peroxide sensitivityof sco1Δ cells arises from the transient accumulation of a pro-oxidant heme a3:Cox1 stalled intermediate and not an oxi-doreductase function [81] (Fig. 7). Heme a/a3 insertion occursin Cox1 prior to the addition of Cox2 in the biogenesis of CcO.A partially accessible channel exists on the IMS side of Cox1through which hemes a/a3 may be inserted. The candidatechannel is sterically blocked upon insertion of Cox2. The glo-bular domain of Cox2 packs onto Cox1 occluding accessibilityto the hemes. Peroxide sensitivity is also observed in cox2Δcells as well as cox20Δ cells that fail to process Cox2. The lackof Cox2 or the inability to fold or mature Cox2 results in vary-

Fig. 7. A pro-oxidant intermediate in the CcO assembly pathway. A pro-oxidantintermediate consisting of heme a3-containing Cox1 accumulates when theassembly pathway is blocked subsequent to Cox1 maturation. The deleteriouseffects of the intermediate can be suppressed by overexpression of Cox11 orSco1 suggesting that these molecules can cap the Cox1 intermediate therebyprecluding peroxide access to the heme a3 catalytic center. Formation of themature core of CcO with Cox2 inserted on Cox1 resolves the potentiallydeleterious Cox1 intermediate.

ing extents of peroxide sensitivity from accumulation of the pro-oxidant Cox1 intermediate [81]. Thus, the structural similarityof Sco1 to thioredoxin may not necessarily imply a redox func-tion for Sco1. If oxidation of Cys residues within Sco1 faci-litates Cu(I) transfer, further evidence is needed to support thispostulate.

The thioredoxin family of proteins often contains a conservedcis-Pro in juxtaposition to the redox-active cysteinyl residues.The cis-Pro was shown recently to preclude metal ion binding[82]. In the absence of the Pro residue, thioredoxin proteins arepoised for metal ion binding either Fe/S cluster or Cu(I)/Zn(II)ions. The corresponding position in Sco1 is the Cu(I) ligand His,consistent with its Cu(I) binding function. The replacement ofthe Pro in human thioredoxin with a His residue enables theprotein to bind Zn(II) or Cu(I) [82]. Thus, the presence of theconserved His in Sco proteins is consistent with the postulatedrole of Sco proteins in Cu(I) transfer.

5.5. Cox11

Formation of the CuB site in Cox1 requires Cox11 [83]. CcOisolated from Rhodobacter sphaeroides cox11Δ cells lackedCuB but contained both hemes, however the environment ofheme a3 was altered [83]. S. cerevisiae cells lacking Cox11have impaired CcO activity and have lower levels of Cox1 [84].Cox11 is tethered to the IM by a single transmembrane helixwith a C-terminal domain protruding into the IMS [85,86]. TheCu(I) binding cysteinyl residues lie within this C-terminaldomain. The cysteinyl residues in this C-terminal domain thatare important for both Cu(I)-binding correlate with in vivofunction, such that when mutated abrogate Cu(I) binding andCcO activity. Removal of the transmembrane domain yieldsa soluble protein that dimerizes upon Cu(I) binding [85]. TheCu(I) sites in each monomer are closely juxtaposed as thedimeric complexes can form a binuclear Cu(I) thiolate cluster atthe dimer interface [85].

Cu(I) transfer from Cox11 to Cox1 forming the CuB site thatlies 13 Å below the membrane surface appears to occur innascent Cox1 chains during its insertion and folding within theIM [86,87]. The CuB site is a heterobimetallic site with heme a3,so CuB site formation is likely concurrent with heme a3 in-sertion. Heme a3 insertion requires Shy1 [88]. Mammalian cellscontaining a mutant Shy1 (SURF1) accumulate a Cox1, Cox4and Cox5A subcomplex suggesting that heme a3 insertion oc-curs in the subcomplex. The CuB site in Cox1 consists of histidylligands, whereas the Cu(I) site in Cox11 consists of cysteinylresidues. Thus, the redox state of Cox11 must be maintainedwithin the IMS to ensure successful Cu(I) transfer.

6. Outlook

Redox control of CcO assembly within the IMS is an im-portant process. Maintanence of reduced thiolates in Cox17,Sco1, Sco2, Cox2 and Cox11 is important for the proposedcopper metallation of CuA and CuB sites. It remains to be es-tablished whether disulfide bond formation in Cox17 or Sco1 isimportant to direct Cu(I) to the CuA site in Cox2. The situation

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may be more complex if the initial report that the IMS Sod1 is ina dynamic redox equilibrium [23]. If the Sod1 postulate issubstantiated, the possibility exists that CcO assembly factorsmay also exist in redox equilibrium that serves a regulatoryrole in CcO biogenesis. Further work is needed to identify themechanism of disulfide bond formation within the IMS ind-ependent of the role of Erv1 in redox control of Mia40. In ad-dition, the identity and regulation of disulfide reductants needs tobe addressed. It remains to be determined whether the redoxpotential of the boundary IMS or cristae lumen differs from thatof the cytoplasm or matrix. A redox proteomic study of the twocompartments would be insightful.

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