1. Introduction

Mitochondria are the major source of cellular energy that is required to sustain life. They are double-membrane organelles in which the process of cellular respiration and ATP production takes place. This process, oxidative phosphorylation, occurs at the electron transport chain (ETC), a series of four protein complexes embedded in the inner mitochondrial membrane (IM). The complexes create a proton gradient by pumping protons from the matrix to the intermembrane space (IMS), which is coupled with electron transfer down the chain. The electrochemical proton gradient thereby produced is used by ATP synthase (complex V) to generate ATP from ADP and phosphate.

Complex IV, or cytochrome c oxidase (COX), is the terminal enzyme of the ETC and is responsible for reducing oxygen to water. Physiologically, the mammalian complex is a dimer, with each monomer composed of 13 tightly bound subunits embedded in the IM, an assembly supported by several crystal structures resolved from COX in bovine heart ^[1][2][1,2]. However, more recently, monomeric crystal structures of COX were also published ^[3][4] [3,4] and monomeric COX was also reported in a supercomplex [5]. It is therefore possible that an equilibrium exists between dimeric and monomeric COX, which could be subject to regulation. In addition, a 14th subunit has been proposed—NDUFA4—which was originally believed to be a subunit of complex I ^[6][7][6,7]. A structural study showed that NDUFA4 appears to be a subunit in the COX monomer, likely adding to the stability of the complex [7]. NDUFA4 as part of the COX monomer is located at the interface of the dimeric complex, where it would prevent or interfere with dimer formation and which could be a reason that the protein was never detected in the dimeric crystal structure. The validity of NDUFA4′s role as a true subunit has been questioned and it was argued that, because NDUFA4 may bind to both complexes I and IV and is not consistently found in COX preparations, it may function as an assembly factor for the respirasome [8].

The three largest subunits are encoded by the mitochondrial genome whereas the other subunits are encoded by the nuclear genome. Among the mitochondrial-encoded subunits, subunits I and II contain the catalytic centers. The latter consist of metal centers that are involved in the electron acceptance from complex III via cytochrome c and the pathway of the electron through the complex itself: electrons received from cytochrome c first reach the Cu_A center in subunit II, are then transferred to heme a in subunit I, and finally reach the heme a₃-Cu_B site of subunit I, where oxygen is reduced to water.

There are various modes of regulation of COX activity [1], summarized in Table 1. The purpose of this review is to explore the regulation of COX through the interaction with proteins of the twin CX₉C family. Members of this protein family have been shown to be important in COX complex assembly and function, as well as for direct regulation of the oxidase ^[9] [9] (Table 2). Note that the 13 tightly bound COX subunits are traditionally distinguished by Roman numerals introduced by the Kadenbach lab, whereas auxiliary proteins are designated with Arabic numerals (yeast nomenclature can be found in Table 2).

The twin CX₉C family of proteins is characterized by its unique motif of two cysteines separated by usually nine amino acid residues. This motif is found in the coiled-coil-helix-coiled-coil-helix (CHCH) domain, where pairs of cysteines form a helix turn helix fold by forming disulfide bonds with one another ^[10][11][12][10,11,12]. Another family of proteins, called the “small Tim” proteins, contains a similar but shorter twin CX₃C motif and plays chaperone roles in the TIM22 pathway for insertion of proteins into the IMS-facing side of the inner membrane (IM) [13]. The CHCH domain is important for the import of the proteins into the intermembrane space (IMS) of the mitochondria. IMS import is facilitated through the Mia40/CHCHD4 redox mechanism ^[14][15][14,15]. The first studies of this family of proteins took place in Saccharomyces cerevisiae, where a detailed study found that 13 of the 14 yeast family members were conserved across species [16]. A follow-up study contained a genome-wide analysis to determine family member functions, with six of the CX₉C proteins determined to be involved in COX assembly [9]. Recently, more information has become available through further research into the function of twin CX₉C family members.

2. COX Regulation through Assembly

The biogenesis and maturation of COX is critical for its proper function. There are multiple steps in this tightly regulated process: the insertion of metal groups in COX I and COX II, the import and folding of nuclear encoded subunits, and the proper assembly of the subunits into the complex. Over 30 auxiliary proteins are involved in the biogenesis of the core enzyme composed of COX I, COX II, and COX III [17]. The hypothesized assembly pathway favors a modular–linear assembly, where subunits are first assembled into module intermediates and then these modules are assembled into the COX monomer (Figure 1) ^[18][19][18,19]. The first step of monomer assembly is the synthesis of mitochondrially encoded COX I, including the insertion of heme a, which is followed by its association with the COX IV and COX Va module ^[18][20][18,20]. The COX II module, which requires the insertion of the Cu_A center into the subunit before assembly can continue ^{[21][22][23][24]}[21,22,23,24], forms a complex intermediate with COX VIc, COX VIIb, COX VIIc, and COX VIIIa. The COX III module consists of COX III, COX VIa, COX VIb, and COX VIIa. These modules are then assembled in a linear fashion, upon which NDUFA4 interacts to assist in the stabilization of the COX monomer [18]. Detailed COX biogenesis and assembly has been reviewed elsewhere [19]; however, summaries of assembly will be provided where necessary.

Figure 1. Assembly of cytochrome c oxidase. The assembly of COX is thought to be both modular and linear. Dark blue, mitochondrial encoded COX core subunits; light blue, nuclear encoded COX subunit; purple, assembly factors; gray, unknown function. *, Twin CX₉C protein. Gray dashed lines represent assembly factor interactions. Black dashed lines represent subunit assembly.

It is important to note that copper metabolism and homeostasis in the mitochondria are important in aerobic respiration of the cell (for detailed reviews, see ^[25][26][25,26]). All of the assembly proteins discussed in this section have been shown to be involved or associated with copper transport and moiety insertion into appropriate subunits during COX assembly, which is an absolute necessity for the complex to function. Chaperones are very important in this process because of the redox sensitivity of the transition metal that, if left unchecked, can become a source of ROS (for review, see [27]).

Most of the twin CX₉C proteins involved in COX assembly were first identified in yeast in a screen to identify proteins that affect OXPHOS; the proteins that were shown to affect OXPHOS were COX17, COX23, COX19, CMC1, and CMC2 [16]. COA4 and COA6 were identified in separate studies ^[28][29][28,29].

3. COX Structural Subunits

COX VIb1/Cox12p

Although not a true twin CX₉C protein, this protein is often considered an “unofficial” family member in the context of COX due to structural similarity and its role in complex IV function. The nuclear encoded Cox12p/COX VIb1 has one CX₉C motif and one CX₁₀C motif. Cox12p in yeast was first shown to be a novel subunit that is required for proper COX activity but not complex assembly ^[30][90]. Cox12p protein is associated specifically with COX II biogenesis, where interaction studies show its physical interaction with COX II, COA6, and SCO1/2 ^[31][81]. The COX6B gene was mapped in human heart and three pseudogenes were identified and subsequently characterized ^[32][33][91,92]. COX6B1 is ubiquitously expressed across tissues whereas its isoform, COX6B2, is testes-specific ^[34][93]. COX VIb1 shares 45% sequence identity with Cox12p (Figure 10). In 1997, the dimeric complex of COX was crystallized from bovine heart and its structure determination confirmed that COX VIb1 is part of the COX complex, sitting on top of the dimer and seemingly forming a bridge on the IMS side of COX that allows the COX monomers to interact [2]. The crystal structure shows that COX VIb1 interacts with core subunits II and III [2] and, based on computer modeling ^[35][94], is part of the cytochrome c binding site of COX.

Figure 10. COX VIb1/Cox12p. Alignment of human and yeast COX VIb1/Cox12p protein sequences. Black boxes indicate conserved residues. Human sequence annotation is as follows: Blue box indicates the CHCH domain. Solid black lines indicate the CX₉C-CX₁₀C motif. Dashed lines indicate the cysteine pairs that form structural disulfide bonds.

Some mutations in COX6B1 have been associated with COX deficiency disease. Two individuals with early-onset leukodystrophic encephalopathy, myopathy, and growth retardation carried a homozygous mutation in COX6B1 resulting in an amino acid residue replacement of arginine to histidine at the N-terminus of the protein ^[36][95]. Another missense mutation, resulting in an arginine to cysteine change at the same position, was mapped in an individual with encephalomyopathy, hydrocephaly, and cardiomyopathy ^[37][96].