Eukaryotic cells necessitate a continuous supply of energy to sustain vital biological processes, wherein adenosine triphosphate (ATP) plays a pivotal role as the primary cellular energy source (1, 2). The energy released from ATP hydrolysis, which yields adenosine diphosphate (ADP) and phosphate (HPO₄²⁻), profoundly drives various nonspontaneous biochemical reactions, including DNA replication, cell division, and chemical synthesis (3). Notably, the conversion of a single glucose molecule through three metabolic pathways—glycolysis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation—can produce 38 ATP molecules (4, 5). Glycolysis partially metabolizes glucose into pyruvates, producing a net of only two ATP molecules per glucose molecule (6). The pyruvate is subsequently converted into acetyl-CoA, which enters the mitochondrial TCA cycle, generating NADH and FADH₂ (7). These electron carriers further fuel oxidative phosphorylation, ultimately resulting in the substantial production of ATP, which characterizes mitochondria as the powerhouse of the cell (8).

To activate ATP synthase, it is imperative to establish an electrochemical gradient across the inner mitochondrial membrane. As illustrated in Fig. 1A, electrons are passed down the electron transport chain proteins, concomitantly pumping protons from the matrix to the intermembrane space. Notably, the involvement of various transition metal ions including copper, iron, zinc, and manganese assumes a critical role as essential cofactors in mitochondrial metalloenzymes to regulate redox catalysis (9). For example, a redox-active heme–copper oxidase or iron–sulfur clusters in electron transport chain could facilitate the electron transport across the inner membrane. In addition, antioxidant metalloenzymes, such as superoxide dismutase (SOD) and catalase, catalytically scavenge oxidative stress originating from reactive oxygen species (ROS).

Among transition metal ions, copper exhibits two oxidation states, i.e., Cu(I) and Cu(II), under physiological conditions, owing to its intrinsic electron configuration, [Ar]3d¹⁰4s¹. The catalytic functions of copper-containing enzymes are closely tied to the redox cycle between Cu(I) and Cu(II) via one-electron transfer, which is essential for substrate oxidation or antioxidant defense coupled to O₂ reduction. Moreover, the distinct coordination geometry of Cu(I/II) plays a significant role in the catalytic reactions of cuproenzymes, considering the reorganization energies of tetrahedral d¹⁰ Cu(I) and square planar d⁹ Cu(II) complexes (10). Notably, copper is the sole 3d transition metal ion that possesses redox activity with the maximum number of d electrons, making it apt for O₂ activation. The d electron density of copper could be readily pushed to O₂ via π-back bonding, ultimately weakening the O–O bond (11, 12). In this mini review, we delve into the current understanding of the coordination chemistry of mitochondrial cuproenzymes, the process of metalation, and copper-related toxicity mechanisms. Moreover, we provide a succinct perspective of potential therapeutic strategies to mitigate the impact of copper-related diseases.

Oxidative phosphorylation stands as the predominant process responsible for producing ATP through cellular respiration (13). This intricate process takes place across the inner membrane of mitochondria, where electrons are shuttled from NADH and FADH₂ along a series of redox reactions of electron transport chain complexes (Fig. 1A): complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome bc₁ complex), and complex IV (cytochrome c oxidase; CcO) (14). Notably positioned as the terminal complex, CcO takes electrons from cytochrome c (Cyt c) and reduces dioxygen (O₂) to form water, translocating protons from the matrix to intermembrane space (15). In particular, the Cu_A site of CcO is localized to the intermembrane space, accepting the electron from Cyt c (16). The nuclear magnetic resonance (NMR) spectroscopic studies revealed the chemical shift perturbation of charged residues (e.g., Lys and Glu) and hydrophobic residues (e.g., Met and Ile) in Cyt c upon treatment of CcO (17). Ishimori and coworkers later predicted the plausible binding interface of Cyt c–CcO complex in silico, and proposed the intermolecular electron transfer reactions between heme c in Cyt c and the Trp104 residue in subunit II of CcO (18).

Coordination sphere of CcO and its electron-transfer process

Electron transport within CcO involves four redox-active metal centers that include Cu_A, Cu_B, and Fe in heme a and heme a₃, each of which receives one electron. The electron transfer process from Cyt c to Cu_A in CcO occurs upon protein–protein interactions (19). As depicted in Fig. 1B, binuclear Cu_A site contains two equivalents of copper, each of which is bridged by Cys residues. These two copper centers are additionally bound by His161, His204, Met207, and the carbonyl backbone of Glu198. The distance between two Cu_A centers in a mixed valent between [Cu₂]³⁺ and [Cu₂]²⁺ is around 2.6 Å, which is suitable for a single electron transfer (20). The electron from Cu_A might be transferred to heme a via hydrogen-bonding network composed of His204, Arg439, and a propionate groups of heme a, although the direct experimental evidence is still limited (21). Distinct from the Cu_A site, the heme a group serves as a type of porphyrin with an iron center, where the four pyrrole units are interconnected through methenyl bridges in a planar conformation to form an aromatic macrocyclic structure. In addition to the four nitrogen donor atoms from the porphyrin ring, imidazole moieties from His61 and His378 are coordinated to the iron center as axial ligands, giving a six-coordinated low-spin state. Quantum mechanics/molecular mechanics (QM/MM) studies later suggested two plausible pathways underlying electron transfer from heme a to heme a₃ through either propionate groups or metal centers of the two hemes (21, 22). The electron can reach the coordination sphere of heme a₃ comprised of porphyrin and one axial His376 in a five-coordinate high-spin state, ensuring structural integrity that efficiently facilitates electron transfer within the enzyme (23, 24). The Cu_B in a trigonal planar coordination composed of His240, His290, and His291 is located parallel to the plane of heme a₃, establishing a binuclear heme a₃/Cu_B site (21, 22, 25, 26). The reduction of Cu_B center is cooperatively coupled to a proton transfer process, ultimately leading to the formation of a water molecule (27).

Proton transfer pathways in CcO

The movement of electrons concomitantly triggers the translocation of protons from the matrix into the intermembrane space (15). In addition to the electron transfer within CcO, extensive research has been dedicated to identifying the possible pathways for proton transfer in the enzyme: K-, D-, and H-channels, as illustrated in Fig. 2. During each turnover of O₂ reduction, a total of eight protons is taken up from the matrix; four of these protons participate in the formation of H₂O, while the remaining four protons are pumped across the membrane, generating electrochemical proton gradients. The K-channel transports two protons into a binuclear center of heme a₃/Cu_B via a conserved Lys362, which is responsible for the catalytic reaction of CcO to reduce O₂ into H₂O. The D-channel starting with Asp91 can facilitate the transfer of two additional protons to the active site of CcO. Moreover, the D-channel can deal with the remaining four protons to be pumped out across the membrane in a vectorial manner (13, 28-31). The proton transfer process is closely related to hydrogen-bonded connection through water molecules, e.g., Asp92, Asn99, and Glu243 (28, 29). The potential of the D-channel to function as a dual pathway for two distinct types of protons, i.e., the substrate and the translocated ones, is still in question (32). Lastly, the third hydrophilic H-channel is another path for the vectorial proton transport in CcO. Separated from K- and D-channels, the proton pathway within H-channel is opened once the heme a₃/Cu_B site is reduced. The binding of O₂ is proposed to close the H-channel via an amide bond gate, suggesting its alternative role as a dielectric well (33). The unidirectional proton transport through the H-pathway is indeed facilitated upon the closure of water channel (27).

Copper insertion of CcO

The metalation of heme and copper centers within CcO is mediated by ferrochelatase and metallochaperone Cox17, respectively. In particular, apo-form of Cox17 is first synthesized in the cytosol, and imported into the mitochondrial intermembrane space through the MIA pathway (34, 35). As illustrated in Fig. 1A, Cox17 subsequently supplies Cu(I) to Cu_A and Cu_B in CcO (36). A metal-binding motif in Cox17 is conserved as Cys-Xxx-Xxx-Cys and Cys-Xxx-Xxx-Met to facilitate the copper binding. Once copper is coordinated to Cys residues in Cox17, it is transferred to downstream proteins Sco1/2 for Cu(I) insertion into Cu_A site in CcO. The NMR structures of Cu(I)-bound Sco1 showed that Cu(I) is coordinated with two Cys from Cys-Pro-Xxx-Xxx-Cys-Pro and one additional His260, each of which is located at the third loop, helix α1, and the seventh β-strand, respectively (37). The Cu(I) binding through different regions contributes to conformational rigidity compared to apo-form, which is presumed to be an important driving force of copper transfer from Cox17 (37). In parallel, Cox17 can transfer Cu(I) into the Cox11 that is anchored to mitochondrial inner membrane for the formation of Cu_B center in CcO. As illustrated in Fig. 1C, the binuclear Cu(I) clusters in Cox11 in a dimeric state are composed of four thiolates from Cys100 and Cys102 from each monomer in a conserved Cys-Xxx-Cys domain (38). Macromolecular complexes, such as Cox17, PET191, and COA6, regulate the metalation to prevent oxidative stress (39). The inner disulfide bond within Cox17 is of particular importance, as it contributes to the structural stabilization of the Cys-His-Cys-His fold, whereas the outer disulfide bond defines the structural environment of its copper-binding site (40). Taken together, the metallochaperones, Cox17, Sco1/2, and Cox11, play pivotal roles in transporting cytosolic copper into mitochondrial cuproenzymes, as well as maintaining redox homeostasis for CcO assembly (36, 41).

During oxidative phosphorylation, the leakage of a few electrons allows one-electron reduction of O₂ and produces superoxide anion (O₂^•−). Even though O₂^•− acts as a signaling molecule, its overproduction leads to oxidative stress that damages cellular lipids, proteins, and nucleic acids, which are found in the pathogenesis of heart failure, hypertension, cancer, neurodegeneration and aging (42-45). O₂^•− is promptly transformed into hydrogen peroxide (H₂O₂), a membrane-permeable ROS, which initiates the Fenton reactions to yield the immensely reactive hydroxyl radical (•OH) species (46). •OH species can damage mitochondrial DNA, as well as prompt carcinogenesis, apoptosis, and necrosis through nucleosome-driven fragmentation, hindering chromatin-related gene transcription regulation (47, 48). SOD as a detoxifying enzyme could maintain the cellular redox balance, protecting cells from oxidative damage (49). The antioxidant SOD family classified as SOD1, SOD2, and SOD3 plays a crucial role in converting O₂^•− into O₂ and H₂O₂, thereby preventing the formation of more reactive ROS, such as •OH (49). Among them, Cu(I/II)- and Zn(II)-containing SOD1 is primarily localized in the cytoplasm, but a fraction of it has also been identified in the intermembrane space of mitochondria (50), which will be further discussed in this review.

Coordination mode of Cu(I/II) and Zn(II) in SOD1

The catalytic functions of SOD1 stem from the coordination of Cu(I/II) and Zn(II) within the active site, each of which is responsible for electron transfer and structural basis, respectively. The Cu(I/II) center in SOD1 demonstrates one-electron redox chemistry between two oxidation states, facilitating the catalytic disproportionation of O₂^•− through ping-pong mechanism (Fig. 3A): (i) O₂^•− into O₂; (ii) O₂^•− into H₂O₂. As illustrated in Fig. 3B, Cu(II) is coordinated with His46, His48, His63, and His120 in a distorted square-planar geometry (51). Zn(II) participates in a distorted tetrahedral coordination with His63, His71, His80, and Asp83 throughout the entire catalytic cycle for SOD1 (52, 53). Notably, such bimetallic coordination is connected through the bridging imidazolate ring from His63, emphasizing its role in the structural flexibility of SOD1 (54). The oxidation of O₂^•− into O₂ is attributed to the reduction of Cu(II) to Cu(I), where Cu(I) can be, in turn, bound to three His residues, i.e., His46, His48, and His120. At the same time, the imidazolate of His63 is protonated until the second O₂^•− is introduced. The reduction of O₂^•− is coupled with proton transfer from the protonated His63 and a water molecule, generating H₂O₂ and reestablishing the imidazolate bridge between Cu(II) and Zn(II). The catalytic prowess of Cu(I/II) arises from its inherent properties of adopting a wide range of oxidation states, accompanied by geometric accommodation (55).

Previous in vivo studies have found the non-metalated SOD1 species in the transgenic mouse models (His46Arg and His48Gln) of amyotrophic lateral sclerosis (ALS) (56). Pertinent to the context of Cu(I/II)-binding residues (i.e., His46 and His48), the metalation state is closely related to the pathogenesis of the disease. On the other hand, Zn(II) primarily assumes a structural role in SOD1; however, this does not imply its exclusion from the catalytic function of the enzyme (57). Notably, a double mutation at Thr135 and Lys136 allosterically disrupting the Zn(II) complexation in SOD1 results in poor antioxidant activity, as well as structural destabilization, underscoring the pathological implication of Zn(II) deficiency in oxidative stress (58). It is worth mentioning that the denaturation of mutant SOD1 (His43Arg) was shown to remove both Cu(I/II) and Zn(II), which in turn exhibited pro-oxidant activity against H₂O₂. This behavior is attributed to the rebound Cu(I/II) with the altered ligand environment, particularly excluding the His120 residue (59). Additionally, the apo-form of the mutant SOD1 (His46Arg) showed pro-oxidant activity following subsequent Cu(II) binding at the Zn(II) coordination sphere (53). These observations collectively highlight that the antioxidant ability of SOD1 emanates from its distinctive coordination chemistry of the binuclear metal centers interconnected by the imidazolate bridging ligand.

Metalation of mitochondrial SOD1

The maturation process of SOD1 encompasses a series of post-translational modifications that include metalation, intramolecular disulfide bond formation, and dimerization. The initial step involves the binding of Zn(II) to the reduced apo-SOD1 monomer through an unknown mechanism, which could facilitate the dimerization, without inducing further aggregation (60). The molecular mechanism for copper insertion into cytosolic SOD1 has been relatively well established: Copper chaperone for SOD1 (abbreviated as CCS in Fig. 1A) composed of three distinct domains mediates the Cu(I) binding and disulfide bond formation in SOD1 (61). Specifically, domain 1 is proposed to be responsible for copper loading in CCS, although its precise role in SOD1 activation remains a subject of debate, compared to other domains (62). The domain 2 of CCS with a significant sequence homology with SOD1 of around 47% enables the chaperone to specifically recognize immature SOD1 through protein–protein interactions (61). On the other hand, the domain 3 with a Cys-Xxx-Cys motif is tasked with transferring Cu(I) into SOD1, aiding in the formation of disulfide bond within the enzyme (61). Previous works by Banci and coworkers utilized in-cell NMR spectroscopy to uncover the progression of SOD1 maturation, including Zn(II) and Cu(I) metalation, homodimer formation, and CCS-dependent disulfide bond formation in human living cells (63). Note that a loss of copper in SOD1 or the removal of free thiol groups (e.g., Cys6Ala, Cys111Ser) has been demonstrated to contribute to the in vitro amyloid fibrillation of the enzyme (64), underscoring the significant role of SOD1 maturation in its conformational state and catalytic function.

In the case of mitochondrial SOD1, the source of copper in the intermembrane space has been less understood, however. The unfolded apo-SOD1 and CCS are imported into the intermembrane space via MIA pathway (34, 35). In parallel, the low-molecular mass Cu(I/II) complexes are proposed to be transported to intermembrane space through outer membrane porins, subsequently interacting with CCS for Cu(I) insertion into SOD1. Cu(I/II) complexes can alternatively be stored in the mitochondrial matrix through mitochondrial phosphate carrier (PiC) at the inner membrane, whose mammalian homolog is solute carrier family 25 member 3 (SLC25A3) (65, 66). Upon transporting the Cu(I/II) complexes back to the intermembrane system through unknown transporters, mitochondrial CCS may contribute to the maturation of mitochondrial SOD1 (67). In the context of the role of SLC25A3 in the storage of mitochondrial Cu(I/II), the knockdown of SLC25A3 significantly reduced the SOD1 activity by 60% (68).

Pathogenic implication of SOD1 aggregates

The misfolding of SOD1 and its accumulation in the spinal cord closely correlate with the onset of ALS (69). In particular, post-translational modifications of SOD1 encompassing metalation, intramolecular disulfide bond formation, and homodimerization could affect its aggregation propensity (69), highlighting the role of wild-type SOD1 aggregates in the pathogenesis of sporadic ALS (70). Depletion of metal ions in wild-type SOD1 or its oxidation can lead to the formation of large, stable, and soluble aggregates, originating from the intermolecular disulfide bond between Cys6 and Cys111 (71, 72). These aggregates may hinder axonal transport, undergo cell-to-cell transmission via exosome, and exhibit pro-oxidant cytotoxic profiles (72-74). Moreover, recent work by Medinas, Hetz, and coworkers demonstrated that endoplasmic reticulum stress could induce SOD1 misfolding and the aggregation through cross-linked disulfide bonds in the transgenic mice overexpressing human wild-type SOD1 (75).

It is noteworthy that over 150 mutations in SOD1 located on chromosome 21 have been implicated in the development of familial ALS (69), where the common mutations include Asp90Ala, Ala4Val, and Glu93Ala (76). Consistent with the coordination chemistry of SOD1 as depicted in Fig. 3, mutations at copper-binding residue, either His46Arg, His48Gln, or both, as well as a quadruple mutant His46Arg/His48Gln/His63Gly/His120Gly, exhibit lowered binding affinities for ⁶⁴Cu, compared to wild-type SOD1 (77). These mutants also showed a failure to adopt native dimeric conformation, potentially emphasizing the importance of copper coordination in stabilizing the SOD1 structure (77). Notably, the monomeric form of SOD1 resulting from dimer destabilization could be susceptible to unfolding and the subsequent formation of oligomeric aggregates (78), where the primary seeding unit is presumed to be a trimeric species (79). These SOD1 oligomers with nonnative quaternary structures differing from the native homodimer conformation could promote cell death in motor neuron-like cells (79). Recent computational work by Rajasekaran also demonstrated that His46Arg mutation on SOD1 leads to the loss of Cu(II) binding, enhancing the formation of toxic aggregates (80).

Copper imbalance, such as overload or deficiency, could directly affect the catalytic activity of mitochondrial cuproenzymes, which are involved in energy metabolism or antioxidant defense. For example, the decrease in systemic copper could inhibit the activity of CcO during oxidative phosphorylation, ultimately declining the ATP levels (81). In addition, the treatment of a copper chelator could establish a copper-deficient environment in living cells, decreasing the mitochondrial SOD1 activity (82). The overload of Cu(II) could inhibit mitochondrial O₂ consumption, and induce phospholipid peroxidation in rat liver mitochondria (83). In addition to the role of Cu(I/II) in ROS scavenging, it could trigger oxidative stress when the balance between the production and removal of ROS is shifted (84). Upon redox cycling of Cu(I/II) and H₂O₂ in the presence of cellular reducing agent ascorbate, Fenton reactions could yield •OH, the most biologically active ROS. The •OH then instantly reacts with surrounding organic molecules, resulting in mitochondrial disruption and caspase cascade activation (85), the latter of which can lead to apoptotic cell death (86-88).

A recent finding by Tsvetkov et al. unveiled a unique type of cell death, known as cuproptosis, which arises from copper accumulation within the mitochondrial matrix (89). Cuproptosis is characterized by the oligomerization of lipoylated mitochondrial proteins and the depletion of iron–sulfur clusters, differing it from other programmed cell death pathways, such as apoptosis, ferroptosis, or necroptosis (89). Although the precise mechanism governing the transfer of cytosolic copper into mitochondria is still unclear, Cu(II) complexed with an exogeneous ionophore elesclomol (ES) has been shown to translocate from the intermembrane space to the mitochondrial matrix through SLC25A3 (vide supra). The dissociation of Cu(II) from ES and its subsequent reduction to Cu(I) can be mediated by mitochondrial matrix reductase ferredoxin-1 (FDX1) (66). The released Cu(I) is proposed to bind to the lipoyl moiety of dihydrolipoamide S-acetyltransferase (DLAT), thereby facilitating the aggregation. While the coordination chemistry of Cu(I) in DLAT has not been elucidated, the conformational and functional impairment of DLAT can be directly linked with mitochondrial dysfunction, given its involvement in pyruvate decarboxylation situated between glycolysis and the TCA cycle (90). Concurrently, Cu(II)–ES complexes may interact with FDX1, ultimately inhibiting the biogenesis of iron–sulfur clusters (91). These clusters function in electron transport through complexes I, II, and III to Cyt c along the electron transport chain; thus, cuproptosis might also contribute to the disruption of ATP synthesis (91). The treatment with copper chelator tetrathiomolybdate shows promise in rescuing cell death induced by Cu(II)–ES, highlighting the role of copper in mitochondrial toxicity (89).

Trace amounts of transition metal ions play crucial roles in various biological processes that encompass energy metabolism, signal transduction, and the synthesis of biomolecules (92, 93). Particularly noteworthy within the context of one-electron transfer, the redox-active Cu(I/II) as a cofactor within CcO and SOD1 contributes to ATP synthesis and antioxidant defense, underscoring the importance of comprehending the ligand environment at the active center. This mini review presents an up-to-date overview of the coordination chemistry of the mitochondrial cuproenzymes, focusing on their catalytic mechanisms. Additionally, we explore how the metalation occurs in the copper-trafficking system, and discuss the impact of copper dyshomeostasis in cell death.

Abnormalities in copper metabolism, often stemming from mutations in copper-transporting P-type ATPases, can give rise to systemic copper imbalances, presenting as either copper deficiency or accumulation. These perturbations are intricately associated with the onset of Menkes disease or Wilson’s disease (94-96). To the best of our knowledge, the prevailing treatment for Menkes disease currently revolves around the parenteral administration of copper–histidine complexes (97). Meanwhile, copper ionophores, including disulfiram, bis(thiosemicarbazone) analogs, and ES, exhibit promise in restoring the activity of mitochondrial cuproenzymes under copper-deficient conditions (98-100), hinting at the therapeutic potential of ionophore-based copper supplementation in ongoing clinical trials. In parallel, copper chelation therapy employing D-penicillamine, triethylenetetramine, or tetrathiomolybdate holds the ability to effectively lower the elevated copper levels, mitigating copper overload toxicity (101). Taken together, advancing our understanding of the coordination chemistry inherent in mitochondrial cuproenzymes holds the potential to unveil novel therapeutic approaches for copper-related disorders in the near future.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00251098).

The authors have no conflicting interests.