Mitochondria contribute to intracellular signaling and regulatory networks in part by coordinating trafficking of metabolites into and out of mitochondria (Cunningham and Rutter, 202020). The mitochondrial carrier family (MCF/Slc25 family) form the largest group of transporters in the IM and are responsible for most of the metabolic flux to, and from, the organelle. In humans there are 53 members that are collectively responsible for the transport of numerous substrates including TCA cycle intermediates, nucleoside di- and triphosphates, amino acids, and metals (Cunningham and Rutter, 202020). Sixteen of the 53 MCFs have no known substrate and the established promiscuity of some of the characterized transporters raises the possibility that even those 37 with known substrates may have additional substrates. Notable members of this family involved in metals and metal cofactor assembly are SLC25A3/Pic2 which is a copper and phosphate transporter (Vest et al., 2013b; Boulet et al., 2018), SLC25A37/MITOFERRIN1/Mrs3 and SLC25A28/MITOFERRIN2/Mrs4 which are involved in mitochondrial iron homeostasis and necessary for iron transport (Shaw et al., 2006; Paradkar et al., 2009; Troadec et al., 2011; Christenson et al., 2018; Seguin et al., 2020), as well as SLC25A38 which transports glycine for heme synthesis (Guernsey et al., 2009).

MCF transporters have a conserved fold consisting of three repeats of approximately 100-amino acids that contain two transmembrane helices connected by a loop with a short α-helix (Ruprecht and Kunji, 2020). This structural insights into the family of transporters is based on the structure determined for ADP/ATP exchanger that was crystalized with inhibitors that stabilized the protein in either an open to the IMS state (c-state) or open to the matrix state (m-state) (Pebay-Peyroula et al., 2003; Ruprecht et al., 2019). The MCF fold is stabilized by salt bridges that form at the closed end of the substrate binding pocket depending on which state the protein adopts. The first,third, and fifth transmembrane helices contains a conserved PX (D/E)XX (R/K) motif that forms salt bridge and hydrogen bonding contacts on the matrix side of the binding pocket. A complementary (Y/F) (D/E) XX (R/K) motif in second,fourth, and sixth helices is found on the IMS side (Ruprecht and Kunji, 2020). The strength of these salt bridge interactions is an important predictor of the requirements and directionality of transport. Two examples exist of single amino-acids changes in MCF that give rise to changes in specificity. In yeast, Rim2 is a bifunctional pyrimidine and iron transporter but E248A mutant specifically disrupts mitochondrial iron transport activity while K299A mutant specifically abrogated pyrimidine nucleotide transport and exchange (Knight et al., 2019). Similarly, a L175A mutation can make the bifunctional copper and phosphate transporter SLC25A3 specific for copper transport (Zhu et al., 2021).

The metal transporting MCF SLC25A3 and SLC25A37 are predicted to be uniporters that import metals into the matrix. Both SLC25A3 and SLC25A37 have been successfully reconstituted into proteoliposomes to demonstrate transport activity (Boulet et al., 2018; Christenson et al., 2018). The yeast homologs, Pic2 and Mrs3 and mammalian SLC25A3 have also been expressed in L. lactis. Expression in L. lactis allows for monitoring import of metals into this organism. Both Pic2 and Mrs3 can transport copper in L. lactis although for Mrs3 to facilitate copper transport iron had to be chelated/limited presumably to prevent competition (Vest et al., 2016). The plant variant of SLC25A37 was also shown to have the ability to transport iron and copper (Bashir et al., 2011a; Bashir et al., 2011b). But our understanding about the interplay between these transporters in vivo is limited.

In addition to a role in transporting metals the MCF family has the potential to form a scaffold for protein-protein interactions to enhance the assembly of the mitochondrial metalloproteome. The MCF proteins have been isolated in many distinct large complexes. The large interactome of these proteins is exemplified by the ADP/ATP exchange proteins (Claypool et al., 2008; Claypool, 2009; Senoo et al., 2020). These proteins play critical role in delivery of the energy currency to the cell and have been isolated in large multiple protein complexes. These ADP/ATP exchanger protein complexes can also include other MCFs including the copper and phosphate carrier (SLC25A3), calcium dependent carriers (SLC25A13, SLC25A24, SLC25A25), the tricarboxylate carrier (SLC25A1), and the carnitine/acylcarnitine carrier (SLC25A20) (Claypool et al., 2008). How these interactions affect and/or regulate the specificity and/or transport activity is unknown due to the difficulties in assessing these biochemical characteristics in vivo or with purified components.

There are 4 mitochondrially-localized ABC-transporters (ABCB6, ABCB7, ABCB8 and ABCB10) which are part of a large superfamily of proteins that mediate nucleotide-dependent transport (Cunningham and Rutter, 202020). ABC-transporters have four core domains for function. Two transmembrane domains that provide specificity by forming a binding site, and two nucleotide binding domains to hydrolyze ATP to facilitate transport of the bound ligand. Conservation is found in the nucleotide binding domains with variability in the transmembrane domains to allow for a broad number of substrates (Wilkens, 2015). The four mitochondrial ABC-transporters are involved in moving Fe-S clusters and heme related metabolites into and out of mitochondria. ABCB6 is localized to the OM (and other cellular locations including the cell surface). ABCB6 has been proposed as porphyrin transporters (Krishnamurthy et al., 2006) and has been shown to specify the Langereis blood group (Helias et al., 2012).

ABCB7, ABCB8 and ABCB10 are all localized to the IM and the exact molecule transported by each is still unclear. ABCB7 links to cofactor assembly are because it was identified as a candidate causative gene for X-linked sideroblastic anemia with spinocerebellar ataxia and the yeast homolog ATM1 is regulated by iron and participates in maintaining Fe-S cluster homeostasis (Pondarre et al., 2007; Lill, 2020). Similarly phenotypic data links ABCB8 to iron homeostasis as a mutation of Abcb8 results in cardiomyopathy with increased mitochondrial iron accumulation with decreased activity of cytosolic Fe-S clusters proteins. Finally, the Abcb10 knockout in mouse is embryonic lethal due to anemia. In support of a role of this transporter in iron homeostasis, Abcb10 forms a complex with MITOFERRIN1 (SLC25A38) and ferrochelatase (FECH) that enhances heme synthesis, Figure 3 (Yamamoto et al., 2014; Medlock et al., 2015; Maio et al., 2019). While the substrate for ABCB10 has not be definitively identified, multiple studies suggest different substrates related to heme synthesis and it is clear this protein plays an important role in iron metabolism. The characterization of a larger complex that includes multiple heme synthesis related proteins is a continuation of the theme observed for the MCF proteins, that complexes of transporters and targets can form a potential regulatory mechanism for metal cofactor assembly or metal homeostasis.

Other notable mitochondrial transporters involved with metals are sideroflexins (Slc56). One of the five sideroflexin proteins, sideroflexin-1, has been shown to have a role in serine transport in mitochondria, and both sideroflexin-1 and sideroflexin-3 have been linked to iron homeostasis and Fe-S cluster biogenesis (Tifoun et al., 2021). Sideroflexins are 5 transmembrane domain proteins that contain a conserved mitochondrial tricarboxylate/iron carrier domain (PFAM: 03820). All members share the same topology with the amino-terminal inside and the carboxy-terminal outside. Sideroflexins 1–4 have sites of post-translational modification including acetylation that regulate activity and stability.

Cellular zinc transporters have been reported to localize to different mitochondrial membrane in some cell types. Zinc transporters can be broadly categorized in two groups ZIP (Irt-like) and ZnT (Kambe et al., 2014). ZIP transporters generally act as in zinc import while ZnT class act as zinc exporters. ZIP family members contain 8 transmembrane domains, with the proposed metal-binding residues embedded in transmembrane helices 4 and 5 and a cytoplasmic (inside/IMS in mitochondria) region between transmembrane 3 and transmembrane 4, that is, important for regulation including protein stability the exact roles of this domain are still under investigation (Kambe et al., 2014). It is also important to recognize that the ZIP family homologs have been shown to transport a variety of divalent ions including but not limited to iron, manganese, copper. Therefore, these transporters could have effects on other metals in mitochondria.

Copper is found within each of the mitochondrial compartments. In the IM, cytochrome c oxidase is the multi-subunit complex that catalyzes the final steps of the electron transport chain (ETC). It accounts for about 25% of mitochondrial copper and is the major cuproenzyme present in this organelle (Cobine et al., 2004). In addition, a small percentage of cellular Cu, Zn superoxide dismutase (Sod1) and its copper chaperone, Ccs1, is localized to the IMS. However, these proteins do not account for a significant proportion of the total mitochondrial Cu. The majority (up to 70%) is found in the matrix and is bound to a biochemically characterized but structurally undefined complex known as the copper ligand (CuL) (Cobine et al., 2021). The copper in the matrix is transported by MCF SLC25A3/Pic2, that is, capable of transporting Cu and CuL as a substrate (Vest et al., 2013b; Boulet et al., 2018; Zhu et al., 2021). The data in multiple experimental models shows that the copper in the matrix is redistributed to the IMS for the assembly of cytochrome c oxidase (COX) and superoxide dismutase (Sod1). The matrix distribution model, that is, based on phenotypic data from mutants of the copper importer and biochemical competition assays in yeast under copper stress. These phenotypes are reproduced in mammalian cell culture without the need for copper restriction (Boulet et al., 2018). However, the identity of the transporter responsible for copper export is unknown and therefore the regulation of this pathway is poorly understood. An alternative pathway that feeds copper directly from the cytosol into these enzymes has been suggested based on purification of copper complexes that are thought to be in the IMS, this alternative pathway could operate under conditions where copper is abundant (Lindahl and Moore, 2016; Cobine et al., 2021).

Copper availability in the IMS is critical for the assembly of COX (Cobine et al., 2021). However, copper levels are restricted and therefore chaperone proteins are required to enhance the assembly process. This evidence for the copper delivery pathway is a combination of in vitro observations and genetic suppression experiments. In the IMS, Cox17 presents copper to Sco1 and Cox11 for assembly of the copper sites in COX called Cu_A and Cu_B sites, respectively (Horng et al., 2004; Cobine et al., 2021). Cox17 is a soluble protein that adopts a coiled coil-helix-coiled coil-helix fold stabilized by two disulfide bonds. The Cox17-Sco1 interaction is the most thoroughly studied copper transfer reaction that occurs in the IMS. Cox17 donates copper to the exposed CxxxC site on Sco1. Studies have shown that the reactions proceed from the Cu-loaded, partially oxidized conformer of human COX17 to SCO1 via transient interactions (Banci et al., 2007; Banci et al., 2008; Banci et al., 2011). This Cu-delivery pathway is further supported by the observation that SCO1 overexpression can rescue the phenotype of a COX17 mutant cell (Glerum et al., 1996). Sco1 subsequently insert the Cu into Cox2 to form the Cu_A site in COX. Multiple redox related steps are involved in the COX assembly process. Mammalian COA6 (and yeast Coa6) and SCO2 are two of the proteins with roles in regulating the reduction state of assembly proteins and the target COX2 (Pacheu-Grau et al., 2015; Soma et al., 2019; Swaminathan and Gohil, 2022). Most recently an additional interplay between the Cu assembly process and heme has been identified with a role discovered for COA7, a heme binding protein, that transiently interacts with the copper metallochaperones SCO1 and SCO2 and catalyzes the reduction of disulfide bonds within these proteins (Formosa et al., 2022). To build the Cu_B site, cells use a different battery of assembly proteins. Cox11 is a membrane bound factor required for the insertion of the Cu in COX1 and is also a recipient of copper from Cox17 (Baker et al., 2017a). Like Sco1 pathway specific accessory factors are required to maintain the redox state of the proteins in the pathway. Cox19 is required for the reduction of two critical copper binding cysteines in Cox11 (Bode et al., 2015). Copper transfer occurs via an interface than, that is, distinct from that used for interaction with Sco1 (Horng et al., 2004; Cobine et al., 2006). The requirement for this multi-step interplay of protein-protein interactions in the IMS to facilitate copper loading reinforces the idea that limited copper availability is a major complication for the correct metalation of cuproenzymes. It is also important to note that mutation in SCO proteins can cause remodeling of cellular copper homeostasis. The mutant cells and mice show copper deficiencies due to inappropriate degradation of the high affinity importer CTR1 or excess activity of the copper exporter ATP7A (Leary et al., 2007; Hlynialuk et al., 2015; Baker et al., 2017a; Baker et al., 2017b). During the cellular copper deficiency mitochondrial copper is maintained suggesting a possible prioritization of the matrix copper under these conditions (Dodani et al., 2011).

To date at least six gene deletions in yeast have been shown to limit matrix copper accumulation based on analysis of the purified mitochondria and mitochondrial targeted copper responsive reporters. The proteins implicated in copper import/maintenance are the MCF proteins: Pic2, Mrs3 and the assembly factors Coa1, Coa4, Coa6 and Shy1 (Pierrel et al., 2007b; Bestwick et al., 2010; Ghosh et al., 2014). Coa6 is a Cx₉C containing protein of the IMS, that is, responsible for regulating the redox state of proteins involved in Cox2 assembly machinery (Ghosh et al., 2016; Soma et al., 2019; Swaminathan and Gohil, 2022). Coa1, Coa4 and Shy1 are all linked to the translation and assembly of Cox1 (Pierrel et al., 2007b). The mammalian homologs of Shy1 (SURF1) and Coa1 (COA1/MITRAC15) are part of the mitochondrial translation regulation assembly intermediate of cytochrome c oxidase (MITRAC) complex (Mick et al., 2012). This complex is another example of a large protein complexes that form in mitochondria to facilitate more efficient processing and/or substrate channeling. The assembly of the respiratory chain complexes proceeds in modular fashion and require complex-specific assembly factors to stabilize the intermediates for the complete maturation of complexes. Because maturation of the enzyme complexes is a sequential process during which new components and cofactors are added, the composition of the MITRAC complex changes during the process (Mick et al., 2012). Since MITRAC is facilitating the translation and assembly of COX1 and this is the heme and copper containing subunit it is intriguing to proposed that this complex could be critical for regulating total mitochondrial copper. It should be noted that only Coa1, Shy1 deletion and not other MITRAC homologs in yeast cause the copper deficiency.

Iron enters mitochondria through multiple mechanisms. The most well-studied example of mitochondrial iron import is via MCF proteins MITOFERRIN1 in mammals and Mrs3 and Mrs4 in yeast (Dietz et al., 2021a). MITOFERRIN and Mrs3 have been shown to modulate the transport of iron in vivo and in vitro while Mrs4 is transcriptionally activated by iron depletion, suggesting a role for homeostasis of iron in mitochondria. Once iron crosses the IM it is used for the synthesis of heme, Fe-S or is bound by other ligands for storage. Genetic experiments suggested that FRAXATIN (or at least the yeast homolog Yfh1) played a role as a chaperone directing iron to produce Fe-S cluster largely based on an iron accumulation phenotype and the activation of Fe-regulated transcription in yeast in YFH1 mutants (Llorens et al., 2019). However additional experimental evidence suggests that this is not a required function of Yfh1. The phenotypes of yfh1∆ can be bypassed completely by a compensatory mutation in the Fe-S cluster scaffold protein IscU (Yoon et al., 2015). This combined with other approaches suggested the primary function of Yfh1 is sulfur delivery to form Fe-S clusters. Once iron reaches the iron-sulfur cluster machinery it is synthesized into 2Fe-2S clusters that are then inserted in mitochondrial targets or exported to the cytosol where the cytosolic iron-sulfur assembly (CIA) machinery matures them to 4Fe-4S clusters and/or inserts them into other targets (Lill, 2020). The other major iron cofactor is heme which is synthesized when iron is inserted into protoporphyrin IX by ferrochelatase (Dailey and Meissner, 2013). Protoporphyrin IX is synthesized by a series of enzymes that are split between the cytosol and the mitochondria with the initial steps of porphyrin synthesis taking place in mitochondria before export of the intermediates for maturation in the cytosol until the final steps occur back in the mitochondrion.

The mechanisms of zinc and manganese uptake into the mitochondrial matrix are not well understood. Zinc is required in the mitochondria for multiple functions including electron transport chain function (COX), ATP synthesis (Atp32), protection against oxidative stress in the IMS (Sod1), lipid transport (Yme1L), mitochondrial dynamics (Oma1, Yme1L), processing peptidase (MPP), and mitochondrial intermediate peptidase (MIP). A genetic screen to identify a transporter using chemical sensors of available zinc uncovered an unexpected connection between Complex III assembly and mitochondrial zinc. A gene designated MZM1 was identified and characterized to show the gene product was required for both zinc maintenance and complex III assembly (Atkinson et al., 2010; Atkinson et al., 2011). However, this did not result in identification of a zinc transporter in mitochondria. Some studies have suggested that zinc can be imported to mitochondria by the calcium uniporter (MCU) (Gazaryan et al., 2007; Ji et al., 2020). While others have reported dual location of zinc transporters suggesting that zinc entry to the IMS could be facilitated by Zip1 that was shown to localize to the OM (Cho et al., 2019). Further still the zinc transporters Zip7 and ZnT7 have also been shown to have dual localization in mitochondria and ER and expression of Zip7 and ZnT7 contribute to cellular zinc exchange between the organelles in certain cell types (Tuncay et al., 2019). Finally Znt9 (SLC30A9) was identified as a zinc exporter in mitochondria (Deng et al., 2021) and SLC25A25 has been implicated in at least regulating uptake of zinc as deletion can reverse phenotypes induced by SLC30A9 deletion (Ma et al., 2022). Perhaps redundancy is the reason for the failure of the yeast genetic screens to yield a single candidate.

Manganese has a single target in the mitochondria in the form of superoxide dismutase 2 (SOD2). This matrix localized enzyme is responsible for protection against a subset of the reactive oxygen species generated. Deletion of SOD2 in mammalian models is embryonic lethal demonstrating its essential role. However, to date no definitive data exists as the identity of the manganese transporter that provides this metal for the matrix for SOD2 assembly and activity. In yeast deletion of the gene MTM1, which encodes a MCF, caused decreased activity of Sod2 but did not prevent manganese accumulation (Luk et al., 2003). It was subsequently shown that deletion of MTM1 changed iron availability resulting in mismetallation and inactivation of Sod2 by iron (Yang et al., 2006; Naranuntarat et al., 2009). Critically this is an example of the complex requirements for essential cofactor assembly in mitochondria and highlights the need for additional research on regulatory mechanisms that change availability.

Heme or Fe-protoporphyrin IX is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans, the heme biosynthesis is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. Key enzymes in the pathway that are found in the mitochondrial matrix are aminolevulinic acid synthase, which utilizes glycine and succinyl-CoA to produce aminolevulinic acid, and FECH, which utilizes iron and protoporphyrin IX to make heme (Phillips, 2019). Overall, the pathway has been well studied and all the biosynthetic enzymes structurally characterized. Nevertheless, understanding of the regulation of heme synthesis in different cells and factors that influence this process remains incomplete. Some details of the transcriptional regulation in the context of erythroid development are known, but transcriptional regulation in most other cell types or cellular conditions is much less understood (Dailey and Meissner, 2013). Recent work supports post-translational mechanisms for the regulation of the mitochondrial heme biosynthesis enzymes (Chung et al., 2017). Studies have shown protein-protein interactions regulate heme precursor levels (Medlock et al., 2015) and the incorporation or disruption of cofactors into key enzymes (Shah et al., 2012; Kardon et al., 2020).

Fe-S clusters are essential cofactors for the ETC and many other biochemical reactions and processes (Lill, 2020). The cofactor assembly systems are required to sequester, chaperone, and regulate delivery of the cofactor. The assembly proteins are not strictly required for building Fe-S cluster, because the clusters will self-assemble in solution, but is thought these proteins are necessary to circumvent the toxicity and indiscriminate reactivity of free iron and sulfide. Defects in the biosynthesis in Fe-S cluster cause dysfunction in mitochondria, neurodegenerative and cardiovascular disease, genomic instability, and the development of aging and cancer.

Mitochondria assemble not only mitochondrial Fe-S clusters but are also involved in the biosynthesis of Fe-S clusters for proteins in the cytosol and in the nucleus (Lill, 2020). These cytosolic and nuclear Fe-S cluster proteins with essential functions for example, ABC protein Rli1, which participates in ribosome assembly and ribosome recycling during termination of polypeptide synthesis (Kispal et al., 2005). Eukaryotic replicative DNA polymerases which also contain a Fe-S cluster in their C-terminal domain (Paul and Lill, 2015). The Fe-S cluster cofactor appears to be indispensable for efficient interaction with their accessory proteins during DNA replication.

Molybdenum is a versatile redox element, that is, used by enzymes to catalyze diverse reactions. While not a focus of this review we will briefly discuss molybdenum cofactor (MoCo) as it is synthesized in mitochondria and has links to iron and copper. MoCo is an ancient cofactor involved in sulfur, drug, and nucleotide metabolism. MoCo synthesis begins in mitochondria with guanosine 5′-triphosphate which is converted via the two proteins to cyclic pyranopterin monophosphate (cPMP). Both mitochondrial enzymes in humans, MOCS1A and MOCS1B, are encoded by a single gene alternatively spliced to yield either protein. For MOCS1A exon one encodes the mitochondrial localization signal, and for MOCS1B localization is designated via exon 10 (Mayr et al., 2020). Interestingly MOCS1A can also be localized to the cytosol in primates via exon 1b, and while it appears to be functional its physiological role in the cytosol is unclear. MOCS1A belongs to the radical SAM enzyme superfamily and has two iron-sulfur clusters (Hänzelmann et al., 2004), specifically 4Fe-4S clusters, thus is connected to iron and iron-sulfur cluster homeostasis. cPMP is then exported across the mitochondria membranes to the cytosol where it is further modified (Mendel, 2013). In plants this is mediated by ATM3 which is the homolog of yeast Atm1 and mammalian ABCB7. Once in the cytosol MPT synthase transfers the two sulfurs to cPMP to create molydopterin (MPT). This intermediate can bind copper, and the copper may act as a protecting group as the cofactor synthesis proceeds (Mendel, 2013). The next step in synthesis is formation of the of adenylated-MPT before the final step of Mo insertion. Therefore, MoCo synthesis has overlap with copper and iron both utilizing cofactors and sharing a transport pathway.

The terminal enzyme of the heme biosynthesis pathway, ferrochelatase, has been identified to be part of a multi-protein complex or metabolon in the mitochondrial matrix. This complex has been studied in both mammalian cells (FECH) (Medlock et al., 2015) as well as yeast (Hem15) (Dietz et al., 2021b). In terms of the mammalian cells, most work has focused on the metabolon in developing erythroid cells, as these cells make a large amount of heme. An interesting finding was that mitochondrial heme metabolon interacted with MICOS proteins including MIC60, Figure 3 (Piel et al., 2016; Dietz et al., 2021b). Further work in yeast showed that loss of MICOS negatively impacts Hem15 activity, it affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic porphyrins (which arise from the heme intermediates porphyrinogens) that may cause oxidative damage. Restoring intermembrane connectivity using a heterologously expressed protein capable of tethering the IM and OM “artificially” in MICOS-deficient cells mitigates these cytotoxic effects (Dietz et al., 2021b). The artificial tether clearly shows the importance of mitochondrial ultrastructure to the function of this enzyme.

The localization of ferrochelatase to MICOS also provides a connection to iron and Fe-S cluster trafficking at these sites. Ferrochelatase was shown to interact with SLC25A37/MITOFERRIN1/Mrs3, ABCB10, and ABCB7 (Taketani et al., 2003; Chen et al., 2010; Medlock et al., 2015; Maio et al., 2019)). The connection with MITOFERRIN and ABCB10 is clear as iron is a substrate for the enzyme and thus can be directly trafficked for heme synthesis. The role of FECH and ABCB7 (Banci et al., 2007; Banci et al., 2008) interaction is less clear, except that FECH itself has an 2Fe-2S cluster (Dailey et al., 1994) and thus may regulate or be regulated by Fe-S cluster flux in the matrix. Overall, these data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme synthesis for the efficient import of heme precursors and export of heme.

In addition to the role Cox17 plays in COX assembly, it also been linked to a copper dependent interaction with the MICOS complex (Vanisova et al., 2019; Chojnacka et al., 2015). Cox17 interacts with Mic60 to modulate MICOS complex integrity (Chojnacka et al., 2015). This interaction does not involve Sco1 or Cox11. However, the Cox17-MICOS interaction is regulated by copper, Figure 4. Cox17 is therefore a factor involved in maintaining the architecture of mitochondria via the MICOS complex. The regulation of the MICOS complex and therefore IM structure by Cox17 and copper was unexpected. However, it further consolidates a link between the mitochondrial morphology, ETC integrity, and metal homeostasis. It was proposed that Cox17 may be facilitating protein-protein interactions that enhance MICOS stability or that it could be delivering of copper to the MICOS complex (Chojnacka et al., 2015). This would require a yet to be identified copper-binding site in the complex. Cryptic sites of copper binding leading to metalloallosteric regulation have recently been described in multiple well studied processes (lipolysis, proliferation, and autophagy) and therefore a precedent exists to identify these new sites (Ge et al., 2022). Another possible model would be that the presence of excess Cu-Cox17 would indicate that COX is maximally metaled and therefore closing of cristae junctions should proceed to allow for enhanced activity of the OXPHOS in the enclosed intercristae space, Figure 4.

The regulation of metal homeostasis is important to avoid the potential formation of reactive oxygen species (ROS). Superoxide dismutase (Sod1) is involved in the scavenging of ROS in the cytosol and in the IMS of mitochondria. Sod1 requires zinc and copper ions and an intramolecular disulfide bond to catalyzes the conversion of superoxide into hydrogen peroxide and water. The activation, localization, and retention (in IMS) of Sod1 is largely dependent upon the copper chaperone Ccs1 (Sturtz et al., 2001; Gross et al., 2011). Ccs1 is involved in the transfer of copper and disulfide bond formation in the maturation of Sod1. The assembly of Sod1 is also dependent on Mia40. Mia40 is responsible for import and oxidation of Ccs1 to the IMS. Ccs1 is responsible to “fold and trap” Sod1 in the IMS. An additional role for mitochondrial ultrastructure in Sod1 activity was uncovered. Mitochondria that are lacking any components of the MICOS complex can create unusual separations in the IMS. MICOS mutants accumulate a disproportionate amount of oxidized Sod1 (Varabyova et al., 2013). The authors speculate that the reduction of the disulfide bond in Sod1 is prevented by mitochondrial ultrastructure/compartmentalization (Varabyova et al., 2013). The metalation state of Sod1 in the MICOS mutants is unknown and perhaps mitochondrial copper status in MICOS mutations play some role in oxidation states of the Sod1. It is known that decreased mitochondrial copper can change the steady state levels of IMS localized Sod1.

Many mutations in human SOD1 are associated with amyotrophic lateral sclerosis (ALS), a nervous system disease that causes loss of muscle control. This debilitating disease has meant that numerous studies have investigated the role of mitochondrial SOD1 is disease progression. The accumulation of wildtype reduced Sod1 was dependent on the cristae architecture controlled by MICOS and mitochondria with MICOS defects showed an increase in mitochondrial accumulation of ALS-related reduced variants of Sod1 (Varabyova et al., 2013). This accumulation resulted in the accumulation of toxic superoxide and mitochondrial dysfunction (Varabyova et al., 2013). Further suggesting a role for correct mitochondrial ultrastructure in this disorder.