The function of the mitochondrial respiratory chain (MRC) depends on a series of redox reactions that transfer electrons from reduced substrates (NADH and FADH₂) to the final acceptor, molecular oxygen (O₂). The electronic transfer is mediated by MRC complexes I-IV and two mobile electron carriers, coenzyme Q (CoQ) and cytochrome c (cyt c), that donate electrons to complex III and complex IV, respectively. Complex IV or cytochrome c oxidase (COX) is the terminal oxidase, where four electrons are transferred from four cyt c molecules to one molecule of oxygen (O₂), reducing it to two molecules of water (H₂O). As part of the family of the heme-copper oxidases, COX catalytic centers contain both iron and copper (Pereira et al., 2001). Cyt c donates electrons to the Cu_A center, which is composed of two closely bound atoms of Cu contained in the core subunit COX2 (MT-CO2). On the other hand, O₂ reduction occurs in the catalytic center within the COX1 (MT-CO1) subunit, composed of a low spin heme a and the binuclear center of heme a ₃ and one Cu atom (Cu_B). The process of electron transfer is coupled to proton pumping from the mitochondrial matrix to the intermembrane space, contributing to the generation of the proton motive force essential for ATP synthesis (Pereira et al., 2008; Wikstrom et al., 2018). All cytochrome c oxidases possess a third core subunit (COX3; MT-CO3) that does not contain an active center but it is believed to be necessary to preserve the catalytic activity (Sharma et al., 2015). In addition to the three mitochondrial DNA-encoded core subunits, COX in animals, including Drosophila melanogaster (Brischigliaro et al., submitted), contains 11 “supernumerary” subunits with no catalytic role but important for the enzyme stability, function and regulation (Pitceathly and Taanman, 2018; Ramzan et al., 2021).

Human COX is assembled in a modular fashion, where each module is defined by each of the core subunits (Vidoni et al., 2017). Upon translation in the proximity of the mitochondrial inner membrane, MT-CO1 binds to a plethora of chaperones that help stabilize the apoprotein and serve as a platform for its metalation (Timon-Gomez et al., 2018). The assembly factor SURF1 is part of this intermediate complex and it is believed to have a role in heme A delivery to the catalytic center, whereas COX10 and COX15, two enzymes also essential for COX function, synthesize heme A (Timon-Gomez et al., 2018). Copper delivery to the Cu_B center in MT-CO1 is mediated by COX17 and COX11 (Cobine et al., 2006). The incorporation of the Cu_A center bound to MT-CO2 requires the participation of several different metallochaperones, namely COX17, COA6, SCO1 and SCO2, that function in the delivery of the two Cu ions to the apoprotein (Jett and Leary, 2018). All of these Cu-delivering proteins are localized in the mitochondrial intermembrane space and contain Cys residues, important for the redox regulation of their import, stability and Cu binding (Geldon et al., 2021). Genetic variants resulting in defects of structural subunits and assembly factors, including those involved in the metalation of COX such as COX10, COX15, SURF1, COA6, SCO1 and SCO2, are the cause of mitochondrial disease associated with COX deficiency in humans (Brischigliaro and Zeviani, 2021). In addition to COX deficiency, mutations in SCO1 and SCO2 produce an overall reduction in the Cu content in cultured cells and in tissues (Leary et al., 2007), related with the loss of the plasma membrane copper transporter CTR1 (Hlynialuk et al., 2015; Baker et al., 2017). Copper transport within the cell and into mitochondria must be accurately controlled due to the toxicity of this metal, which at high levels can determine redox stress and inactivate Fe-containing enzymes (Cobine et al., 2021). Decompensation of cellular Cu levels can lead to various diseases, including neurodegeneration (Cobine et al., 2021; Ruiz et al., 2021). Also, primary Cu deficiencies result in decreased COX activity, most likely contributing to the degenerative clinical course displayed by these patients (Spinazzi et al., 2014). The correlation between altered mitochondrial metal homeostasis and disease is not exclusive for Cu, as dysregulation of Fe is also well known to be associated with neurodegenerative processes (Kozlowski et al., 2009; Dietz et al., 2021; Cheng et al., 2022). In addition, Zn overload can cause neuronal damage principally related with mitochondrial respiratory chain dysfunction (Kozlowski et al., 2009; Liu et al., 2021). This connection might be reciprocal, as complex III deficiency in yeast leads to a depletion of the labile intra-mitochondrial Zn pools (Atkinson et al., 2011).

Drosophila melanogaster models have been useful to understand metal homeostasis in animal cells (Navarro and Schneuwly, 2017), as well as mitochondrial dysfunction (Foriel et al., 2015). In this work we have used well characterized D. melanogaster RNAi strains targeting three COX assembly factors: Coa3/Ccdc56 (Peralta et al., 2012), Scox, which is the single SCO protein homolog present in flies (Porcelli et al., 2010; Nguyen et al., 2014) and Coa8 (Brischigliaro et al., 2019). For this study we have also included a knock-down strain of a COX supernumerary structural subunit cype/COX6C (Szuplewski and Terracol, 2001; Fernandez-Ayala et al., 2009). Using these well-established models of COX deficiency, we have separately analyzed the cytosolic and the mitochondrial content of four of the most biologically-relevant transition metals (Cu, Fe, Mn, and Zn). We conclude that COX deficiency in general produces an imbalance in the compartmentalization of Cu and Zn and, at a lesser extent, of Fe and Mn as well. In addition, these COX-deficient strains showed altered transcript levels of Cu transporters and metallothioneins. We hypothesize that these alterations could be a contributing factor in the pathogenesis of mitochondrial disease associated with COX deficiency.

To investigate the compartmentalization of transition metals in genetic models of COX deficiency, we separated washed mitochondrial and cytosolic (‘post-mitochondrial’ soluble supernatant) fractions from whole homogenates obtained from adults Coa8 RNAi and Coa3 RNAi adults. The analyses of the cype RNAi and Scox RNAi, were performed using third instar larvae as these individuals do not reach the adult stage (Fernandez-Ayala et al., 2009; Nguyen et al., 2014). To determine the extent of the COX defect in these models, we used mitochondrial fractions from the knock-down (KD) flies to perform COX spectrophotometric kinetic enzyme activity measurements (Figure 1A), and COX in-gel-activity assays (Figure 1B). All the KD samples presented COX deficiency to some extent, being the least affected the Coa3 KD adults with a decrease of ∼30% compared with the control, followed by the Coa8 KD adults and cype and Scox larvae, all showing a ∼50% reduction of COX activity, normalized by the activity of citrate synthase (CS). The metal content in both the separated cytosolic and mitochondrial fractions was determined by ICP-MS and normalized by the protein amount in each of the samples (Figures 2, 3).

Mitochondrial enzymatic complex IV (COX) deficiency is a frequent biochemical hallmark in mitochondrial disease, and it can be originated by genetic defects affecting either COX structural components or assembly factors necessary for the correct assembly and function of the enzyme (Fernandez-Vizarra and Zeviani, 2021b; Brischigliaro and Zeviani, 2021). In this work we have used D. melanogaster models deficient in three different assembly factors with a distinct role in the process of COX assembly. Coa3 is a chaperone and/or stabilizing factor of nascent MT-CO1, playing a role in the early assembly stages (Mick et al., 2010; Mick et al., 2012; Peralta et al., 2012; Clemente et al., 2013). The function of Coa8 is still not clear, but evidence collected so far points out to a redox sensing function necessary to promote the middle assembly stages of COX biogenesis (Brischigliaro et al., 2019; Signes et al., 2019). Scox is the D. melanogaster homologue of the human copper-chaperones SCO1 and SCO2, necessary for Cu delivery to the Cu_A center in MT-CO2 (Leary et al., 2004; Leary et al., 2009). In addition, we have included in our analysis a KD strain for cype, the fly homolog of mammalian COX6C subunit. Although mutations in COX6C have not been associated with human mitochondrial disease to date, defects in cype produce severe phenotypes in flies (Szuplewski and Terracol, 2001; Fernandez-Ayala et al., 2009).

The consequences of SCO1 and SCO2 mutations on Cu homeostasis are well documented, resulting in a general cellular Cu deficiency in human and mouse cells and tissues (Leary et al., 2007; Hlynialuk et al., 2015; Baker et al., 2017). This is compatible with the observations in the Scox flies, where the levels of Cu in the mitochondria-enriched fractions were significantly lower than in the controls, without a variation in the cytosolic amounts. When the Cu amounts in the two fractions are added together, a lower global Cu amount occurs in the Scox KD flies. This is also similar to what was observed in a Sco2 KO/KI model, mimicking the most frequently found mutation in humans, where the levels of mitochondrial Cu were reduced in several tissues (Yang et al., 2010). Even though in this study the mitochondria were not purified using density gradients and contaminants from other membranous organelles containing metals could have been present, the simple washing of the crude pellets, as it was done in the samples for this study, eliminates a great proportion of microsomes, peroxisomes and lysosomes (Fernandez-Vizarra et al., 2006). Accordingly, mitochondria purified from adult flies and larvae are devoid of cytoplasmic markers (Supplementary Figure S1B). Therefore, mitochondria are the major contents in these fractions (Supplementary Figure S1B), and the mitochondrial yield in the mitochondrial preparations from the COX deficient models is similar or even higher than in the controls (Supplementary Figure S1A). Furthermore, we found dysregulation of Cu compartmentalization in all the analyzed COX deficient models, including Scox KD. This indicates that not only defects in one of the chaperones directly handling Cu for the delivery to COX produce alterations in Cu homeostasis, but also the failure to assemble COX is associated with lower intra-mitochondrial Cu levels. In fact, there appears to be a direct correlation between COX activity and mitochondrial Cu amounts in the adults as both parameters are around 50% of the control in the Coa8 KD and ca. 70% in the Coa3 KD. In the larvae, the Cu deficiency (∼20% in both models) is more severe than the COX defect (∼50% in the two KD strains). However, even though the nature of the defect is different, i.e., a structural defect (cype KD) vs. a Cu chaperone defect (Scox KD), a similar COX deficiency results in comparable Cu depletion in the larvae. This is compatible with the fact that COX is the most abundant Cu-containing enzyme in the cell types in which mitochondria are present (Ruiz et al., 2021). Cu depletion was also observed in human samples from individuals carrying mutations in COX10, COX15 and SURF1, but Cu cellular levels could be recovered without increasing COX activity by overexpressing the Cu-binding proteins SCO1 and SCO2 (Leary et al., 2007). This observation points out to a general effect of COX deficiency determining Cu depletion inside mitochondria and either no changes or an increase in cytoplasmic Cu concentrations, as observed in our D. melanogaster models.

We were also interested in analyzing the amounts of other transition metals in the mitochondrial and cytosolic fractions of the four COX-deficient models. Iron, which is the other metal present in the catalytic centers of COX, does not appear to be generally altered as a consequence of a generic COX deficiency. We only found an alteration in Fe compartmentalization in the Coa8 KD model, and a modest decrease in the mitochondria of Coa3 KD flies. This could reflect the consequences of the specific COX assembly defect in these models, probably relating to an accumulation of metalated Cox1 in the Coa8 KD flies (Signes et al., 2019), and a reduction in the amounts of Cox1 in the Coa3 KD (Peralta et al., 2012; Clemente et al., 2013). The changes in Mn were also model-specific and rather modest compared with the other metals. On the other hand, some changes in Zn homeostasis were observed in the COX-deficient strains, with increases in mitochondria of the Coa8 KD adults and the Scox and cype KD larvae, and with a very significant elevation in the cytosol of the larval models. Conversely, Zn concentration was lower in both cytosol and mitochondria from Coa3 KD adults. It is difficult to explain why Zn levels are dysregulated in the case of COX deficiency, as Zn is not a component of the enzyme. One could speculate this could be related to changes in the redox state of both mitochondria and cytosol associated with the COX enzymatic and assembly defect (Geldon et al., 2021) or, alternatively, to the tight connection between the metabolism of Cu and Zn observed in yeast, humans and D. melanogaster (Cobine et al., 2004; Kozlowski et al., 2009; Spinazzi et al., 2014; Navarro and Schneuwly, 2017; Barber et al., 2021). The reasons why the alteration in Zn concentrations in the Coa3 KD flies goes in the opposite direction than in the other models is difficult to interpret, being that these same flies show a decrease in intra-mitochondrial Cu and an increase in the cytosol, similar to the other three models. However, this again might reflect early assembly defect driven by Coa3 deficiency, compared with the other factors and structural subunit that have a role in later (middle) stages of COX assembly (Brischigliaro and Zeviani, 2021).

Defects in SCO1 in mouse produce the loss of the copper transporter CTR1 from the plasma membrane by internalization and/or proteasomal degradation (Hlynialuk et al., 2015; Baker et al., 2017), consequently decreasing Cu influx. In the Scox KD flies the transcript levels of Ctr1A, the D. melanogaster homolog of CTR1, were significantly reduced and this was also observed to a lesser extent in cype and Coa3 KD individuals. This may reflect species-specific mechanisms but also suggest a conserved response aimed to reduce Cu uptake at the plasma membrane, likely as a consequence of increased Cu levels in the cytoplasm. In fact, Ctr1A mRNA levels were comparable to the control in the Coa8 KD, where Cu cytoplasmic levels were not increased. In addition, the expression of other genes under the control of MTF-1, such as the ‘low affinity’ Cu transporter Ctr1B and metallothioneins MtnA and MtnD (Egli et al., 2003; Selvaraj et al., 2005; Turski and Thiele, 2007; Günther et al., 2012), were also found altered in the COX-deficient flies. Specifically, the expression of Ctr1B (CTR2 in mammals) was reduced in all the strains except for Scox KD. The role of Ctr1B/CTR2 is not as well defined as that of CTR1 but there seems to be an interplay between the two transporters (Wee et al., 2013). This observation can be interpreted as an attempt to up-regulate Cu uptake, possibly to compensate the reduced activity of Scox. In fact, previous studies demonstrated that increasing copper availability can rescue the COX deficiency arising from SCO2 genetic defects in human cell lines (Jaksch et al., 2001; Salviati et al., 2002; Casarin et al., 2012; Soma et al., 2018). Interestingly, we observed increased expression of metallothioneins MtnA and MtnD in Coa8 and Coa3 KD individuals, which is most likely linked to altered Cu and Zn compartmentalization, and would increase metal trafficking and detoxification (Navarro and Schneuwly, 2017). This effect was strong in adults and negligible at the larval stage, possibly reflecting differences in response to metal overload at different developmental stages in D. melanogaster. Another possibility is that the marked upregulation of Zn levels in the cytoplasm of Scox and cype KD larvae might mitigate the Cu-driven responses, consistent with the fact that increased Zn cellular levels are usually correlated with a decrease in Cu amounts (Spinazzi et al., 2014; Barber et al., 2021).

Mitochondrial diseases are highly heterogeneous in their clinical presentation and additional elements other than ATP deficiency must play a role in the pathogenic mechanisms (Nunnari and Suomalainen, 2012; Suomalainen and Battersby, 2018). Altered metal homeostasis is associated with different pathological states, especially with neurodegeneration (Kozlowski et al., 2009). Although not considered as a main clinical feature in most mitochondrial diseases, iron overload has been reported in GRACILE syndrome, caused by a mutation in the complex III assembly factor BCS1L (Visapaa et al., 2002; Lynn et al., 2012; Kasapkara et al., 2014). In addition, complex III deficiency caused by defects in different biogenetical factors, causes a decrease in mitochondrial Zn pools in yeast (Atkinson et al., 2010; Atkinson et al., 2011). Moreover, evidences of the contribution of altered Fe homeostasis to the pathogenetic mechanisms of Leigh syndrome associated with the loss of the Fe-S binding complex I subunit NDUFS4 have been found (Grillo et al., 2021).

In conclusion, we hypothesize that the disturbance of mitochondrial and, consequently, cellular transition metal content, as well as the responses triggered by these alterations, might play a role in the pathogenesis of the neurodegenerative phenotypes observed in some mitochondrial disorders, especially those involving COX deficiency. Future work is warranted to confirm these observations with larger datasets and additional models in order to explore this hypothesis and investigate the role of transition metal homeostasis in mitochondrial disorders.