**1. Introduction**

The mitochondria are cytosolic organelles of eukaryotic cells in charge of ATP production through the process of oxidative phosphorylation (OXPHOS). However, several other important pathways are associated with mitochondria, such as the citric acid cycle [1,2], the fatty acids oxidation [3] and lipid droplets formation [4], the iron–sulfur (Fe–S) protein biogenesis [5] and amino acids catabolism [6]. Furthermore, mitochondria are implicated in the buffering of cytosolic calcium concentration [7], in generation of reactive oxygen species (ROS) [8], and in regulation and execution of different types of cell death [9]. They are also involved in an array of adaptive responses triggered by perturbations of intracellular homeostasis [10], orchestrating anabolic and catabolic reactions, which are finely adjusted according to different cytosolic conditions. All these interconnected functions are sustained by the activity of the "mitochondrial proteome", estimated to contain at least 1000 (in yeast) [11] to 1500 (in humans) [12] different proteins, 15% of which are directly involved in energy metabolism and the OXPHOS system. Note that recent bioinformatics analysis in yeast provided evidence for more proteins than expected, cryptically localized inside mitochondria [13].

As typically described in the textbooks, mitochondria have two membranes, the outer membrane, which acts a barrier separating mitochondria from the cytoplasm, and the inner membrane surrounding the matrix, where soluble enzymes of intermediary metabolism, ribosomes and the mitochondrial genome (mtDNA) are hosted. The inner mitochondrial membrane is characterized by an extraordinarily high protein content and, in particular,

**Citation:** Rugolo, M.; Zanna, C.; Ghelli, A.M. Organization of the Respiratory Supercomplexes in Cells with Defective Complex III: Structural Features and Metabolic Consequences. *Life* **2021**, *11*, 351. https://doi.org/10.3390/life11040351

Academic Editors: Giorgio Lenaz and Salvatore Nesci

Received: 18 March 2021 Accepted: 14 April 2021 Published: 17 April 2021

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encloses many copies of the respiratory chain components that together with the ATP synthase (named also complex V, CV) form the molecular machinery of OXPHOS, i.e., the ATP production from ADP and inorganic phosphate. The mitochondrial respiratory chain consists of four enzymatic multi-subunit complexes, namely the NADH-coenzyme Q reductase (Complex I, CI), the succinate-Coenzyme Q reductase (Complex II, CII), the ubiquinol-cytochrome *c* reductase (Complex III, CIII), and the cytochrome *c* oxidase (Complex IV, CIV). Two mobile redox-active compounds, the lipophilic coenzyme Q (CoQ) and the hydrophilic cytochrome *c,* connect the enzymatic complexes, thus allowing the electron transfer from soluble reducing equivalents (NADH and FADH2) to molecular oxygen.

Unlike the oxidation of NADH which only occurs via CI, FADH<sup>2</sup> can be oxidized at the inner membrane mainly by CII, but also by other less abundant proteins such as the glycerol-3-phosphate dehydrogenase [14], the electron transfer flavoprotein dehydrogenases [15–17], the dihydroorotate dehydrogenase [18], the choline dehydrogenase [19], the sulfide CoQ reductase [20], and the proline dehydrogenase [21]. All these proteins are able to feed electrons to CoQ and in turn to CIII, which therefore can be considered the central collector delivering electrons through cytochrome *c* to CIV. The electron transport is coupled to proton extrusion from the matrix into the intermembrane space generating a transmembrane proton gradient at the level of CI, CIII and CIV, but not of CII. This later, together with other FAD-linked enzymes, does not contribute to energy conservation.

#### **2. Mitochondrial Proteins Are Encoded by Two Genomes**

Most mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol and then imported into mitochondria by specific targeting mechanisms. However, these organelles are characterized by the presence of an independent genome, the mtDNA. This peculiarity is believed to be due to the evolutionary origin of mitochondria from alpha-proteobacteria integrated into proto-eukaryotic host, of which the details are still debated [22]. Most mitochondrial genes were then transferred to the nucleus, although a few of respiration-competent genes were conserved as an independent genome. The mtDNA is a circular double-stranded DNA, in humans of approximately 16.5 kb, encoding for thirteen polypeptides, all essential subunits of the OXPHOS system, and also for two ribosomal RNAs and twenty-two transfer RNAs, required for the intra-mitochondrial translation of the thirteen proteins. The evolutionary pathways involved in maintaining this transcriptionally active genome in addition to nuclear DNA are still poorly understood. New system biology and bioinformatics approaches have confirmed that the very high hydrophobicity of the proteins encoded by mtDNA is crucial to limit their translocation from the cytoplasm to the mitochondrial membrane and to favour mistargeting to the endoplasmic reticulum. In addition, the high CG content has been shown to increase the thermodynamic stability of the mtDNA, protecting from environmental insults [23]. Of note is that the high GC content might be related also to the surprisingly very high local temperature (about 50 ◦C) recently determined inside mitochondria [24]. Finally, the preferential encoding of components essential for organelle function in the mtDNA would allow localized control of gene expression and therefore the assembly of protein complexes [25].

#### **3. Both Genomes Contribute to the Onset of Mitochondrial Diseases**

Mitochondrial diseases are genetically heterogeneous disorders caused by mutations in nuclear genes encoding OXPHOS structural proteins or assembly factors, which are proteins required for the correct maturation of the complexes, but not contributing to the final structures. Mutations can also affect the molecular machineries of mtDNA replication and maintenance, of mitochondrial transcription and translation, as well as proteins involved in cristae shaping, network dynamics and quality control, composition of membrane lipids or mechanisms of antioxidant defences. Disorders can also be caused by mutations in the mtDNA, encoding structural OXPHOS subunits. In this case the genetic features are very peculiar, since the mtDNA inheritance mode follows the maternal lineage. Furthermore, each cell presents multiple copies (100–1000) of this genome, so that mutated and non-mutated copies can co-exist in the same individual, generating a phenomenon called heteroplasmy. Accordingly, the clinical phenotype and the severity of biochemical dysfunctions are highly variable, and pathology only manifests when the percentage of mutated mtDNA exceeds a threshold, which is variable for each kind of mutation. For a recent exhaustive review on the genetic basis of primary mitochondrial diseases we refer to Fernandez-Vizarra and Zeviani, 2020 [26].

#### **4. The OXPHOS System**

The structures of individual mitochondrial respiratory chain complexes have been determined by X-ray crystallography [27–30] or electron cryo-microscopy [31–35]. The bovine or human mitochondrial CI contains 44 different subunits, forming an L-shaped structure. The minimal functional unit of CI, which is conserved from bacteria to mammals, comprises 14 subunits known as core subunits. Subunits ND1-ND6 and ND4L form the hydrophobic membrane arm, the other seven core subunits form the hydrophilic arm protruding into the matrix and comprising a flavin mononucleotide (FMN) and eight iron– sulfur clusters as redox active prosthetic groups. This latter contains the NADH binding and electron transfer sites, whereas the membrane arm performs the proton translocation. The other supernumerary subunits play significant roles in the assembly, stabilization and regulation of CI [35–37].

CII is composed of four subunits, forming a hydrophilic head, containing a FAD binding protein and an iron–sulfur protein, and the hydrophobic arm with two membraneanchor proteins (CybL and CybS). Three kinds of prosthetic groups, FAD, heme and iron–sulfur clusters, were recognized in CII, coupled with two Q-binding sites [38,39].

The mammalian CIII monomer is composed of three respiratory subunits (cytochrome *b*/MTCYB, cytochrome *c1*/CYC1 and the Rieske iron–sulfur protein/UQCRFS1, two core proteins (UQCRC1, UQCRC2) and six low-molecular-weight proteins (UQCRH/QCR6, UQCRB/QCR7, UQCRQ/QCR8, UQCR10/QCR9, UQCR11/QCR10 and a cleavage product of UQCRFS1). CIII is present as dimer (approx 450 kDa), although it is still controversial whether the two monomers of CIII<sup>2</sup> function cooperatively or independently [40,41].

The CIV monomer has a mass of approximately 200 kDa and is believed to occur in the membrane both as a monomer and a dimer [42]. Each CIV monomer consists of 14 subunits [43], since NDUFA4, which was considered to be a subunit of CI, is actually a subunit of CIV [44]. The four redox-active metal centres constituting the electron transport pathway are heme *a3* and CuB, forming the binuclear centre that binds oxygen, and heme *a*, located in subunit COXI. The CuA center is incorporated in COXII [45]. The remaining subunits (COX4, 5A, 5B, 6A, 6B, 6C, 7A, 7B, 7C, 8A) are thought to have a structural role in the stabilization of the complex [46,47].

#### **5. OXPHOS Optimization by the Inner Membrane Architecture**

The general notion that the inner membrane is heavily folded to form the cristae has been reconsidered after 3D electron microscopy (EM) tomographic analysis in mitochondria from a wide variety of organisms, revealing that the cristae membrane opens toward the intermembrane space through narrow tubular structures, the crista junctions [48]. The molecular components of these cristae junctions have been subsequently identified, the most relevant being the mitochondrial contact site and cristae organizing system (MICOS) [49–51] and the high-molecular-weight GTPase Optic atrophy 1 (OPA1) [52–54] (Figure 1).

Furthermore, the observation of mitochondrial specimens in their native environment by means of the immuno-EM and cryo-EM tomography allowed to demonstrate that dimers of CV are arranged in long rows along the tightly curved ridges of the cristae membrane [55] (Figure 1). These ribbons of CV dimers are involved in the folding of the crista membrane [56]. It seems therefore that the intra-mitochondrial architecture is more complex than initially believed, with MICOS, OPA1 and CV dimers playing a structural

role, together with the abundance of non-bilayer cone-shaped phospholipids, such as cardiolipin and phosphatidylethanolamine [53,57,58].

**Figure 1.** Architecture of the inner membrane and unequal distribution of OXPHOS complexes. CI, CIII and CIV are in the cristae membrane, CV dimers are at the cristae tip, whereas CII is also found in the inner boundary membrane. MICOS and OPA1 stabilize the cristae junctions, providing constrains to membrane mobility of complexes.

> Of note is that the presence of MICOS and other proteins at the cristae junctions provides severe constraints to the mobility of protein complexes along the inner membrane, and therefore two sub-compartments with unequal protein distribution and functions can be envisaged: the cristae membrane, protruding in the matrix, and the inner boundary membrane, facing the outer membrane. The respiratory complexes are mainly found in the cristae membrane [56]. Further, sophisticated analysis of diffusion and directionality of movement of single complexes by super resolution microscopy revealed that all the complexes are trapped in the laminar part of cristae, except CII which is also found in the inner boundary membrane [59] (Figure 1). It has to be noticed that CII is a rather small complex and does not associate in supramolecular assemblies with other complexes. It follows that the specific geometry of the inner membrane, dictated by the cristae junction organization at the bottom and CV dimers on the top of crista, has a critical impact on the energetic function of mitochondria. As summarized in Figure 1, protons ejected by CI, CIII and CIV segregated on the laminar part of the cristae, are enriched in the intracristal space and flow back into the matrix through CV. This arrangement was predicted to be highly efficient in promoting energy conversion [56]. However, the intracristal membrane geometry has not been shown to influence the local pH gradients [60,61], consequently

it has been proposed that the tight packing of the OXPHOS machinery in the cristae membranes favours the kinetic coupling between proton pumping and ATP synthesis [61]. The extraordinary power of super resolution live-cell imaging in combination with EM tomography and genome editing will further illuminate the functional details and dynamic aspects of this important microcompartment.

#### **6. Supramolecular Organization of the Respiratory Complexes**

The unequal protein complex distribution within the inner membrane subcompartments is also favoured by their size and their ability to interact with other complexes to form high-molecular-weight macromolecular aggregates. Differently form CII, which is always found alone, the proton-pumping CI, CIII and CIV can assemble in non-covalent associations defined as respiratory supercomplexes (SCs). Respiratory SCs were first identified by non-denaturing blue native gel electrophoresis (BN-PAGE) of mitochondrial membrane extracts, using the mild detergents Triton X-100 and digitonin, as high-molecular-weight gel bands, showing activity for CI, CIII and CIV [62,63]. The mammalian SCs containing CI, CIII dimer (CIII2), and CIV with different stoichiometry are sometimes referred to as respirasomes, because they contain all the components required to transfer electrons from NADH to molecular oxygen [63–65]. In human cells, the respirasome comprises most of CI (>90%), approximately 40–50% of CIII<sup>2</sup> and 20–30% of CIV. Differently from CI, significant amounts of CIII<sup>2</sup> (50–60%) and CIV (70–80%) can be also found as isolated complexes within the membrane. The CIII+CIV SC represents about 5% of total amount of the complexes [66].
