**1. Introduction: Functions of Mitochondria. The Oxidative Phosphorylation System**

The advancement of molecular medicine has pinpointed the role of mitochondria in the etiology and pathogenesis of most common chronic diseases [1–4], so much that the term "Mitochondrial Medicine" has been proposed [5] and then widely used [6–9].

The early biochemical studies on mitochondria were centered on their role in energy conservation. The energy-transducing membrane-bound enzyme complexes of inner mitochondrial membrane (mtIM) drive biochemical reactions involved in energy transformation or bioenergetics. Oxidation of substrate by respiratory complex and ATP production by ATP synthase are tightly coupled molecular mechanisms in the oxidative phosphorylation (OXPHOS) system as explained by Mitchell's chemiosmotic hypothesis [10].

Once the major aspects of the OXPHOS system were clarified, the interest in mitochondria somewhat decreased. However, in recent years it has been raised again due to a series of novel findings assigning new roles to mitochondria in molecular and cell biology,

**Citation:** Nesci, S.; Trombetti, F.; Pagliarani, A.; Ventrella, V.; Algieri, C.; Tioli, G.; Lenaz, G. Molecular and Supramolecular Structure of the Mitochondrial Oxidative Phosphorylation System: Implications for Pathology. *Life* **2021**, *11*, 242. https://doi.org/ 10.3390/life11030242

Academic Editor: Angela Anna Messina

Received: 1 February 2021 Accepted: 11 March 2021 Published: 15 March 2021

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such as mitochondrial DNA and mitochondrial genetics, the role of mitochondria in generation of reactive oxygen species (ROS) and in cell signaling, in cellular quality control and apoptosis (programmed cell death). Nevertheless, these newly discovered functions are strictly intertwined with the central role of electron transfer and ATP synthesis.

This review intends to outline the major structural and functional aspects of mitochondrial bioenergetics that are at the basis of changes leading to pathology; in particular, we will deal with recent advances of the supramolecular structure of the respiratory chain complexes and of F1FO-ATP synthase/hydrolase; for this reason, this review will not analyze other important aspects in much detail such as the intimate mechanisms of electron transfer and proton translocation on one hand, and the analysis of the individual pathologies on the other.

#### **2. The Respiratory Chain of Mitochondria**

The major mechanism of energy conservation in eukaryotes is OXPHOS, performed by a multi-enzyme system embedded in the mtIM and constituted by two portions: the respiratory chain and the ATP synthase complex.

The membrane electron transfer system (membrane-ETS) is a series of enzymes that collects electrons which stem from the oxidations of intermediary metabolism and drives them downhill to oxygen molecules which are reduced. The free energy fall that accompanies electron flux creates an electrochemical proton gradient (∆µH+) since H<sup>+</sup> are pumped from the mitochondrial the matrix, namely the compartment inside the mitochondrion, to the intermembrane space (IMS) localized between the inner and outer mitochondrial membranes [11]. The energy associated to the proton gradient is then largely used to synthesize ATP from ADP and Pi by the ATP synthase complex. The ATP synthesized is transferred to the cytoplasm in exchange with ADP by the ATP/ADP translocase, also exploiting the H<sup>+</sup> gradient.

As previously revised in [12], it was Hatefi et al. [13] who first isolated from mitochondria four enzyme multi-subunit complexes that concur on the oxidation of NADH and succinate, namely NADH-Coenzyme Q reductase (Complex I, CI), succinate-Coenzyme Q reductase (Complex II, CII), ubiquinol-cytochrome *c* reductase (Complex III, CIII or cytochrome *bc*<sup>1</sup> Complex) and cytochrome *c* oxidase (Complex IV, CIV) [14].

The connection among these enzyme complexes is ensured by two mobile transporters of electrons, i.e., Coenzyme Q (CoQ, ubiquinone) and cytochrome *c* (cyt. *c*) [14]. The former is a lipophilic quinone incorporated in the lipid bilayer of the mtIM, while cyt. *c* is a hydrophilic hemoprotein facing the mitochondrial IMS, in contact with the external surface of the mtIM. The membrane-ETS operates through the following sequence of the respiratory enzyme complexes: (Equations (1) and (2))

$$\text{NADH} \rightarrow \text{CI} \rightarrow \text{CoQ} \rightarrow \text{CII} \rightarrow \text{cyt.} \,\text{c} \rightarrow \text{CIV} \rightarrow \text{O}\_2 \tag{1}$$

or

$$\text{succinate} \to \text{CII} \to \text{CoQ} \to \text{CIII} \to \text{cyt.} \,\varepsilon \to \text{CIV} \to \text{O}\_2 \tag{2}$$

In addition, the membrane-ETS consists of other proteins having electron transfer activity [12] that converge at the CoQ junction. Glycerol-3-phosphate dehydrogenase is involved in a shuttle of reducing equivalents from cytosol to mitochondria [15], electron transfer flavoprotein (ETF) dehydrogenase is involved in fatty acid oxidation [16], dihydroorotate dehydrogenase catalyzes a step of pyrimidine nucleotide biosynthesis [17], choline dehydrogenase is important for the regulation of phospholipid metabolism [18], and sulfide dehydrogenase is involved in the disposal of sulfide [19]. A schematic representation of mammalian respiratory chain is found in Figure 1.

**Figure 1.** A schematic drawing of the respiratory chain depicting the protein complexes and their substrates. Complex I, Complex III and Complex IV are shown in their free form (modified PDB ID: 6YJ4, 2YBB, 1V54). Blue, CI, NADHubiquinone oxidoreductase; yellow, CII, succinate-ubiquinone oxidoreductase (modified PDB ID: 1ZOY); red, CIII, ubiquinolcytochrome *c* oxidoreductase; green, CIV, cytochrome *c* oxidase; AOX, alternative oxidase; CoQ, Coenzyme Q (ubiquinone); Cyt*c*, cytochrome *c*. Four enzymes that reduce CoQ are also shown together with an indication of their metabolic pathways: from the intermembrane space (IMS), glycerol-3.P dehydrogenase (green) and dihydroorotate dehydrogenase (blue); from the matrix, electron transfer flavoprotein (ETF) dehydrogenase (brown) and choline dehydrogenase (purple). (see text for details).

For reviews see, e.g., Sousa et al. [20] and, for individual complexes: Parey et al. [21] for CI, Bezawork-Geleta et al. [22] for CII, Xia et al. [23] for CIII, and Zong et al. [24] for CIV.
