6.1.2. Intrinsic ATP Synthase Uncoupling Due by Amino Acid Changes in the *a* Subunit

The term F-ATPsynthase/ase uncoupling refers to any condition that inhibits the coupling between the catalytic activity carried out by the hydrophilic portion and H<sup>+</sup> translocation by the transmembrane portion F<sup>O</sup> [383]. Frequently, it stems from structural changes that modify the H<sup>+</sup> pathway of the F1FO-ATPase across the mtIM. The tight relationship between structure and function of mitochondrial complexes, described in the previous sections, means that any change in the primary sequence, due even to a mutation which changes a single amino acid can dramatically modify the protein properties, especially when amino acid substitutions involve crucial enzyme domains such as those in the *a* subunit where some amino acids are essential to build the H<sup>+</sup> route. The mitochondrial F1FO-ATPase is a good example of how point mutations in some protein sectors result in severe diseases. Mutations in the nuclear genes that encode F1FO-ATPase subunits are rare and associated with severe diseases nearly incompatible with life. The most known mutations, associated with diseases whose severity is related to mitochondrial heteroplasmy [384], are localized in the mtDNA, which encodes *a* and A6L subunits of the F<sup>O</sup> domain. In mammals, the mtDNA shows a higher mutational rate than nuclear DNA [385].

The most frequent mutations in the F1FO-ATPase associated with human pathologies occur in the mitochondrial *ATP6* gene, which encodes the *a* subunit. The structural arrangement of this subunit which, by positioning specific amino acids and exploiting the chemical properties of their side chains, forms the half-channels for H<sup>+</sup> flow within the mtIM, remained enigmatic for years, thus making it difficult to envisage the link between altered molecular function and pathology. However, recent studies, which highlighted the H<sup>+</sup> route, provided a satisfactory explanation on how point mutations are associated with mitochondrial dysfunctions and depicted a link between the bioenergetic defect and

the syndrome. Up to now, several point mutations have been described [379]. The most severe mutation is the m.T8993>G transversion, namely the substitution of thymine by guanine, which results in the replacement of the hydrophobic leucine by the positively charged arginine, namely a missense mutation (*a*Leu156Arg) [386]. This molecular change is related to pathologies known as Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) or Maternally Inherited Leigh Syndrome (MILS). These diseases are both associated with the same molecular defect, but exhibit various degrees of severity and are differently classified depending on the heteroplasmy degree [384]. The different chemical nature of the two amino acid side chains can satisfactorily explain the bioenergetic defect: since the inserted arginine is close to the crucial electrostatic barrier of *a*Arg-159, the two positive guanidine groups are close to each other to hamper both the H<sup>+</sup> flux across the mtIM and ATP synthesis [140]. The consequence is a severe bioenergetic failure. Accordingly, the F1FO-ATPase becomes unable to pump H<sup>+</sup> in the IMS and re-energize the mtIM, even if the two sectors F<sup>1</sup> and F<sup>O</sup> are still structurally and functionally joined, as proven by the observation that the enzyme complex remains sensitive to the selective inhibitor oligomycin, a clear evidence of the coupling of the two domains [387]. Similarly, the m.T9176>G transversion in the mitochondrial ATP6 gene that changes a conserved leucine into arginine (*a*Leu220Arg) on position 220 of *a* subunit [379] is associated with NARP and MILS diseases. In addition, in this case, as the *a*Leu-220 is close to the essential *a*Arg-159, this transversion changes the situation and makes two Arg residues occur in close positions, thus destabilizing the *a* subunit due to steric hindrance and electrostatic repulsions. Accordingly, the two vicinal Arg would act as a positively charged barrier, which prevents H<sup>+</sup> translocation across the mtIM and decreases ATP synthesis and CIV respiration. Moreover, since ATP hydrolysis becomes uncoupled to H<sup>+</sup> transport, as proven by the oligomycin insensitivity, the membrane potential cannot be restored by the F1FO-ATPase which cannot pump H<sup>+</sup> [388]. Since these two transversions, which cause substitution of the hydrophobic side chain of Leu by a basic and positively charged chain of Arg, deeply alter the protein microenvironment and the H<sup>+</sup> pathway, they cause the bioenergetic failure which constitute the biochemical basis of these severe diseases.

The m.T8993>C transition, which yields *a*Leu156Pro substitution [389], results in a less severe disease, as a result of an increased ROS production. In this case, the functionality is somehow preserved, since the *c*-ring can still slowly rotate, allowing the coupling of H<sup>+</sup> flux to a low ATP synthesis [390]. It is most likely that the insertion of Pro which replaces Leu modifies the protein secondary structure. Accordingly, the Pro five-membered ring may cause a kink in the helices [391] which could slow down H<sup>+</sup> transfer.

The de novo transition (m.G8969>A) in mtDNA which encodes the *ATP6* gene [392] has been recently associated with a rare mitochondriopathy, defined Myopathy, Lactic Acidosis, and Sideroblastic Anemia (MLASA) [392]. The consequent missense mutation Ser148Asn in *a* subunit [393] is localized at one helix turn from the *a*Glu-145 which acts as "H<sup>+</sup> transfer group" in the half-channel which opens in the mitochondrial matrix [151]. The *a*Asn-145 bears a positive charge which makes ionic bond with *a*Glu-145, thus blocking H<sup>+</sup> translocation which requires the –COOH deprotonation of *c*Glu-59 [393].

To sum up, the mutations in *a* subunit, which stepwise addresses H<sup>+</sup> and allow H<sup>+</sup> movement, block or hamper the torque generation in FO, which is essential for ATP synthesis by F1.

As far as we are aware, mutations in A6L subunit leading to pathologies are much less frequent than those in *a* subunit.

#### *6.2. Supercomplexes and ROS Signaling*

The role of mitochondrial ROS in cell signaling has been the subject of excellent reviews (cf. [272,274,394–396]). Here we deal with a possible role of the supramolecular organization of the respiratory chain on ROS signaling.

With ROS being involved in cell signaling, it is expected that their generation is subjected to tight control.

The control of mitochondrial ROS levels depends upon the balance between their rate of generation and of removal, as already considered in Section 5. The steady-state concentrations of the redox species responsible for electron leaking and ROS production are governed by a series of nuclear-encoded protein factors [267] and by the forces directly associated with respiratory activity, which are the redox potential of the NAD+/NADH couple and the ∆*p* [268]. Hoffman and Brookes [292] have investigated the ROS generation by rat liver mitochondria under different substrate and inhibitor conditions and different oxygen tensions, in order to determine the O<sup>2</sup> affinity of the different O2-reacting sites: from such data, the apparent K<sup>m</sup> for O<sup>2</sup> was lowest for CI during forward flow, followed by CI backflow, CIII Q<sup>O</sup> site, and highest for ETF dehydrogenase. They conclude that at physiological O<sup>2</sup> concentration, only CI may be a significant source of ROS.

We first speculated [96] and then obtained experimental demonstration [71] that dissociation of SC I1III<sup>2</sup> occurs upon ROS addition. Therefore, the facilitated electron channeling in the CoQ region is lost. This condition makes electron-transfer necessarily dependent upon random diffusion of the free ubiquinone molecules and collisions with the partner complexes and may elicit further ROS generation.

In fact, SC disorganization eventually leads to destabilization of CI, decreases NADlinked respiration and ATP synthesis and increases superoxide production by CI (cf. also Section 5 for experimental evidence).

The study of Guaras et al. [120] pinpoints another aspect of SCs in relation to ROS formation. Hyper-reduction of the CoQ pool by ETFH<sup>2</sup> oxidation during extensive fatty acid β-oxidation induces reverse electron transfer with a rise in ROS production by CI. Thus, shifting metabolic fuels from NADH-dependent to FADH2-dependent substrates may adjust ROS generation by way of the specific supramolecular assembly of the respiratory complexes involved [119,397].
