H+‐slip correlated to rotor free‐wheeling as cause of F1FO‐ATPase dysfunction in primary mitochondrial disorders

Inborn errors of metabolism are related to mitochondrial disorders caused by dysfunction of the oxidative phosphorylation (OXPHOS) system. Congenital hypermetabolism in the infant is a rare disease belonging to Luft syndrome, nonthyroidal hypermetabolism, arising from a singular example of a defect in OXPHOS. The mitochondria lose coupling of mitochondrial substrates oxidation from the ADP phosphorylation. Since Luft syndrome is due to uncoupled cell respiration responsible for deficient in ATP production that originates in the respiratory complexes, a de novo heterozygous variant in the catalytic subunit of mitochondrial F1FO‐ATPase arises as the main cause of an autosomal dominant syndrome of hypermetabolism associated with dysfunction in ATP production, which does not involve the respiratory complexes. The F1FO‐ATPase works as an embedded molecular machine with a rotary action using two different motor engines. The FO, which is an integral domain in the membrane, dissipates the chemical potential difference for H+, a proton motive force (Δp), across the inner membrane to generate a torsion. The F1 domain—the hydrophilic portion responsible for ATP turnover—is powered by the molecular rotary action to synthesize ATP. The structural and functional coupling of F1 and FO domains support the energy transduction for ATP synthesis. The dissipation of Δp by means of an H+ slip correlated to rotor free‐wheeling of the F1FO‐ATPase has been discovered to cause enzyme dysfunction in primary mitochondrial disorders. In this insight, we try to offer commentary and analysis of the molecular mechanism in these impaired mitochondria.

The numerous harmful mutations in genes that encode structural subunits or supplementary factors of oxidative phosphorylation (OXPHOS) complexes are one of the most common inborn disorders of energy metabolism, which are caused by cell bioenergetics dysfunction. 1,2Defects of oxidation-phosphorylation coupling in OXPHOS occur in inborn metabolic defects and Luft disease is the most well-known example. 3Mitochondrial uncoupling abnormalities, such as Luft syndrome, are distinguished by enhanced mitochondrial respiration that is unrelated to mitochondrial ATP production. 4However, uncoupled respiration that originates outside the F 1 F O -ATPase, the last complex of the OXPHOS system that is able to ATP synthesis, is characterized by a notable decrease of ATP production and respiratory rate in the presence of ADP and nicotine adenine dinucleotide linked substrates or with a flavoprotein-linked substrate.
A case of congenital euthyroid hypermetabolism in two monozygotic twin boys has recently been investigated highlighting a high caloric intake, inability to gain weight, and sporadic hyperthermia. 5The patients presented detailed molecular evidence that such a disorder could be classified as a mitochondrial uncoupling syndrome with a dominant-negative influence on the OXPHOS chemiosmotic coupling.However, the bioenergetic feature of the disease highlights an increased mitochondrial respiration without ATP synthesis resulting in a loss of mitochondrial membrane potential that is independent of cell respiration activity of respiratory complexes, but related to the On the β subunit of F 1 F O -ATPase, the c.1004 T → C transition in the nuclear ATP5F1B gene is a de novo heterozygous mutation causing the change of p.Leu 335 Pro residue in the catalytic site of the enzyme (Figure 1).It is known that F 1 F O -ATPase is a bi-functional enzyme that synthesizes ATP by exploiting the proton motive force (Δp) generated with the mitochondrial respiratory complexes activity driven by substrate oxidation.Conversely, in anaerobiosis pathological or physiological conditions, the F 1 F O -ATPase hydrolyzes ATP to polarize the inner mitochondrial membrane (IMM) working as proton (H + ) pump. 6Structurally the F 1 F O -ATPase is composed of a hydrophilic F 1 domain, responsible for the ATP catalysis, and a membrane-inserted F O domain, involved in the H + transport across the IMM. 7The two domains are functionally and structurally joined by a central stalk rotating inside the F 1 , which mechanically causes a conformational change in the catalytic sites of adenine nucleotides, and a lateral stalk that anchors the F 1 to the stationary membrane-embedded subunits of the F O to avoid the torsion driven by the rotor. 8,9ring ATP synthesis, the H + flow through the IMM is driven by Δp exploiting in F O domain an asymmetric half-channels structure of a subunit inducing the c-ring rotation by a protonation/deprotonation mechanism of the H + binding site of c subunits. 10The torsion generated by H + translocation is conveyed through the central rotor to power the binding change conformation of F 1 domain catalytic sites according to the binding change mechanism (BCM). 7Since the F 1 domain is three-fold symmetric, three ATP molecules are produced with one complete rotor rotation, that is, one molecule every 120°.A hexagonal structure of three catalytic β subunits and three noncatalytic α subunits are arranged in alternation around the asymmetric structure of the central stalk γ subunit, which lies along the central cavity of the α 3 β 3 hexamer. 11The Mg 2+ -ADP and Mg 2+ -ATP binding sites are placed on the three β subunits at the interfaces with the homologous noncatalytic subunit, the α subunits. 12The rotation of the central stalk orients the "γ-bulge" obligating each of the β subunits to adopt the interconversion of different catalytic properties and conformational states designed as β TP , β DP , which contain bound ATP or ADP, and the empty β E structure during the rotor rotation 13 following the BCM model.Therefore, the γ shaft has the role to communicate its rotational position to each β subunit and trigger the catalysis.
The Leu 335 Pro mutation is bioenergetically characterized by increasing the basal oxygen consumption rate approximately to the maximum level of cell respiration.Consequently, ATP5B L335P cells have a drop in mitochondrial membrane potential associated with a low level of ATP production.The inefficiency of the chemiosmotic mechanism of OXPHOS is due to an intrinsic uncoupling of F 1 F O -ATPase energy transduction that causes the Δp dissipation in the absence of a mutual rise in ATP synthesis.Indeed, oligomycin, which is a specific inhibitor of H + translocation in the F O domain and blocks the ATP turnover, 14 restores the IMM polarization decreasing the oxygen consumption rate.Moreover, in wild-type and mutated cells, the protonophore molecules (FCCP or CCCP) maximize mitochondrial respiration. 5Inhibition with oligomycin indicates that the enzyme dysfunction lies in the chemo-mechanical F 1 domain activity, while the protonophore effect and the mutation have a synergistic action in depolarizing the IMM without synthesizing ATP.The damage on β subunits, therefore, suggests a shift in the γ subunit rotation in the catalytic hexamer without inducing the conformational change of BCM stimulating cellular respiration.Consistently, we could also suggest a decrease in the affinity of the catalytic site to adenine nucleotides.Indeed, by adding saturating ADP substrate in fibroblasts of patient the increased oxygen consumption was normalized. 5wever, the Leu 335 Pro mutation on β subunit is placed on the loop connecting regions of the subunit interacting with the γ subunit as the rotor is rotating (Figure 1).Two regions on the β subunit are noticeable for the presence of hydrophobic loop sleeves where the interactions with the γ subunit take place.These are located near the tip of the coiled-coil α-helices of the central stalk, primarily near the C-term of the γ subunit.Two "catch interactions" formed by H-bonds, one in the nucleotide-binding domain of β subunit adopting the empty conformation and the other comprises a critical helix-turn-helix motif arranged in the C-terminal domain of the closed conformation of β subunit ( 394 Asp-Glu-Leu-Ser-Glu-Glu-Asp 400 ) termed "DELSEED," which is supposedly involved in coupling between catalysis and rotation (Figure 1). 15The DELSEED-loop of each of the three β subunits makes contact with the γ subunit playing an important function in coupling between rotor rotation and catalysis of ATP synthesis.Both switches operate as bridges between the catalytic sites of the β subunits and the rotor.Pro is the unique amino acid with cis-trans-isomerization property in the peptide bond involving its imino nitrogen. 16We might consider that the torsional peptide bond by isomerization on the mutate Pro residue could displace the F I G U R E 2 Structural representation of the mechanochemistry of ATP production.F 1 F O -ATPase during ATP synthesis coupled to H + translocation (above) and intrinsic uncoupling of the enzyme caused by Leu 335 Pro mutations that induce a rotor free-wheeling in the energy transduction mechanism of F 1 F O -ATPase (below).[Color figure can be viewed at wileyonlinelibrary.com] hydrophobic portion bearing in the α 3 β 3 hexamer holding spaced the tip of γ shaft.Consequently, the torque generated by the rotor rotation that drives the mechanochemical power of the F 1 -ATPase domain 17 displays a freewheeling of the central stalk bypassing interactions of the γ subunit with the α 3 β 3 subcomplex (Figure 2).The result of intrinsic F 1 F O -ATPase uncoupling means that the enzyme complex itself cannot match ATP production to H + channeling, caused by molecular defects.While mitochondrial uncoupling has a recognized physiological role sustaining the H + leak by uncoupling proteins (UCPs) and ADP/ATP carrier, 18,19 as far as we know the impaired chemo-mechanical energy transduction in F 1 F O -ATPase only represents a basic biochemical symptom of pathologies. 2The enzyme could provide a broad spectrum of therapeutic strategies to counteract at the molecular level diseases whose treatment is still inadequate. 20In conclusion, the H + slip correlated to rotor freewheeling can be considered as the cause of F 1 F O -ATPase dysfunction in primary mitochondrial disorders.

F 1
and F O domains support the energy transduction for ATP synthesis.The dissipation of Δp by means of an H + slip correlated to rotor free-wheeling of the F 1 F O -ATPase has been discovered to cause enzyme dysfunction in primary mitochondrial disorders.In this insight, we try to offer commentary and analysis of the molecular mechanism in these impaired mitochondria.K E Y W O R D S ATP synthase, bioenergetic failure, congenital hypermetabolism, de novo mutation, mitochondria

F I G U R E 1
Molecular organization of the F 1 domain.The F 1 subunits are drawn as ribbon representations obtained from modified Protein Data Bank ID code: 6ZPO.The differently colored letters identify the subunits, drawn in the same color as the letter.The circles in the box on the right highlight the three sites of contact between β and γ subunits.Site 1, Site 2, and Site 3 are composed of the following sequence 273 Gly-Arg-Ile-Pro-Ser-Ala-Val-Gly-Tyr-Gln-Pro 283 , 313 Pro-Ala-Asp-Asp-Leu-Thr-Asp 319 , and 394 Asp-Glu-Leu-Ser-Glu-Glu-Asp 400 , known as DELSSED loop, respectively.The position of βLeu 335 is shown in the dotted red square.The conserved DELSEED motifs and the Leu 335 of the β subunit are shown in the space-filling model.[Color figure can be viewed at wileyonlinelibrary.com]NESCI and ROMEO | 1185