Molecular Regulation of the Mitochondrial F(1)F(o)-ATPsynthase: Physiological and Pathological Significance of the Inhibitory Factor 1 (IF(1))

Abstract

In mammals, the mitochondrial F(1)F(o)-ATPsynthase sets out the energy homeostasis by producing the bulk of cellular ATP. As for every enzyme, the laws of thermodynamics command it; however, it is privileged to have a dedicated molecular regulator that controls its rotation. This is the so-called ATPase Inhibitory Factor 1 (IF(1)) that blocks its reversal to avoid the consumption of cellular ATP when the enzyme acts as an ATP hydrolase. Recent evidence has also demonstrated that IF(1) may control the alignment of the enzyme along the mitochondrial inner membrane, thus increasing the interest for the molecule. We conceived this review to outline the fundamental knowledge of the F(1)F(o)-ATPsynthase and link it to the molecular mechanisms by which IF(1) regulates its way of function, with the ultimate goal to highlight this as an important and possibly unique means to control this indispensable enzyme in both physiological and pathological settings.

Figure 1

Oxidative phosphorylation and the mammalian F₁F_o-ATPsynthase. (a) Scheme of the mitochondrial OXPHOS: it is composed of five complexes, which couple the generation of a proton motive force through the mitochondrial inner membrane (IMM) with ATP synthesis. The first four complexes form the electron-transport chain (ETC), which catalyses the oxidation of NADH and FADH₂ to NAD⁺ and FAD respectively, with the associated reduction of molecular oxygen, to which electrons are transferred, to water. During the process, protons are translocated against a gradient in the intermembrane space by complexes I, III, and IV; the generation of a proton electrochemical potential (Δμ _H ⁺), also called proton motive force (pmf), is achieved, driving the ATP synthesis, which is catalyzed as the final step by the F₁F_o-ATP synthase (Complex V). The supramolecular organization of the respiratory chain, with the F₁F_o-ATPsynthase localized to mitochondrial cristae, where a higher surface density of protons is realized, allows a better enzymatic performance of complex V. (b) Diagram of the structure of mammalian F₁F_o-ATPsynthase. We can divide the enzymatic complex into 4 principal subdomains: a catalytic headpiece (α ₃ β ₃), hosting the three catalytic sites for ATP synthesis (one in each β subunit), a proton channel (ac ₈) and two stalks, the central rotor (γδε) and the peripheral stator (bd(F₆)OSCP) that link the first two subdomains together. While protons flow through the F_o channel from the intermembrane space into the matrix, a rotation of the stator inside the catalytic headpiece is induced, allowing a cyclic change in β-subunits conformation and the synthesis of ATP (N.B. Subunits A6L, e, f, and g are omitted in the scheme).

Figure 2

IF₁: structure and intracellular localization. (a) Schematic representation of bovine IF₁. The mature protein is composed of 84 residues and is α-helical along most of its length; an amine-terminal presequence of 25 aminoacids represents the mitochondrial targeting sequence (MTS) required for the trafficking of IF₁ into the mitochondrial matrix. In complex, IF₁ shows an ordered N-terminal region, which adopts a helix-turn-helix structure (HTH: residues 14–50) and is flanked by two disordered regions. The inhibitory domain (ID) is located at the N-terminus and is part of the minimal inhibitory sequence (MIS: residues 14–47) necessary for a correct interaction with the F₁ domain of the ATP synthase. A calmodulin-binding site (CBS: residues 33–42) have been identified at positions 33–42, followed by a histidine-rich region (HRR: residues 48–70) which is implicated in the pH-sensing mechanism and hence in the dimerization. The dimerization of IF₁ depends on the C-terminal region, which hosts the dimerization domain (DD: residues 37–84), while the oligomerization domain (OD: residues 32–44) is located in the N-terminal region of the protein, so that after oligomerization the inhibitory domain is hidden and the protein inactivated. (b) Immunocytochemical localization of IF₁ in HeLa cells: the preferential mitochondrial matrix compartmentalization of the protein is shown by its colocalization with the ATP synthase. Cells were costained with anti-IF₁ and anti-F₁F_o-ATPsynthase β chain antibodies, while DAPI was used for nuclear counterstaining.

Figure 3

Interaction of IF₁ with the F₁F_o-ATPsynthase. When mitochondria are in normal “energized” conditions (a), the F₁F_o-ATPsynthase can sustain physiological levels of ATP synthesis thanks to the presence of sufficient mitochondrial inner membrane potential; in this situation, the matrix pH is slightly basic, and IF₁ is predominantly present in its inactive, oligomeric form. When the electrochemical H⁺ gradient is lost, the F₁F_o-ATPsynthase starts hydrolysing the ATP imported from the cytosol to pump H⁺ back into the intermembrane space (b), restoring ΔΨ_m. The augmented [H⁺] in the matrix causes a fall in pH that induces the disruption of IF₁ oligomers and the release of free active dimers. The binding of IF₁ dimers at the interface between α- and β-subunits of the F₁ domain is responsible for the selective inhibition of ATP hydrolysis (c–i), while its synthesis is not affected (c–ii). Active IF₁ is able to interact with two F₁ domains at the same time, inducing the dimerization of the F₁F_o-ATPsynthase (c), with subsequent increased enzymatic performance and cristae formation.