Regulation of the F1F0-ATP synthase rotary nanomotor in its monomeric-bacterial and dimeric-mitochondrial forms

Abstract

The F(1)F(0)-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the epsilon subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two alpha-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF(1)) interferes with the intrinsic rotational mechanism of the central gamma subunit and with the opening and closing of the catalytic beta-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF(1) protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F(1)F(0) mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F(1) portion. In addition, F(1)F(0) dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.

Fig. 1

Rotor–stator subunit distribution in the mitochondrial F₁F₀-ATP synthase. Only the core subunits present in bacteria and mitochondria are shown for simplicity. Rotating subunits are shown in red (γ), orange (ε), and yellow (ring of c subunits) whereas static subunits are in dark blue (β), blue (α), and cyan (subunits a and b). The arrows indicate the reversible rotation of γ–ε–c subunits relative to α and β catalytic subunits of F₁ that takes place during ATP synthesis (“clockwise” or right direction) and hydrolysis (“counterclockwise” or left direction). Bidirectional proton flow at the c-ring–sub a interface occurs associated with the gyration of the rotor as indicated by the red arrow. The second-stalk structure is simplified as two cyan subunits (a and b) that work as stator to anchor the catalytic α₃β₃ to the membranous a subunit. Image is generated in RasMol 2.6 from the mitochondrial F₁F₀ structure of S. cerevisiae (PDB code 1Q01) and edited as shown

Fig. 2

Comparison of the bovine (left) and E. coli (right) F₁F₀-ATP synthases at the central stalk domain. Crystallographic structures of MF₁ and EF₁ central stalks are shown in the same orientation. Homologous subunits are drawn in the same color, γ (blue), ε subunits (green). For clarity, only one α subunit (red) and one β subunit (yellow) are shown. The structure shown on the right is a composite of the E. coli γ–ε structure [18] and the bovine MF₁ structure [19], constructed by aligning segments of γ present in both structures. Segments of MF₁ γ subunit are shown in darker blue, and those of E. coli γ subunit are shown in lighter blue. This figure was modified from an original courtesy of Dr. Andrew J.W. Rodgers

Fig. 3

Model and crystal structures of the F₁–IF₁ complex from bovine heart mitochondria. Top three panels, our model: we positioned the IF₁ N-terminal domain at an entrance-binding site (α_E–β_E interface) at about 12-Å cross-linking distance from γ and ε subunits as we found [44]. From the side (left and right) and “bottom” (center) views, it was clearly shown and proposed for the first time that IF₁ is close enough to the rotor of the enzyme to block gyration of the central stalk as part of its inhibitory mechanism [44]. Bottom panels, the crystal structure from the F₁–IF₁ crystal with a nondimerizing fragment of IF₁ [21]: the same IF₁ N-terminal side was resolved and observed actually bound to the γ subunit at an α_DP–β_DP interface [21, 22]. The top structure depicts the entrance site of IF₁, whereas the bottom structure shows the final inhibited structure where IF₁ is locked into the same α_E–β_E interface that became α_DP–β_DP after two counterclockwise 120° gyration steps (shift from top to bottom panels). IF₁ therefore inhibits rotation of the central stalk and the opening and closing conformational changes of a single catalytic interface

Fig. 4

Model of the dimeric-mitochondrial ATP synthase: possible localization of the IF₁ protein and its movements to allow rotation of the central stalk during ATP synthesis. The model depicts the overall shape of the dimeric ATP synthase molecule that we observed for the bovine mitochondrial enzyme [54]. The dimeric interface involves F₀ subunits (e and g) and two protein bridges, one at the F₀–F₀ side of unknown composition (question mark) and another at the F₁–F₁ interface where the second stalks (not shown for clarity) and the IF₁ protein (red) are likely to be located. The C-terminal side of the IF₁ molecule is assumed to cross the dimer interface and to stabilize the dimer by interacting with subunits OSCP [65] and possibly subunits of the second stalk. The N-terminal inhibitory domain that in the absence of the proton gradient blocks rotation of the central stalk by entering at an α–β–γ interface (Fig. 2) is removed from this position and exposed into the media after establishment of a transmembrane proton gradient, thus allowing rotation of the central stalk during ATP synthesis. The F₁ structures were constructed from the bovine F₁-DCCD coordinates available (PDF code 1E79)

Fig. 5

Possible arrangement of the ATP synthase helical polymer that wraps and gives shape to the mitochondrial tubular cristae. a Inner view of the polymer from the interior of the cristae (membrane is transparent); the green spheres represent the F₀ channels connected by the protein bridge we observed [54]; the blue spheres are the F₁ heads. b The ATP synthase polymer is depicted from the outer surface of a single cristae (yellow). The F₁ particles are in blue and connected with a protein bridge that could be composed by the IF₁ and second-stalk subunits. The original model is from Allen et al. [75]