Catalytic and mechanical cycles in F-ATP synthases. Fourth in the Cycles Review Series

Abstract

Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F1F(o) ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F(o) sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated.

Figure 1

Structure and function of a bacterial ATP synthase in a biological membrane. F₁ (subunits α₃β₃γδɛ) and F_o (subunits ab₂c_10–15) are two motors that exchange energy by rotational coupling. The rotary subunits (γɛc_10–15) are shown in blue and the membrane-anchored and cytoplasmic stator subunits (ab₂α₃β₃δ) are shown in orange and green. During ATP synthesis, coupling ions (orange) pass through the F_o motor from the periplasm to the cytoplasm inducing rotation and enabling the F₁ motor to synthesize ATP.

Figure 2

Energy conversions by ion-cycling across membranes. (A) Proton cycle coupling respiration to ATP synthesis in mitochondria or aerobic bacteria. In the respiratory chain from NADH to O₂, the number of protons pumped across the membrane by the chain complexes I, III and IV is sufficient for the synthesis of approximately three molecules of ATP by the ATP synthase. (B) Na⁺ ion-cycle coupling ATP synthesis to the decarboxylation of methylmalonyl-CoA during succinate fermentation in Propionigenium modestum.

Figure 3

Structure of the I. tartaricus c₁₁ ring in ribbon form. (A) Individual subunits are shown in different colors. The grey spheres indicate the bound Na⁺ ions. The structure shows a cylindrical, hourglass-shaped protein complex with an outer diameter of ∼45 Å in the middle of the membrane and ∼50 Å at the top and bottom. The ring has a height of ∼70 Å and therefore protrudes out of the membrane on both sides. A hydrophobic cavity with a diameter of ∼17 Å at its narrowest part is located in the centre of the ring. (B) Close-up of the Na⁺ binding site formed by the inner (N1) and outer helix (C1) of one c-subunit and the outer helix (C2) of the neighbouring c-subunit. Na⁺ coordination and selected hydrogen bonds are indicated with dashed lines. This structure shows the locked conformation. During opening, the side chain of Y70 might relocate into a cavity underneath the binding site, thus destabilizing the hydrogen bonding network and allowing unloading and loading of the binding site to and from subunit a.

Figure 4

Model for ion translocation in the a/c subunit interface. The c ring (blue) rotates counterclockwise against subunit a (orange) during ATP synthesis. (1) The Na⁺ ion (orange) is coordinated by the E65, which is on the outer helix distal to subunit a, closing the horizontal gate towards subunit a. (2) On reaching the aR227 in the a/c subunit interface, electrostatic interactions stabilize the closed conformation of the glutamate and simultaneously repel the Na⁺ ion vertically out of its site. (3) After passing aR227, the electrostatic attraction between cE65 and aR227 is retained. This opens the gate and allows a Na⁺ ion to enter the site from subunit a. (4) On coordination of the incoming Na⁺ ion, the E65 switches back into the closed conformation and the cycle can start again.

Figure 5

Model for torque generation in the H⁺- and Na⁺-translocating F_o motor. (A) Two-channel model with a ratchet-type mechanism for H⁺-dependent enzymes. The crucial events during ion translocation in ATP synthesis direction in the a/c interface can be divided into four different zones. (1) The occupied rotor site enters the interface and releases its coupling ion through the outlet channel into the cytoplasm with high pH. The deprotonation of the binding site prevents backwards rotation into the lipid phase and acts as a molecular ratchet. (2) The negative charge of the binding site is compensated by the stator arginine. In this functionally symmetric state, Brownian back-and-forth motions towards either channel are possible. (3) As the inlet channel, which is in contact with the periplasm where there is a low pH, contains more protons than the outlet channel, which is in contact with the cytoplasm, the binding site is more frequently protonated from the periplasm. Therefore, the ΔpH determines the direction of rotation. (4) The loaded binding site can now move out of the interface into the lipid bilayer, whereby the next binding site enters the interface and experiences the events described in (1). (B) Push-and-pull model for Na⁺-dependent enzymes. The upper part shows the events taking place in the a/c subunit interface and the lower part shows the calculated free energy (ΔG) profiles of an empty or occupied site during ion translocation. Arrows indicate where a Na⁺ ion is released or taken up, respectively. (1) In ATP synthesis direction, an occupied rotor site enters the interface from the left and releases its bound Na⁺ ion towards the cytoplasm. This process is aided by the stator arginine. (2) The stator arginine compensates for the now negatively charged empty binding site. The horizontal component of the membrane potential, however, pulls the arginine to the left and pushes the glutamate to the right. Therefore, the electrical component of the ion motive force determines the direction of rotation from left to right. (3) The hydration of the binding site within the inlet channel stabilizes this conformation and allows loading of the binding site from the periplasm. Movement of the binding site from zone 2 to 3 pulls the next rotor site into the a/c interface as described in (1). (4) The binding site that has been occupied from the periplasm is allowed to rotate out of the interface into the lipid phase. This event is aided by a push mechanism during the events described in (2).