Directed proton transfer from Fo to F1 extends the multifaceted proton functions in ATP synthase

Abstract

The role of protons in ATP synthase is typically considered to be energy storage in the form of an electrochemical potential, as well as an operating element proving rotation. However, this review emphasizes that protons also act as activators of conformational changes in F₁ and as direct participants in phosphorylation reaction. The protons transferred through F_o do not immediately leave to the bulk aqueous phase, but instead provide for the formation of a pH gradient between acidifying F_o and alkalizing F₁. It facilitates a directed inter-subunit proton transfer to F₁, where they are used in the ATP synthesis reaction. This ensures that the enzyme activity is not limited by a lack of protons in the alkaline mitochondrial matrix or chloroplast stroma. Up to one hundred protons bind to the carboxyl groups of the F₁ subunit, altering the electrical interactions between the amino acids of the enzyme. This removes the inhibition of ATP synthase caused by the electrostatic attraction of charged amino acids of the stator and rotor and also makes the enzyme more prone to conformational changes. Protonation occurs during ATP synthesis initiation and during phosphorylation, while deprotonation blocks the rotation inhibiting both synthesis and hydrolysis. Thus, protons participate in the functioning of all main components of ATP synthase molecular machine making it effectively a proton-driven electric machine. The review highlights the key role of protons as a coupling factor in ATP synthase with multifaceted functions, including charge and energy transport, torque generation, facilitation of conformational changes, and participation in the ATP synthesis reaction.

Keywords: F1Fo ATP synthase; H+ ions; Oxidative phosphorylation system; Protein conformational changes; Proton transport.

Fig. 1

Demonstration of the ATP synthase structure is shown through the example of spinach chloroplasts ATP synthase, which was obtained by single particle cryo-EM in lipid nanodiscs (Hahn et al. 2018). (a) General view displays the main subunits and functional parts (PDB 6FKF). (b, c, d) Slices through the nucleotide-binding centers of F₁ show three different positions of the rotor subunit γ varying on 120°. (b) Slice of PDB structure 6FKF. (c) Slice of PDB structure 6FKH. (d) Slice of PDB structure 6FKI. The beta subunit, which is tightly connected to the rotor (γ-subunit, blue color), does not bind the nucleotide (open conformation) in accordance with mechanochemical coupling model

Fig. 2

The binding change mechanism (Boyer et al. 1973). For clarity, it is shown only one direction of catalysis — ATP synthesis reaction. The transition between forms 2a and 2b goes isoenergetically

Fig. 3

The structure of the catalytic center in the transitional state, binding ADP, Mg²⁺, and P_i, is illustrated using the example of Bacillus sp. PS3 F₁ subunit (PDB 7L1Q). The P-loop (from Gly159 to Thr165) and some other charged residues in the vicinity of ADP and phosphate binding sites are shown. The positions of water molecules are not identified. Oxygen is red, phosphorus orange, nitrogen blue, carbons of β and α subunits are gray and white correspondingly

Fig. 4

Proton path within the membrane-embedded F_o factor. a Schematic representation. b Illustration of proton path in three-dimensional structure (PDB 6FKF). Alpha-helices of the c-ring and a-subunit are shown as tubes and are surrounded by membrane lipids, which are not shown. The proton inside the protein partially loses its hydrate shell and should regain it in or after the exit channel. After binding to the glutamate of c-ring, the proton moves with the rotation of the c-ring to the exit channel

Fig. 5

The buffering capacity β of lettuce leaf chloroplasts was evaluated in both light and dark conditions, utilizing data from (Walz et al. 1974). It is seen that beta increases in the 7–9 pH range under illumination, while simultaneously decreases to a similar value in the 4–6 range. The inset displays the buffering capacity of pea leaves chloroplasts in the light with and without DCCD, based on the data from (Zolotareva et al. 1986)

Fig. 6

The comparison of F₁F_o ATP synthases from different organisms is presented. Charged amino acids on the water-accessible surface of the enzymes, which have a significant role in proton binding at varying pH, are highlighted with different colors as per the legend. The ATP synthase structures of spinach chloroplast (PDB 6FKF), thermophilic bacterium Bacillus sp. PS3 (PDB 6N2Y), yeast Saccharomyces cerevisiae mitochondria (PDB 6CP6), and bovine mitochondria (PDB 5ARA) are displayed

Fig. 7

Proton functioning and cycling in ATP synthesis system. Protons are transferred from F_o to F₁ subunit of ATP synthase in the presence of an electrochemical potential and induce local increase of H⁺ activity in the interface