Fundamentals of Molecular Biology. Part 1: Molecular Biology of the Cell - A. N. Ogurtsov 2011

Generation of Proton-Motive Force and ATP Synthesis
Oxidative Phosphorylation

8.4.1. Oxidative Phosphorylation in Bacteria. Although bacteria lack internal membranes, aerobic bacteria possess oxidative phosphorylation systems similar to those described above for eukaryotic Cell/35.html">Mitochondria.

The Enzymes catalyzing the reactions of both Glycolysis and the citrate cycle are located in the bacterial Cytosol, whereas the enzymes that oxidize NADH to NAD^⊕ and ultimately transfer electrons to O₂ are situated in the bacterial Plasma Membrane. This electron transfer is coupled with the outward pumping of protons from The Cell via proton pumps (Figure 146).

The reverse influx of protons from the exoplasm into the cell driven by the proton gradient is coupled with ATP synthesis. The Structure and function of bacterial F₀F₁ complexes are identical to those of mitochondria (Figure 132), but they are much easier to isolate and study. The proton-motive force across the bacterial plasma membrane is also utilized for the Transmembrane Translocation of nutrients such as sugars, employing proton-sugar symporters (transport Membrane Proteins), as well as for the Rotation of the bacterial flagellum (Figure 143).

Primitive aerobic bacteria appear to have been the precursors of Introduction/5.html">Eukaryotic Cell mitochondria (Figure 147).

8.4.2. F₀F₁ Complex. The F₀F₁ complex, or ATP synthase, consists of two main components, F₀ and F₁ (Figure 170). The F₀F₁ complex is sometimes referred to as Complex V because ATP synthase is directly linked to the Respiratory Chain.

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Figure 170 - Schematic diagram of ATP synthase in the bacterial plasma membrane

The membrane component F₀ consists of Three types of integral membrane proteins designated as a, b, and c. In bacterial and Yeast Cells, the F₀ moiety consists of one a subunit, two b subunits, and ten c subunits; therefore, the COMPOSITION OF THE F₀ component is denoted as a₁b₂c₁₀.

The F₀ component of animal cell mitochondria contains 12 c subunits, whereas that of plant METABOLISM/14.html">Chloroplasts contains 14 c subunits. The c subunits form a ring within the plane of the membrane. The a and b subunits are tightly bound to each other but do not interact with the c subunit ring.

The cytosolic component F₁ is a Water-soluble complex of five distinct Polypeptides with the structure a₃ß₃γδε.

At the bottom of F₁ lies the ε subunit, which is firmly attached both to the γ subunit and to several c subunits of the F₀ component.

The a and ß subunits of the F₁ component are arranged in alternation to form a annular hexamer aßaßaß or (aß)₃, which sits atop the γ subunit. The δ subunit anchors this hexamer to the b subunits of the F₀ component.

Consequently, the a and b subunits of the F₀ component, together with the δ subunit and the (aß)₃ hexamer of the F₁ component, form a tightly bound membrane-anchored structure—a stator of sorts that does not participate in mechanical motion and relative to which the rotor rotates, consisting of the c subunits of the F₀ component and the γ and ε subunits of the F₁ component.

8.4.3. ATP Synthesis. A proton passes through proton hemichannel I in the a subunit to reach the c subunit and binds to the Asp61 residue of this subunit. Following the rotation of the c-ring, this proton reaches hemichannel II, which allows the proton to dissociate from the aspartate and enter the cytosol.

The rotation of the γ subunit driven by this proton flux relative to the stationary a and ß subunits cyclically stimulates conformational transitions in the catalytic ß subunits.

Each catalytic ß subunit sequentially adopts three Conformations (Figure 171):

1. The O (open) conformation, which binds ADP and P_i weakly and does not bind ATP;

2. The L (loose) conformation, which binds ADP and P_i more tightly;

3. The T (tight) conformation, in which ADP and P_i are bound so tightly that they spontaneously form ATP.

Further rotation of the γ subunit returns the catalytic subunit to its initial O conformation, in which the newly formed ATP molecule is released and the cycle begins anew.

Figure 171 - Catalytic cycle of ATP synthase

Suppose ADP and P_i bind to the ß₁ subunit in the O conformation (Figure 171(a)). The proton current rotates the ATP synthase rotor (and the γ subunit) by 120° relative to the stationary ß subunits (Figure 171(a) → (b)).

As a result of this rotation, the affinity of the ß₁ subunit for ADP and P_i increases from O to L, that of the ß₃ subunit increases from L to T, and that of the ß₂ subunit decreases from T to O, leading to the release of the synthesized ATP molecule from the ß₂ subunit.

In the next step (Figure 171(b) → (c)), ADP and P_i within the ß₃ subunit, which now adopts the T conformation, combine into a molecule of

ATP, and to the ß₂ subunit (in the O conformation) bind ADP and P_i. As a result (Figure 171(b)), an F₁-metabolite complex identical to the initial one (Figure 171(a)) is formed, but rotated by 120°.

At the next stage (Figure 171(c) → (d)), the y-subunit undergoes another 120° rotation, which once again triggers the O→L→T→O Conformational Changes in the ß subunits described above. Repeating steps (a)→(b) and (b)→(c) yields three ATP molecules per "full 360° revolution" of the y-subunit.

ATP or ADP molecules also bind to regulatory (or allosteric) sites on the three a-subunits. Such binding modulates The rate of ATP Synthesis in response to changing ATP concentrations in the matrix, but does not directly affect the synthesis of ATP from ADP and P_i.

8.4.4. Rotation of the ATP Synthase Rotor. The currently most accepted working model of the F₀ motor, out of the various hypotheses proposed, relies on rotational diffusion and is illustrated in Figure 172.

Figure 172 - Operating principle of the F₀ motor

The cylindrical transmembrane rotor of ATP synthase features negatively charged proton-binding sites (aspartic acid residues Asp61). Because the rotor is embedded in the membrane, it can only rotate when these sites are neutralized by protons.

Protons reach the rotor via one half-channel in the stator (Figure 31 schematically shows the stator domain adjacent to the right side of the rotor) and, having completed a full circle along with the rotor, exit through another half-channel in the stator body on the opposite side of the membrane.

Currently, two Variants of the F₀ motor driven by proton or sodium ion gradients have been studied in detail.

In principle, the F₀ rotor can undergo random Thermal Fluctuations in both directions. To ensure unidirectional rotation, it is necessary to permit rotation in the desired direction while blocking reverse rotation. In other words, out of random thermal motions, the motor must "select" only "favorable" events, acting as a "Brownian ratchet" (or "Brownian sieve"). The negatively charged aspartic acid residues at the proton-binding sites prevent the rotor from spinning freely within the membrane. Protons flow from the high-concentration region to the rotor via a half-channel in the stator (the upper half-channel in Figure 172). In the region shielded from the membrane by the stator, the rotor aspartates become protonated, thereby neutralizing their negative charge. These neutral rotor domains can then rotate (counterclockwise, as shown in the figure) within the membrane. Upon completing a revolution, the protonated aspartate re-enters the region shielded from the membrane by the stator. Here, a second half-channel leads to the opposite side of the membrane, where the proton concentration is lower.

The proton dissociates from the aspartate and exits through the half-channel, while the remaining charged aspartate prevents the rotor from spinning backward (acting as a molecular ratchet that Functions as a Brownian sieve).

This entire process is reversible.

Spontaneously, the F₀ motor will capture protons from the half-channel where their concentration is high and release them into the half-channel where the proton concentration is low.

However, if a constant torque is applied to the rotor in the opposite direction, the rotor will be unable to rotate (now clockwise, opposite to the direction indicated by the arrow in Figure 172) until it captures a proton from the low-concentration half-channel.

Upon completing a revolution, the rotor releases the proton into the high-concentration half-channel. In this scenario, driven by an external force, the motor functions as a proton pump, actively transporting protons across the membrane.