BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 16. ENERGY METABOLISM

16.2. Oxidative Phosphorylation of ADP

16.2.1. Mechanism of Coupling between Oxidation and Phosphorylation

How is the coupling of these two processes achieved? The most well-founded answer is provided by Peter Mitchell's chemiosmotic theory, proposed in 1961. The core principles were subsequently confirmed and elaborated in detail through the joint efforts of many researchers over the following years.

Proton Gradient and Electrochemical Potential. Electron transport along the Respiratory Chain from NADH to oxygen is accompanied by the pumping of protons from the mitochondrial matrix across the inner membrane into the intermembrane space. This work consumes a portion of the energy carried by electrons moving along the Electron Transport Chain (ETC).

Protons transferred from the matrix to the intermembrane space cannot return to the matrix because the inner membrane is impermeable to protons. Consequently, a proton gradient is established, wherein the proton concentration in the intermembrane space is higher and the pH is lower than in the matrix. Furthermore, each proton carries a positive charge, resulting in a potential difference across both sides of the membrane: a negative charge on the inner side and a positive charge on the outer side. Together, the electrical and concentration gradients of H⁺ constitute the electrochemical potential gradient ΔμH⁺—the energy source for ATP synthesis. The most active proton transport in the intermembrane space, required for the generation of ΔμH, occurs at the ETC segments corresponding to complexes I, III, and IV. These segments are referred to as the sites of coupling between Respiration and phosphorylation (Fig. 16.9).

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Fig. 16.9. Coupling of respiration and ATP Synthesis in Cell/35.html">Mitochondria:

I - NADH dehydrogenase; II - succinate dehydrogenase;

III - QH₂ dehydrogenase; IV - cytochrome c oxidase; V - ATP synthase. The energy of the proton potential (electrochemical potential ΔμH) is utilized for ATP synthesis when protons return to the matrix through the Ion Channels of ATP synthase

The Mechanism of proton Transport Across the mitochondrial membrane at the coupling sites remains insufficiently clear. However, it has been established that KoQ plays a crucial role in this process. The mechanism of proton transfer involving KoQ has been studied in detail at the level of complex III (Fig. 16.10).

Fig. 16.10. Coupling of electron transfer through respiratory complex III with H⁺ transport across the membrane: reduced ubiquinone (QH₂) interacts with Fe³⁺ of heme b₁, and by reducing it, releases a proton into the aqueous phase and is converted into semiquinone (HQ^.). The electron from heme b₁ is transferred to Fe³⁺ of heme b₂. HQ^. donates the second electron to the FeS center located closer to the outer surface of the membrane, whereupon the second proton enters the intermembrane space, the electron passes to cytochrome c₁, and subsequently to cytochrome c. Oxidized Q diffuses to the inner surface of the membrane, where it receives an electron from heme b₂ and a proton from the matrix, converting into HQ^.. HQ^. receives an electron from complex I and a proton from the matrix; QH is formed within the membrane, and the entire process repeats

KoQ transfers electrons from complex I to complex III and protons from the matrix into the intermembrane space, undergoing specific cyclic transformations known as Q-cycles. The electron donor for complex III is reduced ubiquinone (QH₂), and the acceptor is cytochrome c. The latter is located on the outer side of The inner mitochondrial membrane, alongside the active center of cytochrome c₁, from which electrons are transferred to cytochrome c.

A stationary total Q/QH₂ pool exists within the membrane, from which each QH₂ molecule in one cycle ensures The transfer of protons from the matrix to the intermembrane space and electrons that ultimately reach oxygen. The work performed during proton pumping consumes a portion of the Free energy released during electron transfer down the redox potential gradient. The energy of the electrochemical potential (ΔμH⁺) is used for ATP synthesis when protons return to the matrix through the ion channels of ATP synthase.

Structure of ATP Synthase and ATP Synthesis. ATP synthase (H⁺-ATPase) is an integral protein of the inner mitochondrial membrane, located in close proximity to the respiratory chain. ATP synthase consists of two Protein Complexes designated as F₀ and F₁ (Fig. 16.11).

Fig. 16.11. Structure and Mechanisms of action of ATP synthase:

A - ATP synthase complexes. B - Catalytic cycle of ATP synthesis:

1 - binding of ADP and H₃PO₄; 2 - Formation of the phosphoanhydride bond of ATP;

3 - release of the end product. With each proton transfer through the F₀ channel into the matrix, all 3 active centers catalyze the successive phase of the cycle. The energy of the electrochemical potential is expended on the Rotation of the stalk, resulting in cyclic conformational changes of the α- and β-subunits and the synthesis of ATP

The hydrophobic F₀ complex is embedded in the membrane. It acts as a base that anchors ATP synthase within the membrane. The F₀ complex consists of several subunits that form a channel through which protons are translocated into the matrix.

The F₁ complex projects into the mitochondrial matrix. It consists of nine subunits (3α, 3β, γ, ε, δ). The α and β subunits are arranged in alternating pairs to form a "HEAD"; three active centers where ATP synthesis takes place are located between the α and β subunits, while the γ, ε, and δ subunits link the F₁ complex to F₀.

An increase in proton concentration within the intermembrane space activates ATP synthase. The electrochemical potential ΔμH⁺ drives protons to move through the ATP synthase channel into the matrix. Concurrently, under the action of ΔμH⁺, conformational changes occur in the pairs of α and β subunits of the F₁ protein, resulting in The formation of ATP from ADP and inorganic phosphate. The electrochemical potential generated at each of the three coupling sites in the ETC is utilized to synthesize one ATP molecule.

Oxidative Phosphorylation coefficient. Oxidation of a NADH molecule in the ETC is accompanied by the formation of three ATP molecules; electrons from FAD-dependent dehydrogenases enter the ETC at KoQ, bypassing the first coupling site. Therefore, only two molecules of ATP are formed. The ratio of The amount of phosphoric acid (P) used for the phosphorylation of ADP to the oxygen atom (O) consumed in THE RESPIRATORY PROCESS is called the oxidative phosphorylation coefficient and is designated as P/O. Thus, for NADH P/O = 3, and for succinate P/O = 2. These values represent the theoretical maximum of ATP synthesis; in reality, this value is lower.

Respiratory Control. The oxidation of substrates and the phosphorylation of ADP in mitochondria are tightly coupled. The rate of ATP utilization regulates the rate of electron flow through the ETC. If ATP is not utilized and its concentration in Cells increases, the flow of electrons to oxygen also stops. Conversely, ATP consumption and its conversion into ADP increases substrate oxidation and oxygen uptake. The dependence of mitochondrial respiration intensity on ADP concentration is known as respiratory control. The mechanism of respiratory control is highly precise and plays a vital role, as it ensures that the rate of ATP synthesis matches The Cell's energy demands. There are no stored reserves of ATP in the cell. The relative concentrations of ATP/ADP in Tissues vary within narrow limits, whereas cellular energy consumption—that is, the turnover rate of the ATP-ADP cycle—can vary significantly (by tens of times).

The total ATP content in the body is 30-50 g, but each ATP molecule in the cell "lives" for less than a minute. Over the course of a day, a human synthesizes 40-60 kg of ATP and breaks down the exact same amount. An increase in ADP concentration instantly leads to an acceleration of respiration and phosphorylation.