BIOCHEMISTRY: A TEXTBOOK FOR UNIVERSITIES - E. S. Severin - 2004

CHAPTER 6. ENERGY METABOLISM

II. Oxidative Phosphorylation of ADP

Since electrons naturally tend to flow from electronegative to electropositive systems, their transport along the Electron Transport Chain (ETC) to oxygen is accompanied by a decrease in Free energy.

Comparing the electrochemical potentials of electron carriers (Table 6-3) shows that free energy drops at each step of the ETC, meaning electron energy is released in discrete portions.

At the same time, three segments in the Respiratory Chain can be identified where Electron transport is accompanied by a relatively large drop in free energy (Fig. 6-11). These stages are capable of driving ATP synthesis, as The amount of released free energy is approximately equal to the energy required to synthesize ATP from ADP and phosphate. It has been experimentally confirmed that Electron transport along the ETC and ATP synthesis are energetically coupled.

The first process—The transfer of electrons from the reduced Coenzymes NADH and FADH₂ through the ETC to oxygen—is exergonic. For example,

NADH + Н⁺ + 1/2 O₂ —> NАD⁺ + Н₂O + 52 kcal/mol (~220 kJ/mol). (1)

The second process—the phosphorylation of ADP, or ATP synthesis—is endergonic:

АДФ + Н₃РO₄ + 7,3 ккал/моль (30,5 кДж/моль) = АТФ + Н₂O. (2)

The synthesis of ATP from ADP and H₃PO₄ driven by the energy of electron transport along the ETC is termed Oxidative Phosphorylation.

A. Mechanism of coupling between Oxidation and Phosphorylation

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

1. Proton Gradient and Electrochemical Potential

The transfer of electrons 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 the electrons moving through the ETC.

Protons transferred from the matrix into the intermembrane space cannot return to the matrix because the inner membrane is impermeable to protons. Thus, 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, generating 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 form the electrochemical potential ∆μН⁺, which serves as the energy source for ATP synthesis. Since the most active proton transport into the intermembrane space—required for generating ∆μH⁺—occurs at the ETC segments corresponding to the locations of complexes I, III, and IV, these regions are referred to as the sites of coupling between Respiration and phosphorylation (Figs. 6-11, 6-13).

Class="center">Fig. 6-13. 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 ∆μH⁺) 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 CoQ plays a crucial role in this process. The mechanism of proton transfer facilitated by CoQ has been studied in greatest detail at the level of complex III (Fig. 6-14).

Fig. 6-14. Coupling of electron transfer through respiratory complex III with H⁺ transport across the membrane. Reduced ubiquinone (QH₂) interacts with Fe³⁺ of heme b₁ and, reducing it, releases a proton into the aqueous phase, converting into a semiquinone (HQ*). The electron from heme b₁ is transferred to Fe³⁺ of heme b₂. HQ* releases its second electron to the FeS center located near the outer surface of the membrane; simultaneously, the second proton enters the intermembrane space, and the electron is transferred to cytochrome c₁ and subsequently to cytochrome c. Oxidized Q diffuses to the inner side of the membrane, where it accepts an electron from heme b₂ and a proton from the matrix, becoming HQ*. HQ* receives an electron from complex I and a proton from the matrix; QH₂ is formed in the membrane, and the entire cycle repeats.

CoQ transfers electrons from complex I to complex III and protons from the matrix to the intermembrane space through distinct cyclic transformations known as Q-cycles. The electron donor for complex III is reduced ubiquinone (QH₂), while the acceptor is cytochrome c. Cytochrome c is located on the outer side of The inner mitochondrial membrane, which also houses the Active Site of cytochrome c1, from where electrons are transferred to cytochrome c.

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

2. Structure of ATP Synthase and ATP Synthesis

ATP synthase (H⁺-ATPase) is an integral protein of the inner mitochondrial membrane. It is located in close proximity to the respiratory chain. ATP synthase consists of two Protein Complexes, designated as F₀ and F₁ (Fig. 6-15).

Fig. 6-15. Structure and MECHANISM OF ACTION of ATP synthase. A — F₀ and F₁ represent the complexes of ATP synthase. F₀ is composed of polypeptide chains that form a transmembrane channel, allowing protons to return from the intermembrane space into the matrix; the F₁ protein protrudes into the matrix from the inner side of the membrane and contains 9 subunits, 6 of which form 3 α and β pairs (the "HEAD") shielding the central stalk, which consists of 3 subunits (y, δ, and ε). The y and ε subunits are mobile, forming a stalk that rotates within the stationary head and links to the F₀ complex. The active sites, formed by pairs of α and β subunits, are responsible for binding ADP, inorganic phosphate (P_i), and ATP. B — The catalytic cycle of ATP synthesis consists of 3 sequential phases occurring alternately across the 3 active sites: 1 — binding of ADP and H₃PO₄; 2 — Formation of the phosphoanhydride bond of ATP; 3 — release of the final product. With each transfer of protons through the F₀ channel into the matrix, all 3 active sites catalyze the next phase of the cycle. The energy of the electrochemical potential drives the Rotation of the stalk, which induces a cyclic conformational change in the α and β subunits, thereby driving ATP synthesis.

The hydrophobic F₀ complex is embedded in the membrane and acts as the anchor that fixes ATP synthase in place. F₀ consists of several subunits that form a channel through which protons are translocated into the matrix.

The F₁ complex protrudes into the mitochondrial matrix. It consists of 9 subunits (3α, 3β, y, ε, δ). The α and β subunits are arranged in pairs to form the "head"; the 3 active sites where ATP synthesis takes place are located between the α and β subunits, while the y, ε, and δ subunits link the F₁ complex to F₀.

An elevated proton concentration in the intermembrane space activates ATP synthase. The electrochemical potential ∆μH⁺ forces protons to flow through the ATP synthase channel into the matrix. Concurrently, driven by ∆μH⁺, conformational changes occur within the α and β subunit pairs of the F₁ protein, resulting in The formation of ATP from ADP and inorganic phosphate. The electrochemical potential generated at each of the 3 coupling sites in the ETC is utilized to synthesize a single molecule of ATP.

3. Oxidative Phosphorylation Ratio

The oxidation of an NADH molecule in the ETC is accompanied by the formation of 3 molecules of ATP; electrons from FAD-dependent dehydrogenases enter the ETC at CoQ, bypassing the first coupling site. Consequently, only 2 ATP molecules are produced. The ratio of the amount of phosphoric acid (P) used to phosphorylate ADP to the amount of oxygen atoms (O) consumed during respiration is termed the oxidative phosphorylation ratio and is denoted as P/O. Thus, for NADH, P/O = 3, and for succinate, P/O = 2. These values represent the theoretical maximum for ATP synthesis; in reality, this figure is lower.

4. Respiratory Control

Substrate oxidation and ADP phosphorylation in mitochondria are tightly coupled. The rate of ATP consumption regulates the rate of electron flow through the ETC. If ATP is not consumed and its intracellular concentration rises, the flow of electrons to oxygen ceases. Conversely, ATP consumption and its conversion to ADP accelerate substrate oxidation and oxygen uptake. The dependence of mitochondrial respiration intensity on ADP concentration is referred to 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 energy demands of The Cell. There are no significant ATP reserves in the cell. The relative ATP/ADP concentrations in Tissues vary within a narrow range, whereas cellular energy consumption—i.e., the turnover rate of the ATP–ADP cycle—can fluctuate dozens of times over.

The total body content of ATP is 30–50 g, yet each ATP molecule in the cell has a "lifespan" of less than a minute. In a single day, The Human Body synthesizes and degrades 40–60 kg of ATP. An increase in ADP concentration immediately accelerates respiration and phosphorylation.

B. Transport of ATP and ADP Across Mitochondrial Membranes

In most Eukaryotic Cells, the bulk of ATP synthesis occurs inside the mitochondria, whereas the primary ATP consumers are located outside. Conversely, an adequate concentration of ADP must be maintained within the mitochondrial matrix. These charged molecules cannot cross The Lipid Bilayer on their own. The inner membrane is impermeable to charged and hydrophilic substances, yet it contains a specific set of transporters that selectively shuttle such molecules between the Cytosol and the matrix.

The membrane contains the ATP/ADP antiporter protein, which facilitates the Transmembrane Transport of these metabolites (Fig. 6-16). An ADP molecule can enter the mitochondrial matrix only in exchange for an ATP molecule exiting the matrix.

Fig. 6-16. Diagram of transmembrane substance transport driven by the energy of ∆μH⁺. Flows of various substances (ATP, ADP, H₃PO₄, Ca²⁺) occur via specific transporters, consuming the energy of the membrane's electrochemical potential.

The driving force for this exchange is the Membrane Potential generated by electron transport along the ETC. Calculations indicate that about a quarter of the Free energy of the proton motive force is expended on The transport of ATP and ADP. Other transporters can also harness the energy of the electrochemical gradient. For instance, inorganic phosphate, required for ATP synthesis, is transported into the mitochondria in this manner. Furthermore, the direct source of free energy for the transport of Ca²⁺ into the matrix is the proton gradient rather than ATP energy.

C. Uncoupling of respiration and Phosphorylation

Certain chemical agents (protonophores) can transport protons, while others (ionophores) transport other ions from the intermembrane space across the membrane into the matrix, bypassing the proton channels of ATP synthase. As a result, the electrochemical potential dissipates and ATP synthesis halts. This phenomenon is known as the uncoupling of respiration and phosphorylation. Consequently, ATP levels decrease while ADP levels increase. Under these conditions, the rate of NADH and NADH₂ oxidation rises, along with oxygen consumption; however, the energy is released entirely as heat, and the P/O ratio drops sharply. Typically, uncouplers are lipophilic compounds that readily cross the lipid bilayer of the membrane. One such compound is 2,4-dinitrophenol (Fig. 6-17), which easily shifts between ionized and non-ionized forms, picking up a proton in the intermembrane space and carrying it into the matrix.

Fig. 6-17. Mechanism of uncoupling of respiration and phosphorylation. The protonated form of 2,4-dinitrophenol transports protons across the inner mitochondrial membrane, preventing the formation of the proton gradient.

Other Examples of uncouplers include certain medications, such as the anticoagulant dicumarol (see Section 14), as well as endogenous metabolites like bilirubin, a heme Catabolism product (see Section 13), and thyroxine, a thyroid hormone (see Section 11). All of these substances exhibit uncoupling effects only at high concentrations.

D. Thermoregulatory function of the ETC

Roughly 40–45% of the total energy of electrons transferred along the ETC is utilized for ATP synthesis, and about 25% is spent on transmembrane transport work. The remaining energy is dissipated as heat and used by warm-blooded animals to maintain body Temperature. Additionally, extra heat can be generated through the uncoupling of respiration and phosphorylation. The uncoupling of oxidative phosphorylation can be biologically beneficial, as it generates heat to maintain body temperature in newborns, hibernating animals, and mammals adapting to cold environments. Newborns and hibernating animals possess a specialized tissue dedicated to heat production via uncoupling—brown adipose tissue (brown fat). Brown fat is rich in mitochondria, where the inner mitochondrial membrane contains a vast excess of respiratory Enzymes relative to ATP synthase. About 10% of all mitochondrial Proteins consist of the so-called uncoupling protein (UCP-1), or thermogenin. Structurally, thermogenin is similar to the ATP/ADP antiporter; however, it is incapable of nucleotide transport, though it retains The ability to transport fatty acid anions, which act as uncouplers (Fig. 6-18).

Fig. 6-18. Mechanism of the uncoupling action of Fatty acids. 1 — proton pumping by the respiratory chain; 2 — protonation of the fatty acid anion; 3 — diffusion of the protonated fatty acid toward the inner membrane surface; 4 — dissociation of RCOOH into RCOO^- and an H⁺ ion; 5 — transport of RCOO^- by the ATP/ADP antiporter or uncoupling protein to the outer surface of the mitochondrial membrane.

On the outer side of the membrane, the fatty acid anion binds a proton and crosses the membrane in this form; on the inner side of the membrane, it dissociates, releasing a proton into the matrix and thereby reducing the proton gradient. The resulting anion is transported back to the outer side of the membrane by the ATP/ADP antiporter.

Cooling stimulates the release of noradrenaline from sympathetic nerve terminals. This results in the activation of lipase in adipose tissue and the mobilization of fat from fat depots (see Section 8). The resulting free fatty acids serve not only as "fuel," but also as a crucial regulator of the uncoupling of respiration and phosphorylation.