Fundamentals of Biochemistry - Filippovich, Y. B. 1999

Biological Oxidation
Oxidation coupled with ADP phosphorylation

Substrate-level phosphorylation. Examples of the Coupling of oxidation with phosphorylation at the substrate level include The oxidation of 3-phosphoglyceraldehyde to 1,3-diphosphoglyceric acid, 2-phosphoglyceric acid to 2-phosphoenolpyruvic acid, and a-ketoglutaric acid to succinic acid (here GDP is phosphorylated — see Fig. 117). The resulting compounds contain a high-energy phosphate bond that is readily transferred to ADP (or GDP). One example of such coupling and The Mechanism of activated phosphate transfer to ADP is discussed in detail above (see Fig. 114). However, Substrate-Level Phosphorylation Reactions yield a relatively small amount of ATP (Fig. 128).

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Fig. 128. Localization of ATP synthesis during substrate-level and Oxidative Phosphorylation (explanations in the text)

Oxidative phosphorylation. The bulk of ATP in aerobic organisms is synthesized via oxidative phosphorylation in Cell/35.html">Mitochondria—the true powerhouses of The Cell.

Hydrogen atoms removed from substrates in the dicarboxylic and Tricarboxylic Acid Cycle, during the ß-Oxidation of Higher Fatty acids, as well as in Pyruvate dehydrogenase, Glutamate dehydrogenase, and several other reactions, enter the Respiratory Chain of Enzymes located in The inner mitochondrial membrane. NADH serves as the universal hydrogen atom donor for the enzyme respiratory chain.

Fig. 129. Components of the mitochondrial respiratory chain:

E'₀ — oxidation-reduction potentials of the respiratory chain components; ∆Е₀ — potential difference between respiratory chain components at the sites coupled with ADP phosphorylation (underlined with a thick black line); I, II, III — coupling sites. The mitochondrial respiratory chain is more complex than shown in the figure. Under certain conditions, it can be separated into 4 complexes, each characterized by its molecular weight, polypeptide composition, and oxidation-reduction potential values (E'₀). Complex I: M = 700—900 kDa, 2S Polypeptides; Complex II: M = 140 kDa, 4–5 polypeptides; Complex III: M = 250 kDa, 9–10 polypeptides; Complex IV: M = 160–170 kDa, S polypeptides. The respiratory chain is adjacent to the ATP synthase complex (Complex V: M = 500 kDa, 12–14 polypeptides), shown in Figs. 130 and 131. Thus, energy transformation in mitochondria involves about 70 different polypeptides and 6 types of Phospholipids in the coupling membrane (see the figure "Block Structure OF THE Mitochondrial Chain" on the endpaper at the beginning of the textbook)

Consequently, in this case as well, H atoms are transferred to NAD⁺ prior to entering the respiratory chain (Fig. 128).

Another primary source of hydrogen atoms and electrons in the respiratory chain is reduced flavoprotein, when it acts as the primary dehydrogenase, as, for example, in the oxidation of succinic acid in the tricarboxylic and Dicarboxylic Acid Cycle (see Fig. 117). A flavoprotein of a slightly different nature serves as an intermediary for transferring hydrogen atoms and electrons from NADH to the ubiquinone protein of the respiratory chain.

Fig. 129 illustrates the respiratory chain of mitochondrial membrane enzymes. Naturally, it begins with NADH, from which H atoms are transferred to the first protein component of the respiratory chain—a flavoprotein carrying flavin mononucleotide (FMN) as a coenzyme. The remaining components of the respiratory chain are arranged in order of increasing normal oxidation-reduction potentials (E'₀) (measured at a 1 M concentration, a Temperature of 25° C, denoted by the index E₀, and pH 7.0, and marked with a prime symbol '), which ensure the orderly transfer of hydrogen atoms and electrons along this redox chain.

The most remarkable feature of the enzyme respiratory chain is the presence of segments where adjacent components differ sharply in their oxidation-reduction potential values (∆E₀):

Standard Oxidation-Reduction Potentials of Compounds Involved in Biological Oxidation

It is precisely here that oxidation is coupled with ADP phosphorylation (Fig. 129), since the difference in Energy Levels of an electron transported at an immense speed (about 1 ms from one carrier to another) is quite sufficient for the synthesis of a high-energy bond, amounting to 51 kJ for coupling site I, 36 kJ for site II, and 80.7 kJ for site III. Overall, The rate of Oxidative phosphorylation is determined by The energy charge, i.e., The ratio of adenosine mono-, di-, and triphosphates:

Fig. 130. Structure of a mitochondrion (A) and schematic arrangement of respiratory chain enzymes and the ATP synthase complex in its inner membrane (B):

FMN — flavoprotein with flavin mononucleotide as a coenzyme; FeS — iron-sulfur Proteins; Q — ubiquinone protein; b, c₁ and c — Cytochromes; a — cytochrome oxidase

Fig. 131. Transmembrane electron and proton transport and its coupling with ATP synthesis (designations are the same as in Fig. 129)

Membranes containing the enzymes of Electron Transport and coupled phosphorylation are referred to as coupling membranes. These include the inner mitochondrial membrane, the thylakoid membrane of green plant METABOLISM/14.html">Chloroplasts, the chromatophore membrane of photosynthetic Bacteria, and the cell membranes of aerobic bacteria exhibiting a respiratory type of Energy Metabolism. They are characterized by a thickness of 7.0–9.0 nm, a protein-to-lipid ratio favoring proteins (2:1), a low Cholesterol content, and the presence of cardiolipin; approximately one-third of their constituent proteins belong to respiratory chain enzymes assembled into complexes (Fig. 129)—for example, several thousand such complexes are present in each mitochondrion of rat Liver Cells.

The Structure and function of the mitochondrial coupling membrane have been studied most thoroughly. Alongside the respiratory enzyme complex, it houses the ATP synthase complex responsible for ATP generation. How are these arranged within the mitochondrion? Fig. 130 provides the answer. Both enzyme complexes are localized in the inner mitochondrial membrane (Fig. 130, B), with the ATP synthase complex forming so-called mushroom-like projections that stud the inner membrane and face the matrix of mitochondrial particles.

The problem of coupling oxidation with phosphorylation is extraordinarily complex and still far from final resolution. Early hypotheses on this subject—the chemical intermediate hypothesis (E. Slater, 1953) and the conformational hypothesis (P. Boyer, 1964)—are now of purely historical interest, although certain elements of both are reflected to some extent in Peter Mitchell's currently accepted chemiosmotic hypothesis, which was supported and further developed in our country through the works of V.P. Skulachev and coworkers.

According to the chemiosmotic hypothesis, it is the Structural and functional Features of the coupling membrane (a term introduced into Bioenergetics by V. P. Skulachev) that drive ATP Biosynthesis. As the enzyme respiratory chain Functions in the mitochondrial coupling membrane—which is impermeable to both NADH and protons—an Active Transport of six H⁺ from the matrix into the intermembrane space occurs (Fig. 131) for every pair of electrons passing through the Electron Transport Chain. Various opinions have been expressed regarding the mechanism of this transport. The Essence of some of these is clear from Fig. 131. It is also hypothesized that proton translocation involves proton translocases—specific proteins localized in the coupling membrane that couple proton transfer with electron transport via a protein complex, as, for example, in the case of cytochrome c oxidase (Fig. 132) or NADH:β-oxidoreductase.

Fig. 132. Proton pump coupled with cytochrome c oxidase of the mitochondrial membrane

At M = 140,000, cytochrome c oxidase consists of 7 subunits containing 2 Cu atoms and 2 Fe atoms (within the heme group, shown by the oval circle), connected by coordination bonds to the nitrogen atoms of Histidine radicals. Since the Cu and Fe atoms of cytochrome a · a₃ are separated by a distance of 3.5 nm, only electron tunneling between them is possible. By undergoing conformational changes during electron transfer, cytochrome oxidase either functions as a proton pump itself or relies on a closely adjacent protein. As can be seen from the figure, the mechanism of molecular oxygen activation by cytochrome oxidase is quite analogous to that of Dioxygenases and certain Monooxygenases—that is, it is accomplished by transferring electrons from Fe²⁺ and Cu⁺ to oxygen.

As a result, a transmembrane potential difference is created, since H⁺ ions accumulate on the outer side of the inner mitochondrial membrane (in the intermembrane space), while OH^- ions accumulate on its inner side (in the matrix) (see Fig. 131). This gives rise to an electrochemical proton gradient (denoted as ∆μ_H+). It is composed of the electrical potential gradient (∆ψ) and the hydrogen ion concentration gradient (∆pH), and serves as the driving force for ATP synthesis.

Naturally, the H⁺ ions that have accumulated in the intermembrane space of the mitochondrion—having been transported there at the expense of energy lost by electrons during their passage through the enzyme Respiratory Chain and their transition from a higher energy level in the oxidized substrate to a lower energy level in the activated oxygen molecule (see Fig. 129)—tend to return to the mitochondrial matrix. The energized, electrically charged inner mitochondrial membrane is capable of deenergizing, or discharging. This process is mediated by proton ATPase.

Proton ATPase (H⁺-ATPase) is a lipoprotein complex that hydrolyzes ATP coupled with the Transmembrane Transport of hydrogen ions against their electrochemical gradient (∆μ_H+). The energy for this uphill transport of H⁺ is derived from the Cleavage of the high-energy bond in the ATP molecule during its Hydrolysis. When the integrity of the F₀F₁ complex of mitochondria is disrupted (Fig. 132), H⁺-ATPase accelerates precisely this process, driving the reverse transport of H⁺ and the generation of ∆μ_H+. However, within an energized mitochondrial membrane, with a normal F₀ · F₁ complex, the function of proton ATPase is not to transport hydrogen ions from the matrix to the intermembrane space, but rather in the opposite direction: to transport protons into the mitochondrion, dissipate the electrochemical H⁺ gradient, and—naturally—synthesize ATP (coupled with The transfer of H⁺ from the outer to the inner side of the coupling membrane). Therefore, it is also referred to as ATP synthase, which emphasizes its true function in the mitochondrial membrane.

Fig. 133. Structure of proton ATPase (explained in the text)

ATP synthase (proton ATPase) consists of two Protein Complexes, which in turn are composed of subunits (Fig. 133, A). The first of these, completely embedded in the coupling membrane and spanning it entirely, consists of Three types of hydrophobic polypeptide chains (with molecular weights on the order of 19,000–24,000, 13,500–18,000, and 5,400–8,400, depending on the source, in ratios of 1:2:5 or close to it) and is designated as F₀. Its function is to deliver protons from the intermembrane space—where it opens—to the second protein complex, which is tightly attached to it.

The second complex includes five different proteins and protrudes as a mushroom-like projection, being partially embedded in the coupling membrane. This is the F₁ factor, or coupling factor, which is directly responsible for ATP biosynthesis. Its molecular weight, varying slightly depending on the isolation source, averages 368,000, and its subunits are represented by polypeptides with M ~ 57,000 (α), 53,000 (β), 34,000 (γ), 17,000 (δ), and 10,000 (ε). According to several authors, the subunit COMPOSITION OF THE coupling factor obeys the formula α₃β₃γε. It is believed that the catalytic center accelerating the synthesis of ATP from ADP and H₃PO₄ is located on the β-subunit, while the α-subunit shields it from the constituents of the mitochondrial matrix. There is also the view that the ε-subunit regulates proton ATPase activity by inhibiting its ability to hydrolyze ATP. Chloroplast and bacterial H⁺-ATPases are structured and function similarly.

How is ATP generated via ATP synthase? There is as yet no exhaustive answer to this question, but several noteworthy concepts have been proposed.

The first of these assumes that protons flowing through the proton-conducting channel of F₀ activate inorganic phosphate (P_i) bound to the active center of the β-subunit by abstracting an OH group from it (a Water-elimination reaction). Simultaneously, the OH group of the terminal phosphate of ADP—also attached to the active center of the β-subunit—loses a proton through interaction with an OH^- group of the matrix (where OH^- groups accumulate as a result of H⁺ transport into the intermembrane space, see Fig. 131). The activated phosphate and ADP combine to form ATP (Fig. 133, B, 1).

The second concept assumes that H⁺ ions in the active center of the coupling factor activate phosphate and a carboxyl group of one of the F₁ factor subunits, resulting in The formation of a phosphoenzyme in which the phosphate is attached by a high-energy bond. Subsequent interaction between ADP and the phosphoenzyme yields ATP (Fig. 133, B, 2). This represents a modified version of the carrier hypothesis.

The third concept stems from the hypothesis that The Role of protons translocated into the F₁ factor is to alter its conformation. Possessing at least two binding sites for ADP and inorganic phosphate, the F₁ factor is capable of synthesizing ATP from ADP and P_i when the binding center is in a closed state. In this state, ADP and P_i are surrounded by amino acid radicals that facilitate the removal of a water molecule and the synthesis of ATP (Fig. 133, B, 3, right side of the F₁ factor). Upon proton translocation, the binding site opens, and ATP is released from it into the matrix (left side of the F₁ factor in Fig. 133, B, 3), while its place is taken by ADP and P_i. A new cycle of conformational rearrangements transitions this binding site back to the closed state, simultaneously releasing the synthesized ATP from the other binding center, which transitions to the open state. It is easy to see that this explanation of the ATP biosynthesis mechanism incorporates ideas from the conformational hypothesis of oxidative phosphorylation coupling.

Recently, new Perspectives on the mechanism of the ATP synthase reaction have emerged. A. D. Vinogradov proposed a kinetic model according to which ATP Synthesis in the ATPase complex and ATP hydrolysis by F₁-ATPase follow different pathways and are catalytically accelerated by different forms of the enzyme, with the synthase centers localized on the α-subunit and the hydrolase centers on the β-subunit. L. F. Dmitriev substantiated a variant of the chemical hypothesis in which an energized lipid radical of the coupling membrane plays the role of an intermediate, taking into account the role of the Electrochemical Potential and intramembrane protons in The process of oxidative phosphorylation coupling.

Fig. 134. Giant branched mitochondria in renal tubule cells

In addition to ATP biosynthesis, the electrochemical potential ∆μ_H+ generated across the coupling membrane—which shifts it into an energetically charged, energized state—serves as an energy source for mechanical work (e.g., flagellar rotation in bacterial mutants that have lost ATP synthesis via oxidative phosphorylation coupling, chloroplast rotation in Characeae Algae, burrowing of cyanobacteria into silt, etc.), for maintaining osmotic pressure and transporting substances against concentration gradients, for heat production when mitochondria lose Respiratory Control (e.g., during animal supercooling, or when flowers evaporate Essential Oils to attract insects, etc.), for the reversal of electron transport in the enzyme respiratory chain, and for the synthesis of pyrophosphate and polyphosphates (as high-energy compounds).

The source of the membrane's energized state can also be the generation of an electrochemical potential ∆μ_Na+, which is characteristic of certain marine bacteria. In these organisms, ∆μ_Na+ is used to drive flagellar rotation, generate salt gradients, and, most importantly, synthesize ATP via a Na⁺-dependent ATPase (Na⁺-ATP synthase). In this regard, the question arises regarding the status of other metal-dependent ATPases (Na⁺,K⁺-ATPase, Ca²⁺-ATPase), and it is not excluded that this area of research will become a "hot spot" in bioenergetics in the future.

According to V. P. Skulachev, this suggests that the generation of ∆μ_H+—and in some cases ∆μ_Na+—across the coupling membrane is a universal mechanism of cellular energy storage that preceded its conservation in the high-energy bonds of ATP.

Moreover, modern observations indicate that in cells such as Muscle cells, There is a mitochondrial reticulum through which mitochondria are linked into a single network or form a single giant branched mitochondrion. Along its energized membrane, ∆μ_H+ can be transmitted over considerable distances to ensure the Synthesis of the required amount of ATP at the target site or to perform other functions inherent to the membrane electrochemical potential (Fig. 134).