Plant Physiology - Musienko M. M. 2001

Respiration
Respiratory chain. Oxidative phosphorylation

The third and final stage of Respiration is associated with The transport of electrons to molecular oxygen. All NAD×H and FAD×H₂ molecules generated during Glycolysis and the Krebs cycle

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Fig. 93. Regulation of The Tricarboxylic Acid Cycle via Oxidative Decarboxylation of Pyruvate: 1 — pyruvate dehydrogenase, 2 — citrate synthase, 3 — isocitrate dehydrogenase, 4 — α-ketoglutarate dehydrogenase, 5 — succinate dehydrogenase, 6 — malate dehydrogenase

carry a significant energy reserve because each contains a pair of high-potential electrons. The mitochondrial system of redox reactions, in which the hydrogen removed from substrates is oxidized, is called the Respiratory Chain (Fig. 94). It should be considered as a crucial mechanism of Biological Oxidation. This multienzyme system is located on The inner mitochondrial membrane. The terminal Electron Transport Chain consists of a series of compounds that readily switch between their oxidized and reduced forms. Redox components are arranged in order of decreasing negative and increasing positive E₀

Fig. 94. The electron transport respiratory chain of the inner mitochondrial membrane

(transport occurs from NAD×H/NAD⁺ with E₀ of -0.32 V to the oxidant 1/O₂/H₂O with E_o of +0.81 V (Fig. 95).

Fig. 95. The terminal electron transport chain and Free energy conservation sites in plant Cell/35.html">Mitochondria. FP* — flavoprotein component of the pyruvate and 2-oxoglutarate dehydrogenase complexes; FP_np and FP_vp — flavoprotein dehydrogenases localized on the outer and inner surfaces of the inner mitochondrial membrane, respectively; FP_C — succinate dehydrogenase; FP_ha — flavoprotein with a high (positive) E₀ value and a large absorption change upon redox state transition; Fes — iron-sulfur protein; three-digit numbers in the indices of Cytochromes a, b, c represent the wavelength in nm of the absorption maximum in the a-region of the respective cytochrome at room Temperature; points 1, 2, 3 indicate free energy conservation sites

With the exception of ubiquinone, all Components of the ETC are Proteins with characteristic prosthetic groups. The chain includes Three types of proteins:

· Flavoproteins — with a FAD or FMN prosthetic group;

· cytochromes — with a heme prosthetic group;

· iron-sulfur proteins (Non-heme iron proteins), in which the prosthetic group consists of non-heme iron complexed with inorganic sulfur or Cysteine sulfur.

In the electron transport chain, from ubiquinone to O₂, there are 5 different classes of cytochromes whose redox potentials increase in the following sequence:

These cytochromes differ in Structure and properties. Since the purpose of the electron transport chain is to transfer electrons from a high-energy reductant to O₂, all its components must act as electron carriers. Only some of them carry protons along with electrons: flavoproteins and ubiquinone are hydrogen and electron carriers, whereas cytochromes and iron-sulfur proteins carry electrons only. Moreover, flavoproteins and ubiquinone are two-electron carriers, while cytochromes are one-electron carriers.

Thus, 2e are transferred along the ETC through a series of components with a gradual decrease in electrical potential until they reach the final component of the chain — cytochrome c oxidase. This enzyme uses these two electrons (2e^-) together with two protons (2H⁺) to reduce one oxygen molecule to H₂O. Therefore, each ETC component must interact simultaneously with two electrons. This poses no problem for two-electron carriers. However,

one-electron carriers must work in pairs; thus, to transfer 2e received from ubiquinone, the chain requires two molecules of each cytochrome type.

Most ETC components are embedded in the inner mitochondrial membrane, whereas cytochrome c is loosely bound to the outer surface of the inner membrane. Only NAD and NADP dehydrogenases, representing the first link of the ETC, are loosely associated with the mitochondrial membrane.

Plant cell mitochondria differ from animal mitochondria in possessing an NAD×H dehydrogenase located on the outer surface of the inner mitochondrial membrane. This enzyme likely catalyzes The transfer of electrons and H⁺ from an NAD×H molecule in the intermembrane space to a ubiquinone molecule. Consequently, it is responsible for The oxidation of cytosolic NAD×H. The fact is that NAD⁺ and NAD×H cannot freely penetrate intact mitochondria, though they readily cross the outer mitochondrial membrane.

Therefore, hydrogen generated in various cytoplasmic oxidation processes is bound by specific substrates and transported across the membrane into the mitochondrion. Mitochondrial dehydrogenases deliver this hydrogen (as NAD×H) directly to the site of oxidation.

It is believed that the mitochondrial respiratory chain consists of four multienzyme complexes: NAD×H — coenzyme Q oxidoreductase, succinate-ubiquinone oxidoreductase, ubiquinone, and ubiquinol-cytochrome c oxidoreductase. Each multienzyme complex can be considered a distinct structural unit integrated into the single ETC (Table 11).

It should be noted that the ETC sequence in higher plant mitochondria discussed here cannot be considered final. As our knowledge deepens, structural details of the electron transport chain are subject to revision.

Table 11. Components of the plant Mitochondrial Electron Transport chain and their standard redox potentials (E₀)

Oxidative Phosphorylation. Molecular O₂ is not involved in any of the reactions discussed above, whereas oxygen uptake is a hallmark of the respiration process. The demand for oxygen arises only at the Third Stage of respiration. As noted above, a significant portion of the energy previously stored in the hexose molecule now resides in the reduced carriers NADH and FADH₂. This energy must now be released during The final stage of respiration, when these carriers are re-oxidized by donating their electrons to free O₂. Because these carriers contain substantial energy reserves, this energy must be released gradually. Consequently, the electron transport chain (ETC) contains several loci where electron transfer releases energy that is then captured in the form of ATP (Fig. 96).

Fig. 96. Energy transfer along the electron carrier chain in the mitochondrial membrane. Electrons from NADH pass from one carrier to the next, each time dropping to a lower energy level, and ultimately reduce 1/2O₂ to H₂O. During this transfer, three molecules of ADP are phosphorylated to yield ATP, a high-energy compound utilized in other reactions. The sites where phosphorylation occurs are currently tentative; proton translocation may potentially also take place here.

Furthermore, for every molecule of NADH transferring its electrons to the ETC, 3 ATP molecules are synthesized, compared to 2 ATP molecules for each FADH₂ molecule. Because ATP is generated As a result of the oxidation of each successive carrier, with electrons ultimately transferred to O₂, this specific process is termed oxidative phosphorylation.

The driving force of Oxidative phosphorylation is the electron transfer potential characteristic of NADH and FADH₂. The redox potential change of the respiratory chain during the oxidation of NADH by O₂ can be calculated. As is well known,

The magnitude of the redox potential difference will be: (+0.82 V) - (-0.32 V) = +1.14

V, and The amount of Free energy of oxidation for this reaction is calculated using the formula:

where n is the number of electrons, equal to 2; F is the Faraday constant = 96,633.97 J; ΔE is the redox potential difference between sections of the ETC from -0.32 to +0.82 = 1.14; ΔG is the Standard Free Energy change; ΔG = 2 × 96,633.97 J × 1.14 = 220.8 kJ.

Fig. 97. Scheme of mitochondrial METABOLISM

Thus, the flow of electrons in the system from NADH to O₂ represents an exergonic process in which the free energy change is approximately 220.8 kJ (Fig. 97). The free energy of ATP Hydrolysis is 30.6 kJ. Given that the transfer of a pair of electrons from NADH to oxygen releases 220.8 kJ, one might theoretically predict the synthesis of 7 ATP molecules. However, it has been demonstrated that the passage of a pair of electrons through this system yields only 3 ATP molecules, meaning there are three phosphorylation sites in the electron transport chain.

The complexity of this process requires a highly organized mitochondrial structure. Each enzyme must be positioned to receive its specific substrate and pass the processed product directly to the next enzyme in the sequence. A bottleneck at any point in the ETC can completely halt electron transport. ATP synthesis is carried out by a specialized molecular complex located in the inner mitochondrial membrane (Fig. 98), which is structurally and functionally analogous to the one we examined in the thylakoid membrane (see Fig. 98).

Fig. 98. Schematic of the ATP synthase complex. Part of the CF₀ component is embedded in and spans the inner membrane, while the CF₁ component, consisting of nine subunits, protrudes into the mitochondrial matrix: 1 — external environment, 2 — inner mitochondrial membrane, 3 — mitochondrial matrix

According to Mitchell's chemiosmotic theory, Electron transport along the ETC drives the expulsion of protons from the matrix into the intermembrane space of the mitochondria, where the concentration of H⁺ ions consequently increases. This generates a Membrane Potential with a positive charge on the cytoplasmic side of the membrane, which in turn drives adenosine triphosphate synthesis by the ATP synthase complex. This requires that the carriers in the Respiratory Chain and the ATP synthase possess a vectorial Organization (Fig. 99).

Fig. 99. Generation of membrane potential on the outer side of the mitochondrial membrane

In addition, the inner mitochondrial membrane must be entirely impermeable to protons, as a closed compartment is essential for establishing a proton gradient. The primary energy-storing event is the translocation of protons across the inner mitochondrial membrane. In reality, electron carriers are arranged asymmetrically on the inner side of the mitochondrial membranes. Because some of these carriers transport both electrons and protons across the Two Sides of the inner membrane, a proton concentration gradient arises. Notably, the pH outside the membrane is 1.4 units lower than that inside, generating a potential of 0.14 V. Similar processes occur in the thylakoid membrane of Chloroplasts. The only difference is that protons are transported from the inside out, making the inner side of the mitochondrial membrane more alkaline and the outer side more acidic. The transfer

of each pair of electrons along the ETC from NADH to 1/2O₂ results in the translocation of 6 H⁺ ions across the mitochondrial membrane. This creates a significant pH gradient across the membrane sides, which serves as a potential energy source (Fig. 100).

The proton gradient is generated at three distinct sites along the path of electron flow through the respiratory chain from NADH to O₂, namely:

Fig. 100. Model of phosphorylation in the respiratory chain According to the chemiosmotic theory: 1 — matrix, 2 — inner membrane, 3 — outer membrane, 4 — crista

NADH–Q reductase complex, QH₂–cytochrome c reductase complex, and cytochrome c oxidase complex.

It has been proven that all three of these complex systems thoroughly span the inner mitochondrial membrane. It is likely that electron transport alternately induces conformational changes that facilitate the translocation of protons from the matrix to the cytoplasmic side of the membrane.

The energy released during the reverse transport of protons—that is, their movement back into the mitochondria via specialized channels within the stalked particles (oxysomes)—is utilized, with the participation of a specific cofactor (coupling factor), to synthesize ATP from ADP and P_i (inorganic phosphate).

Mitchell's hypothesis posits that the electrochemical H⁺ ion gradient across the inner membrane acts as the driving force for ATP synthase, which catalyzes the phosphorylation of ADP.

This force has been termed the proton-motive force. It comprises a pH gradient and an electrical potential gradient resulting from the accumulation of H⁺ ions on the outer surface of the inner membrane and anions (such as OH^-) on the inner surface. A pH gradient of 1.5 generates an electrical potential difference of 1.5 V.

How exactly does the electrochemical H⁺ ion gradient drive phosphorylation?

Essentially, the synthesis of ATP from ADP and inorganic phosphate involves the removal of water elements from these two substrates: ADP loses an H⁺, while orthophosphate loses an OH^-. This reaction is catalyzed by ATP synthase, which is embedded in the inner mitochondrial membrane. Recall that ATP synthase is a multi-protein complex consisting of a spherical HEAD (the CF₁ component), a short stalk, and a transmembrane portion (the CF₀ component). The Physiological Role of CF₁ is to catalyze ATP synthesis (Fig. 101).

According to Mitchell's theory, when ADP and P_i (originating from the matrix) bind to the Active Site of ATP synthase, H⁺ and OH^- released during ATP synthesis are transported in opposite directions driven by the electrochemical gradient. Specifically, H⁺ is translocated into the matrix, pulled by the excess of OH^- ions and the negative electrical potential of the matrix. Meanwhile, the OH^- ions

Fig. 101. ATP Synthesis in mitochondria

remain in the intermembrane space, and the newly formed ATP is released into the matrix. The majority of biochemists consider this theory to be the closest to the actual Mechanism of Oxidative phosphorylation (Fig. 102).

It should be noted that ATP and ADP cannot freely diffuse across the inner mitochondrial membrane; furthermore, ADP enters the mitochondrial matrix only in exchange for an ATP molecule leaving

Fig. 102. Mechanism of ATP generation during oxidative phosphorylation: A — proton pump; B — ATP-synthase-mediated phosphorylation: a — outer side of the membrane, b — inner side of the membrane

it, and vice versa. This exchange is mediated by ATP-ADP translocase, a dimer consisting of identical subunits with a mass of 29 kDa each. It accounts for up to 6% of the total protein in the inner mitochondrial membrane. In addition, mitochondria possess numerous other transport systems for ions and metabolites (Fig. 103). Typically, electrons are transferred along the respiratory chain only when coupled with the simultaneous phosphorylation of ADP to ATP. The Regulation of the rate of oxidative

Fig. 103. Key translocators of the inner mitochondrial membrane. Phosphate and ATP-ADP translocators are shown in Fig. 101.

phosphorylation of ADP is referred to as Respiratory Control. In the absence of demand for ATP synthesis, electron transfer to oxygen does not occur.

In some cases, the direct utilization of proton gradient energy (ΔμН⁺) is possible. Indeed, because the inner side of the membrane is electronegative, a transmembrane potential is generated. Driven by electrical attraction, cations continuously enter and accumulate within the mitochondrial interior. Evidence suggests that the proton gradient can even drive the uptake of CARBOHYDRATES, such as sucrose loading into sieve tubes. Thus, the proton gradient performs osmotic work and mediates the transport of certain substances against their concentration gradient. Importantly, (ΔμН⁺) can also serve as a transportable form of energy transmitted along the membrane.

Consequently, The Cell utilizes two forms of energy: ATP and (ΔμН⁺). ATP is a high-energy compound soluble in water, making it readily available for use in the aqueous phase. The energy of the proton gradient (ΔμН⁺) is electrochemical and inextricably linked to the membrane. Both forms of energy are interconvertible. For instance, ATP synthesis utilizes the energy of (ΔμН⁺), whereas ATP hydrolysis results in the accumulation of energy as (ΔμН⁺):