OXIDATIVE PHOSPHORYLATION: STAGES, FUNCTIONS AND INHIBITORS - BIOLOGY - 2025

The oxidative phosphorylation is a process where molecules are synthesized ATP from ADP and P _i (inorganic phosphate). This mechanism is carried out by bacteria and eukaryotic cells. In eukaryotic cells, phosphorylation takes place in the mitochondrial matrix of non-photosynthetic cells.

ATP production is driven by the transfer of electrons from the coenzymes NADH or FADH ₂ to O ₂. This process represents the major energy production in the cell and is derived from the breakdown of carbohydrates and fats.

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The energy stored in the charge and pH gradients, also known as the proton motive force, enables this process to take place. The proton gradient that is generated causes the outer part of the membrane to have a positive charge due to the concentration of protons (H ⁺) and the mitochondrial matrix to be negative.

Where does oxidative phosphorylation occur?

The processes of electron transport and oxidative phosphorylation are associated with a membrane. In prokaryotes, these mechanisms take place through the plasma membrane. In eukaryotic cells they associate with the mitochondrial membrane.

The number of mitochondria found in cells varies depending on the type of cell. For example, in mammals, erythrocytes lack these organelles, while other cell types, such as muscle cells, can have up to millions of them.

The mitochondrial membrane consists of a simple outer membrane, a somewhat more complex inner membrane, and in between them the intermembrane space, where many ATP-dependent enzymes are located.

The outer membrane contains a protein called porin that forms the channels for the simple diffusion of small molecules. This membrane is responsible for maintaining the structure and shape of the mitochondria.

The inner membrane has a higher density and is rich in proteins. It is also impermeable for molecules and ions so, to cross it, they need intermembrane proteins to transport them.

Inside the matrix, folds of the inner membrane extend, forming ridges that allow it to have a large area in a small volume.

Cell power plant

The mitochondria is considered as the cellular energy producer. It contains the enzymes involved in the processes of the citric acid cycle, fatty acid oxidation, and the redox enzymes and proteins involved in electron transport and the phosphorylation of ADP.

The proton concentration gradient (pH gradient) and the charge gradient or electric potential in the inner membrane of the mitochondria are responsible for the proton motive force. The low permeability of the inner membrane for ions (other than H ⁺) allows the mitochondria to have a stable voltage gradient.

Electronic transport, proton pumping, and ATP production occur simultaneously in the mitochondria, thanks to the proton motive force. The pH gradient maintains acidic conditions in the intermembrane and in the mitochondrial matrix with alkaline conditions.

For every two electrons transferred to O ₂ about 10 protons are pumped through the membrane, creating an electrochemical gradient. The energy released in this process is produced gradually by the passage of electrons through the transport chain.

Stages

The energy released during the oxidation-reduction reactions of NADH and FADH ₂ is considerably high (around 53 kcal / mol for each pair of electrons), so to be used in the manufacture of ATP molecules, it must be produced gradually with the passage of electrons through transporters.

These are organized into four complexes located on the inner mitochondrial membrane. The coupling of these reactions to the synthesis of ATP is carried out in a fifth complex.

Electron transport chain

NADH transfers a pair of electrons that enter complex I of the electron transport chain. The electrons are transferred to the flavin mononucleotide, and then to ubiquinone (coenzyme Q) through an iron-sulfur transporter. This process releases a large amount of energy (16.6 kcal / mol).

Ubiquinone transports electrons across the membrane to complex III. In this complex the electrons pass through cytochromes b and c ₁ thanks to an iron-sulfur transporter.

Electrons pass from complex III to complex IV (cytochrome c oxidase), transferred one by one in cytochrome c (peripheral membrane protein). In complex IV the electrons pass through a pair of copper ions (Cu _a ²⁺), then to cytochrome c _a, then to another pair of copper ions (Cu _b ²⁺) and from this to cytochrome a ₃.

Finally, the electrons are transferred to O ₂ which is the last acceptor and forms a water molecule (H ₂ O) for each pair of electrons received. The passage of electrons from complex IV to O ₂ also generates a large amount of free energy (25.8 kcal / mol).

Succinate CoQ reductase

Complex II (succinate CoQ reductase) receives a pair of electrons from the citric acid cycle, through the oxidation of a succinate molecule to fumarate. These electrons are transferred to the FAD, passing through an iron-sulfur group, to ubiquinone. From this coenzyme they go to complex III and follow the route previously described.

The energy released in the electron transfer reaction to the FAD is not enough to drive the protons through the membrane, so no proton motive force is generated in this step of the chain, and consequently the FADH yields less H ⁺ than NADH.

Coupling or transduction of energy

The energy generated in the previously described electron transport process must be able to be used for the production of ATP, a reaction catalyzed by the enzyme ATP synthase or complex V. The conservation of this energy is known as energy coupling, and the mechanism has been difficult to characterize.

Several hypotheses have been described to describe this energy transduction. The best accepted is the chemosmotic coupling hypothesis, described below.

Chemosmotic coupling

This mechanism proposes that the energy used for ATP synthesis comes from a proton gradient in cell membranes. This process intervenes in the mitochondria, chloroplasts and bacteria and is linked to the transport of electrons.

Complexes I and IV of electron transport act as proton pumps. These undergo conformational changes that allow them to pump protons into the intermembrane space. In complex IV, for each pair of electrons, two protons are pumped out of the membrane and two more remain in the matrix, forming H ₂ O.

Ubiquinone in complex III accepts protons from complexes I and II and releases them to the outside of the membrane. Complexes I and III each allow the passage of four protons for each pair of transported electrons.

The mitochondrial matrix has a low concentration of protons and a negative electric potential, while the intermembrane space presents the inverse conditions. The flow of protons through this membrane represents the electrochemical gradient that stores the necessary energy (± 5 kcal / mol per proton) for the synthesis of ATP.

ATP synthesis

The enzyme ATP synthetase is the fifth complex involved in oxidative phosphorylation. It is responsible for harnessing the energy of the electrochemical gradient to form ATP.

This transmembrane protein consists of two components: F ₀ and F ₁. The F ₀ component allows the return of protons to the mitochondrial matrix, functioning as a channel and F ₁ catalyzes the synthesis of ATP through ADP and P _i, using the energy of said return.

The ATP synthesis process requires a structural change in F ₁ and the assembly of components F ₀ and F ₁. Proton translocation through F ₀ causes conformational changes in three subunits of F ₁, allowing it to act as a motor of rotation, directing the formation of ATP.

The subunit responsible for the binding of ADP with P _i changes from a weak state (L) to an active one (T). When ATP is formed, a second subunit goes into an open state (O) that allows the release of this molecule. After ATP is released, this subunit goes from the open state to an inactive state (L).

The ADP and P _i molecules bind to a subunit that has passed from an O state to a L state.

products

The electron transport chain and phosphorylation produce ATP molecules. The oxidation of NADH produces about 52.12 kcal / mol (218 kJ / mol) of free energy.

The overall reaction for the oxidation of NADH is:

NADH + 1⁄2 O ₂ + H ⁺ ↔ H ₂ O + NAD ⁺

The transfer of electrons from NADH and FADH ₂ occurs through various complexes, allowing the free energy change ΔG ° to break down into smaller energy "packets", which are coupled to ATP synthesis.

The oxidation of one molecule of NADH generates the synthesis of three molecules of ATP. While the oxidation of a molecule of FADH ₂ is coupled to the synthesis of two ATP.

These coenzymes come from the glycolysis and citric acid cycle processes. For each molecule of glucose degraded they end up producing 36 or 38 molecules of ATP, depending on the location of the cells. In the brain and skeletal muscle 36 ATP are produced while in muscle tissue 38 ATP are produced.

Features

All organisms, unicellular and multicellular, need minimal energy in their cells to carry out the processes within them, and in turn maintain vital functions in the entire organism.

Metabolic processes require energy to take place. Most of the usable energy is obtained from the breakdown of carbohydrates and fats. This energy is derived from the oxidative phosphorylation process.

Control of oxidative phosphorylation

The ATP utilization rate in cells controls its synthesis, and in turn, due to the coupling of oxidative phosphorylation with the electron transport chain, it also generally regulates the rate of electron transport.

Oxidative phosphorylation has a strict control that ensures that ATP is not generated faster than it is consumed. There are certain steps in the process of electron transport and coupled phosphorylation that regulate the rate of energy production.

Coordinated control of ATP production

The main pathways of energy production (cellular ATP) are glycolysis, the citric acid cycle, and oxidative phosphorylation. The coordinated control of these three processes regulates the synthesis of ATP.

The control of phosphorylation by the mass action ratio of ATP depends on the precise supply of electrons in the transport chain. This in turn depends on the / ratio, which is kept high by the action of glycolysis and the citric acid cycle.

This coordinated control is carried out by regulating the glycolysis control points (citrate inhibited PFK) and the citric acid cycle (pyruvate dehydrogenase, citrate tapease, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase).

Control by acceptor

Complex IV (cytochrome c oxidase) is an enzyme regulated by one of its substrates, that is, its activity is controlled by reduced cytochrome c (c ²⁺), which in turn is in equilibrium with the concentration ratio between / and the mass action ratio of / +.

The higher the / ratio and the lower the / +, the higher the concentration of cytochrome and the higher the complex IV activity. This is interpreted, for example, if we compare organisms with different resting and high activity activities.

In an individual with high physical activity, the consumption of ATP and therefore its hydrolysis to ADP + P _i will be very high, generating a difference in the mass action ratio that causes an increase in and therefore an increase in the synthesis of ATP. In an individual at rest, the reverse situation occurs.

Ultimately, the rate of oxidative phosphorylation increases with the concentration of ADP within the mitochondria. Said concentration depends on the ADP-ATP translocators responsible for the transport of adenine nucleotides and P _i from the cytosol to the mitochondrial matrix.

Uncoupling agents

Oxidative phosphorylation is influenced by certain chemical agents, which allow electron transport to continue without the phosphorylation of ADP, uncoupling energy production and conservation.

These agents stimulate the oxygen consumption rate of the mitochondria in the absence of ADP, also causing an increase in ATP hydrolysis. They work by removing an intermediate or breaking an energy state in the electron transport chain.

2,4-dinitrophenol, a weak acid that passes through the mitochondrial membranes, is responsible for dissipating the proton gradient, as they bind to them on the acidic side and release them on the basic side.

This compound was used as a "diet pill" as it was found to produce an increase in respiration, therefore an increase in metabolic rate and associated weight loss. However, it was shown that its negative effect could even cause death.

The dissipation of the proton gradient produces heat. Cells in brown adipose tissue use hormonally controlled uncoupling to produce heat. Hibernating mammals and newborns that lack hair consist of this tissue that serves as a kind of thermal blanket.

Inhibitors

The inhibitory compounds or agents prevent both O ₂ consumption (electron transport) and associated oxidative phosphorylation. These agents prevent the formation of ATP through the use of energy produced in electronic transport. Therefore, the transport chain stops when said energy consumption is not available.

The antibiotic oligomycin functions as a phosphorylation inhibitor in many bacteria, preventing the stimulation of ADP to ATP synthesis.

There are also ionophore agents, which form fat-soluble complexes with cations such as K ⁺ and Na ⁺, and pass through the mitochondrial membrane with these cations. The mitochondria then use the energy produced in electronic transport to pump cations instead of synthesizing ATP.

References

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