Oxidative Phosphorylation and Substrate-level phosphorylation

Oxidative phosphorylation is defined as the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) using energy or electrons derived from the electron transport chain (ETC). The electron transport chain or system is a series of membrane-associated electron carriers (including NADH, FADH2, and ubiquinone or coenzyme Q) that functions in an integrated manner to carry electrons from the primary electron donors as aforementioned to terminal electron acceptors such as oxygen (O2). In the final reaction of the ETC, O2 is reduced to water (H2O), and ATP production is driven in the process. Oxidative phosphorylation begins with the entry of electrons into the respiratory chain i.e. the electron transport chain. Oxidative phosphorylation is the process by which electrons from the ETC is used to make ATP (Figure 1). Electrons are usually transported in the respiratory chain in the form of nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD+) which are all referred to as electron acceptors.

The term “phosphorylation” simply means the formation of a phosphate derivative of a molecule (e.g. ATP). And it is usually carried out by enzymatic reactions in which a phosphate molecule or phosphate group is transferred or attached to another molecule to generate a new molecule. In oxidative phosphorylation, ATP is generated for cellular and metabolic activities of the cell in a redox reaction that is often coupled to the electron transport chain or any other oxidation-reduction reaction that is driven by energy derived from a proton motive force (as is obtainable in the ETC). Proton motive force (PMF) is the inherent energy produced within the inner mitochondrial membrane of the mitochondria during oxidative phosphorylation, and which is vital for several mechanical and chemical works in the cell (Figure 2). As aforementioned, the PMF is usually the energized state of a membrane (inclusive of the inner mitochondrial membrane) created by a proton gradient in which electrons are translocated within the cell and one which is usually formed through the activities of the ETC. Oxidative phosphorylation involves the reduction of oxygen (O2) to water (H2O) with the help of electrons donated by nicotinamide adenine dinucleotide hydrogenase (NADH) and flavin adenine dinucleotide hydrogenase (FADH2) which are both electron carriers of the ETC. NADH and FADH2 are the reduced forms of NAD+ and FAD+ respectively.

Electron acceptors are molecules that accept electrons in the course of an oxidation-reduction (redox) reaction while electron donors are those molecules that donate electrons in a redox reaction. Redox reactions which can also be called oxidation reduction reactions are chemical reactions in which electrons are transferred from one molecule to another i.e., from electron donors (i.e. the oxidized compounds or molecules) to electron acceptors (i.e., the reduced molecules). In oxidation-reduction reactions, the reduced substance (e.g., oxygen) is referred to as the electron acceptor while the oxidized substance is known as the electron donor (Figure 3). Typical example of a redox reaction is a chemical reaction in which hydrogen donates its electrons to oxygen to from water as shown in Figure 3. Generally, redox reactions are a paired-type of chemical reactions involving two molecules which are the electron acceptors (oxidizing agents) and the electron donors (reducing agents).

Figure 1: Illustration of ATP production in the cell. Pi = inorganic phosphate molecule. ADP is an important precursor for the biosynthesis of ATP in the cell.

Figure 2: Schematic illustration of the mitochondrion.

Figure 3: Formation of water from oxygen (an electron acceptor) and hydrogen (an electron donor)

Generally, the production of ATP for the cell via oxidative phosphorylation is dependent on the electron transport chain unlike the substrate level phosphorylation in which ATP is generated independent of the ETC. Since it is dependent on the ETC, oxidative phosphorylation can also be called electron transport phosphorylation. The enzyme that drives the synthesis of ATP from ADP and inorganic phosphate (Pi) during oxidative phosphorylation in the mitochondrion is ATP synthase. ATPase is the enzyme that brings about the hydrolysis of ATP to ADP and inorganic phosphate (Pi). The energy produced by the electron transport chain is mainly channeled towards the manufacture or production of ATP (the energy currency of the cell); and this biosynthesis of ATP with the cooperation of energy derived from the ETC occurs in the mitochondrion of eukaryotic cells. However, in prokaryotic cells (e.g., bacteria), this process occurs in the cell (plasma) membrane.

Since viruses are obligate intracellular parasites and rarely exist outside a living host cell; viral particles (i.e., virions) take over the cellular machinery of their host cell to generate their own energy. Electron transport within the mitochondrion of eukaryotic cells is usually driven or catalyzed by anaerobic or aerobic respiration occurring in the cell. Oxidative phosphorylation occurs within the inner membrane of the mitochondrion of eukaryotic cells. The mitochondrion (plural: mitochondria) is a membrane-bound organelle that occurs in eukaryotic cells; and this organelle is composed of two membranes which are the outer mitochondrial membrane and the inner mitochondrial membrane that both bounds the organelle (Figure 4). The mitochondrion is the powerhouse of the cell because of its main responsibility of energy generation in the cell. The energy generated within the inner membrane of the mitochondrion is channeled and used for the performance of other vital cellular and metabolic activities of the cell. While the inner mitochondrial membrane is impermeable to small molecules and ions, and also responsible for energy generation; the outer mitochondrial membrane is permeable to ions and other small molecules, and it takes no part in energy generation.

Another important part of the mitochondria is the mitochondrial matrix which is enclosed by the inner mitochondrial membrane. The mitochondrial matrix contains the nucleic acid (i.e., DNA), ribosomes, and other granules of the mitochondria. Within the mitochondrial matrix are series of coordinated redox reaction that stimulate energy generation for the cell. Aside oxidative phosphorylation, the activities of the tricarboxylic acid (TCA) cycle is another vital metabolic pathway that occurs in the mitochondrion. It is within the inner mitochondrial membrane that the electron carriers (e.g. NADH and FADH2) and enzymes that are directly involved in oxidative phosphorylation and the electron transport reaction are situated. The mitochondrion is responsible for energy generation by the TCA cycle, electron transport and oxidative phosphorylation (Figure 4). Oxidative phosphorylation and electron transport within the inner mitochondrial membrane can be inhibited by chemicals such as cyanide, carbon monoxide and antimycin (an antibiotic) which kills the cell at high concentration. Substrate-level phosphorylation is the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) independent of the electron transport chain (ETC). In substrate level phosphorylation, ATP is produced directly from an energy-rich compound such as glucose when the energy-rich compound or intermediate is being hydrolyzed or broken down in its catabolic pathway. In oxidative phosphorylation, ATP is generated in the cell using electrons derived from the ETC; and this makes ATP generation via oxidative phosphorylation to be dependent on the ETC. ATP is synthesized at the expense of the proton motive force (PMF) through the process of oxidative phosphorylation. This is however not the case in substrate level phosphorylation – in which ATP is basically generated independent of the electrons from the ETC.

Photophosphorylation is another mechanism of ATP generation that is driven by energy derived from sunlight. In photophosphorylation, ATP synthesis occurs via light energy; and it is only common in phototropic organisms (i.e. organisms that carryout photosynthesis). Thus the three mechanisms via which ATP can be generated in living systems are oxidative phosphorylation, photophosphorylation and substrate level phosphorylation. In substrate-level phosphorylation, ATP is generally produced through the partial breakdown or oxidation of a substrate. A substrate is a molecule that undergoes a specific reaction with an enzyme; and they specifically binds to the active sites of enzymes in the course of the reaction. Glucose is a typical example of a substrate molecule that is required for several metabolic activities in the cell; and this molecule is also oxidized to generate energy for the cell as well. Phosphorus, sulphur and other organic and inorganic molecules can also undergo oxidation to produce energy via substrate-level phosphorylation. Both oxidative phosphorylation and substrate-level phosphorylation generate energy (i.e., ATP) for the cell, but oxidative phosphorylation generates more energy for cellular and metabolic activities of the cell unlike substrate-level phosphorylation. And both substrate-level phosphorylation and oxidative phosphorylation occurs in anaerobic and aerobic conditions. It also occurs in fermentation reactions.

Figure 4: Schematic illustration of the flow of electron in the electron transport chain for ATP generation. Ubiquinone or coenzyme Q (coQ) is a hydrophobic benzoquinone that carries electrons and protons into the inner mitochondrial membrane. Cytochromes (cyt) are iron-containing proteins that also carry electrons; and they include cyt a, cyt b and cyt c. In addition to NAD and FAD; cytochromes and ubiquinone are electron carriers of the ETC. MicroDok.

Substrate-level phosphorylation is a typical example of what happens in the glycolytic pathway – in which glucose molecule is metabolized or broken down in the glycolytic pathway (i.e., glycolysis). In the glycolytic pathway, glucose (a high energy substrate molecule) is oxidized to form pyruvate as its final product. However, adenosine diphosphate which is produced in the process is phosphorylated to ATP; and because the phosphorylation of ADP to ATP is connected to the exergonic breakdown of glucose molecule (a high energy substrate molecule as aforementioned); it is generally known as substrate-level phosphorylation. Substrate level phosphorylation occurs at several stages of the glycolytic pathway. Exergonic reactions are reactions that occur without the input of external energy; and such reactions generally lead to the production of energy. In endergonic reactions, an input of energy is required for the chemical reaction to occur or progress. It is worthy of note that microorganisms that lack cytochromes and ubiquinones (key precursors or electron carrier molecules of the ETC) mainly generate their own energy or ATP through substrate-level phosphorylation. Though ATP may be produced at a faster or higher rate in substrate-level phosphorylation without the help of oxygen (an external electron acceptor) unlike in oxidative phosphorylation, oxidative phosphorylation as aforementioned still produces more ATP than the former. Substrate-level phosphorylation occurs outside the mitochondrion of the cell while oxidative phosphorylation is restricted to the inner membrane of the mitochondrion. It is in the cytoplasmic space or cytoplasm that substrate-level phosphorylation occurs since it occurs independent of the ETC and has no need for an external electron acceptor (e.g., oxygen).          


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