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Oxidative Phosphorylation Introduction: Oxidative phosphorylation is the culmination of a series of energy transformations that are called cellular respiration or simply respiration in their entirety. Oxidative phosphorylation is a highly efficient method of producing large amounts of ATP, the basic unit of energy for metabolic processes. During this process electrons are exchanged between molecules, which creates a chemical gradient that allows for the production of ATP. The most vital part of this process is the electron transport chain, which produces more ATP than any other part of cellular respiration. In oxidative phosphorylation the oxidation of catabolic intermediates by molecular oxygen occurs via a highly ordered series of substances that act as hydrogen and electron carriers. They constitute the electron transfer system, or respiratory chain. In most animals, plants, and fungi, the electron transfer system is fixed in the membranes of mitochondria; in bacteria (which have no mitochondria) this system is incorporated into the plasma membrane. Sufficient free energy is released to allow the synthesis of ATP by a process described below. However, it is necessary to consider the nature of the respiratory chain. First, carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer potential. Then, this electron- motive force is converted into a proton-motive force and, finally, the proton-motive force is converted into phosphoryl transfer potential. The conversion of electron-motive force into proton-motive force is carried out by three electron-driven proton pumps—NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase. These large transmembrane complexes contain multiple oxidation-reduction centers, including quinones, flavins, iron-sulfur clusters, hemes, and copper ions. The final phase of oxidative phosphorylation is carried out by ATP synthase, an ATP-synthesizing assembly that is driven by the
flow of protons back into the mitochondrial matrix. Components of this remarkable enzyme rotate as part of its catalytic mechanism. Oxidative phosphorylation vividly shows that proton gradients are an interconvertible currency of free energy in biological systems. Oxidative phosphorylation is conceptually simple and mechanistically complex. Indeed, the unraveling of the mechanism of oxidative phosphorylation has been one of the most challenging problems of biochemistry. The flow of electrons from NADH or FADH 2 to O 2 through protein complexes located in the mitochondrial inner membrane leads to the pumping of protons out of the mitochondrial matrix. The resulting uneven distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a proton- motive force. ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex. Thus, the oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane. Why do we need oxygen? You, like many other organisms, need oxygen to live. As you know if you’ve ever tried to hold your breath for too long, lack of oxygen can make you feel dizzy or even black out, and prolonged lack of oxygen can even cause death. But have you ever wondered why that’s the case, or what exactly your body does with all that oxygen? As it turns out, the reason you need oxygen is so your cells can use this molecule during oxidative phosphorylation, the final stage of cellular respiration. Oxidative phosphorylation is made up of two closely connected components: the electron transport chain and chemiosmosis. In the electron transport chain, electrons are passed from one molecule to another, and energy released in these electron transfers is used to form
avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress- associated disorders. Oxidation-reduction status (redox) is an important regulator of various metabolic functions of the cell. Perturbations in the redox status of cells by external or internal stimuli elicit distinct responses, resulting in alteration of cell function. Glutathione and thioredoxin are two major reducing systems of the eukaryotic cell that maintain redox balance, as well as interact with various transducer and effector molecules to bring about specific responses. However, these two systems differ greatly in their functions and responses to various types of stress. Oxidative stress profoundly impacts them both by direct or indirect oxidation of sulfhydryl groups. Glutathione is a small tripeptide with a single cysteine residue that undergoes oxidation-reduction. Thioredoxin (Trx) is an approximately 12-kD protein that contains five cysteine residues (two catalytic and three structural). These cysteines undergo oxidation- reduction reactions in response to oxidants or reductants in the environment. Mitochondria are the main intracellular location for fuel generation; however, they are not just power plants but involved in a range of other intracellular functions including regulation of redox homeostasis and cell fate. Dysfunction of mitochondria will result in oxidative stress which is one of the underlying causal factors for a variety of diseases including neurodegenerative diseases, diabetes, cardiovascular diseases, and cancer. They are also involved in many other cellular functions including redox homeostasis maintenance. Mitochondria are the major sites for free radical species production, including both reactive oxygen species (ROS) and reactive nitrogen species (RNS). On one hand, free radical species are indispensable for proper cell signaling; on the other hand,
excessive generation of ROS results in cell/tissue injury and death. Since mitochondria are major sources for ROS production, it is not surprising that they are well equipped with antioxidant defenses, including a large pool of glutathione, glutathione peroxidase, glutathione reductase, MnSOD, catalase, and the thioredoxin system. Although excessive levels of ROS will lead to protein oxidation and lipid peroxidation causing damage to mitochondrial membrane, proteins, and DNA, especially when the mitochondrial DNA is not protected with associated histones, lower levels of ROS have been demonstrated to be essential signaling molecules. A new concept is now emerging that mitochondrial ROS production is likely to be highly regulated as a part of physiological mitochondrial functions and the underlying molecular mechanisms are being gradually uncovered [7]. In this paper, a few mitochondrial proteins that act as redox regulators will be discussed as examples, including the antiapoptotic protein Bcl-2, cytochrome c oxidase (COX), and the small GTPase Rac1. Mitochondria : Mitochondria are membrane-bound cell organelles (mitochondrion, singular) that generate most of the chemical energy needed to power the cell's biochemical reactions. Chemical energy produced by the mitochondria is stored in a small molecule called adenosine triphosphate(ATP). Mitochondria contain their own small chromosomes. Generally, mitochondria, and therefore mitochondrial DNA, are inherited only from the mother. Mitochondria are membrane-bound organelles, but they're membrane- bound with two different membranes. And that's quite unusual for an intercellular organelle. Those membranes function in the purpose of mitochondria, which is essentially to produce energy. That energy is produced by having chemicals within the cell go through pathways, in other words, be converted. And the process of that conversion produces energy in the form of ATP, because the phosphate is a high-energy bond
Mitochondrial Respiratory Chain : The mitochondrial respiratory chain ( electron transport chain) is a series of proteins and organic molecules found in the inner membrane of the mitochondria. Electrons are passed from one member of the transport chain to another in a series of redox reactions. Energy released in these reactions is captured as a proton gradient, which is then used to make ATP in a process called chemiosmosis. Together, the electron transport chain and chemiosmosis make up oxidative phosphorylation. The key steps of this process, shown in simplified form in the diagram above, include:
- Delivery of electrons by NADH and FADH 2. Reduced electron carriers (NADH and FADH 2 ) from other steps of cellular respiration transfer their electrons to molecules near the beginning of the transport chain. In the process, they turn back into NAD+^ and FAD, which can be reused in other steps of cellular respiration.
- Electron transfer and proton pumping. As electrons are passed down the chain, they move from a higher to a lower energy level, releasing energy. Some of the energy is used to pump H+^ ions, moving them out of the matrix and into the intermembrane space. This pumping establishes an electrochemical gradient.
- Splitting of oxygen to form water. At the end of the electron transport chain, electrons are transferred to molecular oxygen, which splits in half and takes up H + to form water.
- Gradient-driven synthesis of ATP. As H+^ ions flow down their gradient and back into the matrix, they pass through an enzyme called ATP synthase, which harnesses the flow of protons to synthesize ATP.
Four types of hydrogen or electron carriers are known to participate in the respiratory chain, in which they serve to transfer two reducing equivalents (2H) from reduced substrate ( A H 2 ) to molecular oxygen; the products are the oxidized substrate ( A ) and water (H 2 O). The carriers are NAD
and the flavoproteins FAD and FMN (flavin mononucleotide); ubiquinone (or coenzyme Q); and several types of cytochromes. Each carrier has an oxidized and reduced form (e.g., FAD and FADH 2 , respectively), the two forms constituting an oxidation- reduction, or redox, couple. Within the respiratory chain, each redox couple undergoes cyclic oxidation-reduction; i.e., the oxidized component of the couple accepts reducing equivalents from either a substrate or a reduced carrier preceding it in the series and in turn donates these reducing equivalents to the next oxidized carrier in the sequence. Reducing equivalents are thus transferred from substrates to molecular oxygen by a number of sequential redox reactions. Most oxidizable catabolic intermediates initially undergo a dehydrogenation reaction, during which a dehydrogenase enzyme transfers the equivalent of a hydride ion (H+^ + 2 e − , with e − representing an electron) to its coenzyme, NAD
. The reduced NAD
thus produced (usually written as NADH + H
or NADPH + H+) diffuses to the membrane-bound respiratory chain to be oxidized by an enzyme known as NADH dehydrogenase; the enzyme has as its coenzyme FMN. A few substrates (e.g., acyl coenzyme A and succinate) bypass this reaction and instead undergo immediate dehydrogenation by specific membrane-bound dehydrogenase enzymes. During the reaction, the coenzyme FAD accepts two hydrogen atoms and two electrons (2H + 2 e − ). The reduced flavoproteins (i.e., FMNH 2 and FADH 2 ) donate their two hydrogen atoms to the lipid carrier ubiquinone, which is thus reduced.
The electron transport chain is the final component of aerobic respiration and is the only part of glucose metabolism that uses atmospheric oxygen. Electron transport is a series of redox reactions that resemble a relay race. Electrons are passed rapidly from one component to the next to the endpoint of the chain, where the electrons reduce molecular oxygen, producing water. The electron transport chain is a collection of membrane-embedded proteins and organic molecules, most of them organized into four large complexes labeled I to IV. In eukaryotes, many copies of these molecules are found in the inner mitochondrial membrane. In prokaryotes, the electron transport chain components are found in the plasma membrane. As the electrons travel through the chain, they go from a higher to a lower energy level, moving from less electron-hungry to more electron- hungry molecules. Energy is released in these “downhill” electron transfers, and several of the protein complexes use the released energy to pump protons from the mitochondrial matrix to the intermembrane space, forming a proton gradient. A complex is a structure consisting of a central atom, molecule, or protein weakly connected to surrounding atoms, molecules, or proteins. The electron transport chain is an aggregation of four of these complexes (labeled I through IV), together with associated mobile electron carriers. The electron transport chain is present in multiple copies in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. All of the electrons that enter the transport chain come from NADH and FADH 2 molecules produced during earlier stages of cellular respiration: glycolysis, pyruvate oxidation, and the citric acid cycle.
- NADH is very good at donating electrons in redox reactions (that is, its electrons are at a high energy level), so it can transfer its electrons directly to complex I, turning back into NAD^++start superscript, plus, end superscript. As electrons move through complex I in a series of redox reactions, energy is released, and the complex uses this energy to pump protons from the matrix into the intermembrane space.
- FADH 2 is not as good at donating electrons as NADH (that is, its electrons are at a lower energy level), so it cannot transfer its electrons to complex I. Instead, it feeds them into the transport chain through complex II, which does not pump protons across the membrane. Because of this "bypass," each FADH 2 molecule causes fewer protons to be pumped (and contributes less to the proton gradient) than an NADH. Complex I To start, two electrons are carried to the first complex aboard NADH. Complex I is composed of flavin mononucleotide (FMN) and an enzyme containing iron-sulfur (Fe-S). FMN, which is derived from vitamin B 2 (also called riboflavin), is one of several prosthetic groups or co- factors in the electron transport chain. A prosthetic group is a non- protein molecule required for the activity of a protein. Prosthetic groups can be organic or inorganic and are non-peptide molecules bound to a protein that facilitate its function. Prosthetic groups include co-enzymes, which are the prosthetic groups of enzymes. The enzyme in complex I is NADH dehydrogenase, a very large protein containing 45 amino acid chains. Complex I can pump four hydrogen ions across the membrane from the matrix into the intermembrane space; it is in this way that the hydrogen ion gradient is established and maintained between the two compartments separated by the inner mitochondrial membrane.
Complex IV The fourth complex is composed of cytochrome proteins c, a, and a 3. This complex contains two heme groups (one in each of the cytochromes a and a 3 ) and three copper ions (a pair of CuA and one CuB in cytochrome a 3 ). The cytochromes hold an oxygen molecule very tightly between the iron and copper ions until the oxygen is completely reduced. The reduced oxygen then picks up two hydrogen ions from the surrounding medium to produce water (H 2 O). The removal of the hydrogen ions from the system also contributes to the ion gradient used in the process of chemiosmosis. Beyond the first two complexes, electrons from NADH and FADH 2 travel exactly the same route. Both complex I and complex II pass their electrons to a small, mobile electron carrier called ubiquinone ( Q ), which is reduced to form QH 2 and travels through the membrane, delivering the electrons to complex III. As electrons move through complex III, more H
ions are pumped across the membrane, and the electrons are ultimately delivered to another mobile carrier called cytochrome C ( cyt C ). Cyt C carries the electrons to complex IV, where a final batch of H+^ ions is pumped across the membrane. Complex IV passes the electrons to O 2 , which splits into two oxygen atoms and accepts protons from the matrix to form water. Four electrons are required to reduce each molecule of O 2 , and two water molecules are formed in the process.
Overall, what does the electron transport chain do for the cell? It has two important functions:
- Regenerates electron carriers. NADH and FADH 2 pass their electrons to the electron transport chain, turning back into NAD + and FAD. This is important because the oxidized forms of these electron carriers are used in glycolysis and the citric acid cycle and must be available to keep these processes running.
- Makes a proton gradient. The transport chain builds a proton gradient across the inner mitochondrial membrane, with a higher concentration of H + in the intermembrane space and a lower concentration in the matrix. This gradient represents a stored form of energy and it can be used to make ATP.
Complexes I, III, and IV of the electron transport chain are proton pumps. As electrons move energetically downhill, the complexes capture the released energy and use it to pump H
ions from the matrix to the intermembrane space. This pumping forms an electrochemical gradient across the inner mitochondrial membrane. The gradient is sometimes called the proton-motive force , and you can think of it as a form of stored energy, kind of like a battery. Like many other ions, protons can't pass directly through the phospholipid bilayer of the membrane because its core is too hydrophobic. Instead, H
ions can move down their concentration gradient only with the help of channel proteins that form hydrophilic tunnels across the membrane. In the inner mitochondrial membrane, H+^ ions have just one channel available: a membrane-spanning protein known as ATP synthase. Conceptually, ATP synthase is a lot like a turbine in a hydroelectric power plant. Instead of being turned by water, it’s turned by the flow of H+^ ions moving down their electrochemical gradient. As ATP synthase turns, it catalyzes the addition of a phosphate to ADP, capturing energy from the proton gradient as ATP. This process, in which energy from a proton gradient is used to make ATP, is called chemiosmosis. More broadly, chemiosmosis can refer to any process in which energy stored in a proton gradient is used to do work. Although chemiosmosis accounts for over 80% of ATP made during glucose breakdown in cellular respiration, it’s not unique to cellular respiration. For instance, chemiosmosis is also involved in the light reactions of photosynthesis. What would happen to the energy stored in the proton gradient if it weren't used to synthesize ATP or do other cellular work? It would be
released as heat, and interestingly enough, some types of cells deliberately use the proton gradient for heat generation rather than ATP synthesis. This might seem wasteful, but it's an important strategy for animals that need to keep warm. For instance, hibernating mammals (such as bears) have specialized cells known as brown fat cells. In the brown fat cells, uncoupling proteins are produced and inserted into the inner mitochondrial membrane. These proteins are simply channels that allow protons to pass from the intermembrane space to the matrix without traveling through ATP synthase. By providing an alternate route for protons to flow back into the matrix, the uncoupling proteins allow the energy of the gradient to be dissipated as heat.
electrical energy. Attached to the crista is a complex enzyme (ATP synthetase) that binds ATP, ADP, and P i. It has nine polypeptide chain subunits of five different kinds in a cluster and a unit of at least three more membrane proteins composing the attachment point of ADP and P i. This complex forms a specific proton pore in the membrane. When ADP and P i are bound to ATP synthetase, the excess of protons (H
) that has formed outside of the mitochondria (an H+^ gradient) moves back into the mitochondrion through the enzyme complex. The energy released is used to convert ADP and P i to ATP. In this process, electrical energy is converted to chemical energy, and it is the supply of ADP that limits the rate of this process. The precise mechanism by which the ATP synthetase complex converts the energy stored in the electrical H
gradient to the chemical bond energy in ATP is not well understood. The H+^ gradient may power other endergonic (energy-requiring) processes besides ATP synthesis, such as the movement of bacterial cells and the transport of carbon substrates or ions. How many ATP do we get per glucose in cellular respiration? The most current sources estimate that the maximum ATP yield for a molecule of glucose is around 30 - 32 ATP. This range is lower than previous estimates because it accounts for the necessary transport of ADP into, and ATP out of, the mitochondrion. Where does the figure of 30-32 ATP come from? Two net ATP are made in glycolysis, and another two ATP (or energetically equivalent GTP) are made in the citric acid cycle. Beyond those four, the remaining ATP all come from oxidative phosphorylation. Based on a lot of experimental work, it appears that four H
ions must flow back into the matrix through ATP synthase to power the synthesis of one ATP molecule. When electrons from NADH move through the transport chain, about 10 H
ions are pumped from the matrix to the intermembrane space, so each NADH yields about 2.5 ATP. Electrons
from FADH 2 , which enter the chain at a later stage, drive pumping of only 6 H
, leading to production of about 1.5 ATP. ATP Synthase: ATP is synthesized from its precursor, ADP, by ATP synthases. These enzymes are found in the cristae and the inner membrane of mitochondria. ATP synthesis is the most widespread chemical reaction inside the biological world. ATP synthase is the very last enzyme in oxidative phosphorylation pathway that makes use of electrochemical energy to power ATP synthesis. ATP synthase is one of the most ubiquitous and plentiful protein on the earth, accountable for the reversible catalysis of ATP to ADP and Pi. The mitochondrial ATP synthase is a multi-subunit protein complex having an approximate molecular weight of 550 kDa. The human mitochondrial ATP synthase or F1/ F0 ATPase or complex V is the fifth component of oxidative phosphorylation chain. This enzyme is the smallest known biological nanomotor and plays a crucial role in ATP generation. The ATP synthase, also called Complex V, has two major subunits designated F 0 and F 1. The F 0 part, bound to inner mitochondrial membrane is involved in proton translocation, whereas the F 1 part found in the mitochondrial matrix is the water soluble catalytic domain. F 1 is the first factor recognized and isolated from bovine heart mitochondria and is involved in oxidative phosphorylation. It was named so from the term ‘Fraction 1’. F 0 was named so as it is a factor that conferred oligomycin sensitivity to soluble F 1. The structure of enzyme ATP synthase mimics an assembly of two motors with a shared common rotor shaft and stabilized by a peripheral stator stalk. The F1 part of ATP synthase is made up of 8 subunits, 3α, 3β, γ, δ and ε, where the γ, δ and ε subunits add up to the central stalk (or the rotor shaft) and an alternate arrangement of 3α and 3β form a hexameric ring with a central cavity. The γ subunit inserted in the central cavity protrudes out to meet ε which binds on its side and together they bind the F 0. Eukaryotic F 0 has
