Introduction

While the large quantity of NADH resulting from TCA cycle activity can be used for reductive biosynthesis, the reducing potential of mitochondrial NADH is most often used to supply the energy for ATP synthesis via oxidative phosphorylation. Oxidation of NADH with phosphorylation of ADP to form ATP are processes supported by the mitochondrial electron transport assembly and ATP synthase, which are integral protein complexes of the inner mitochondrial membrane. The electron transport assembly is comprised of a series of protein complexes that catalyze sequential oxidation reduction reactions; some of these reactions are thermodynamically competent to support ATP production via ATP synthase provided a coupling mechanism, such as a common intermediate, is available. Proton translocation and the development of a transmembrane proton gradient provides the required coupling mechanism.

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Principals of Reduction/Oxidation (Redox) Reactions

Redox reactions involve the transfer of electrons from one chemical species to another. The oxidized plus the reduced form of each chemical species is referred to as an electrochemical half cell. Two half cells having at least one common intermediate comprise a complete, coupled, redox reaction. Coupled electrochemical half cells have the thermodynamic properties of other coupled chemical reactions. If one half cell is far from electrochemical equilibrium, its tendency to achieve equilibrium (i.e., to gain or lose electrons) can be used to alter the equilibrium position of a coupled half cell. An example of a coupled redox reaction is the oxidation of NADH by the electron transport chain:

NADH  +  (1/2)O₂  +  H⁺  ----->  NAD⁺  +  H₂O

The thermodynamic potential of a chemical reaction is calculated from equilibrium constants and concentrations of reactants and products. Because it is not practical to measure electron concentrations directly, the electron energy potential of a redox system is determined from the electrical potential or voltage of the individual half cells, relative to a standard half cell. When the reactants and products of a half cell are in their standard state and the voltage is determined relative to a standard hydrogen half cell (whose voltage, by convention, is zero), the potential observed is defined as the standard electrode potential, E₀. If the pH of a standard cell is in the biological range, pH 7, its potential is defined as the standard biological electrode potential and designated E₀^'. By convention, standard electrode potentials are written as potentials for reduction reactions of half cells. The free energy of a typical reaction is calculated directly from its E₀^' by the Nernst equation as shown below, where n is the number of electrons involved in the reaction and F is the Faraday constant (23.06 kcal/volt/mol or 94.4 kJ/volt/mol):

DG^0'  =  -nFDE₀^'

For the oxidation of NADH, the standard biological reduction potential is -52.6 kcal/mole. With a free energy change of -52.6 kcal/mole, it is clear that NADH oxidation has the potential for driving the synthesis of a number of ATPs since the standard free energy for the reaction below is +7.3kcal/mole:

ADP  +  P_i   ------>   ATP

Classically, the description of ATP synthesis through oxidation of reduced electron carriers indicated 3 moles of ATP could be generated for every mole of NADH and 2 moles for every mole of FADH₂. However, direct chemical analysis has shown that for every 2 electrons transferred from NADH to oxygen, 2.5 equivalents of ATP are synthesized and 1.5 for FADH₂.

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Complexes of the Electron Transport Chain

NADH is oxidized by a series of catalytic redox carriers that are integral proteins of the inner mitochondrial membrane. The free energy change in several of these steps is very exergonic. Coupled to these oxidation reduction steps is a transport process in which protons (H⁺) from the mitochondrial matrix are translocated to the space between the inner and outer mitochondrial membranes. The redistribution of protons leads to formation of a proton gradient across the mitochondrial membrane. The size of the gradient is proportional to the free energy change of the electron transfer reactions. The result of these reactions is that the redox energy of NADH is converted to the energy of the proton gradient. In the presence of ADP, protons flow down their thermodynamic gradient from outside the mitochondrion back into the mitochondrial matrix. This process is facilitated by a proton carrier in the inner mitochondrial membrane known as ATP synthase. As its name implies, this carrier is coupled to ATP synthesis.

Electron flow through the mitochondrial electron transport assembly is carried out through several enzyme complexes. Electrons enter the transport chain primarily from cytosolic NADH to mitochondrial NADH but can also be supplied by succinate (to mitochondrial FADH₂) or by the glycerol phosphate shuttle via mitochondrial FADH₂.

 

With the exception of NADH, succinate, and CoQ, all of the components of the pathway are integral proteins of the inner mitochondrial membrane whose cofactors undergo redox reactions. NADH and succinate are soluble in the mitochondrial matrix, while CoQ is a small, mobile carrier that transfers electrons between the primary dehydrogenases and cytochrome b. CoQ is also restricted to the membrane phase because of its hydrophobic character.

The mitochondrial electron transport proteins are clustered into complexes (as shown above) known as Complexes I, II, III, and IV. Complex I, also known as NADH:CoQ oxidoreductase, is composed of NADH dehydrogenase with FMN as cofactor, plus non-heme-iron proteins having at least 1 iron sulfur center. Complex I is responsible for transferring electrons from NADH to CoQ. The DE₀^' for the latter transfer is 0.42V, corresponding to a DG' of -19 kcal/mol of electrons transferred. With its highly exergonic free energy change, the flow of electrons through Complex I is more than adequate to drive ATP synthesis. Complex II is also known as succinate dehydrogenase or succinate:CoQ oxidoreductase. The DE₀^' for electron flow through Complex II is about 0.05 V, corresponding to a DG' of -2.3 kcal/mol of electrons transferred, which is insufficient to drive ATP synthesis. The difference in free energy, of electron flow through Complexes I and II, accounts for the fact that a pair of electrons originating from NADH and passing to oxygen supports production of 3 equivalents of ATP, while 2 electrons from succinate support the production of only 2 equivalents of ATP.

Reduced CoQ (CoQH₂) diffuses in the lipid phase of the membrane and donates its electrons to Complex III, whose principal components are the heme proteins known as cytochromes b and c1 and a non-heme-iron protein, known as the Rieske iron sulfur protein. In contrast to the heme of hemoglobin and myoglobin, the heme iron of all cytochromes participates in the cyclic redox reactions of electron transport, alternating between the oxidized (Fe⁺³) and reduced (Fe⁺²) forms. The electron carrier from Complex III to Complex IV is the smallest of the cytochromes, cytochrome c (molecular weight 12,000). Complex IV, also known as cytochrome oxidase, contains the hemeproteins known as cytochrome a and cytochrome a3, as well as copper-containing proteins in which the copper undergoes a transition from Cu⁺ to Cu²⁺ during the transfer of electrons through the complex to molecular oxygen. Oxygen is the final electron acceptor, with water being the final product of oxygen reduction.

Normal oxidation of NADH or succinate is always a 2-electron reaction, with the transfer of 2 hydride ions to a flavin. A hydride ion is composed of 1 proton and 1 electron. Unlike NADH and succinate, flavins can participate in either 1-electron or 2-electron reactions; thus, flavin that is fully reduced by the dehydrogenase reactions can subsequently be oxidized by 2 sequential 1-hydride reactions. The fully reduced form of a flavin is known as the quinol form and the fully oxidized form is known as the quinone form; the intermediate containing a single electron is known as the semiquinone or semiquinol form.

Like flavins, CoQ (also known as ubiquinone) can undergo either 1- or 2-electron reactions leading to formation of the reduced quinol, the oxidized quinone, and the semiquinone intermediate. The ability of flavins and CoQ to form semiquinone intermediates is a key feature of the mitochondrial electron transport systems, since these cofactors link the obligatory 2-electron reactions of NADH and succinate with the obligatory 1-electron reactions of the cytochromes.

The cytochromes are heme proteins. Like hemoglobin and myoglobin, the cytochromes generally contain 1 heme group per polypeptide, except for cytochrome b which has 2 heme residues in 1 polypeptide chain. There are 3 forms of heme found in heme proteins, each of which are derived from iron-protoporphyrin IX also called heme b.

Cytochromes vary in the structure of the heme and in its binding to apoprotein. Cytochromes of the c type contain a modified iron protoporphyrin IX known as heme c. In heme c the 2 vinyl (C=C) side chains are covalently bonded to cysteine sulfhydryl residues of the apoprotein. Only cytochromes of the c type contain covalently bound heme. Heme a is also a modified iron protoporphyrin IX. Heme a is found in cytochromes of the a type and in the chlorophyll of green plants.

Oxidative Phosphorylation

The free energy available as a consequence of transferring 2 electrons from NADH or succinate to molecular oxygen is -57 and -36 kcal/mol, respectively. Oxidative phosphorylation traps this energy as the high-energy phosphate of ATP. In order for oxidative phosphorylation to proceed, two principal conditions must be met. First, the inner mitochondrial membrane must be physically intact so that protons can only reenter the mitochondrion by a process coupled to ATP synthesis. Second, a high concentration of protons must be developed on the outside of the inner membrane.

The energy of the proton gradient is known as the chemiosmotic potential, or proton motive force (PMF). This potential is the sum of the concentration difference of protons across the membrane and the difference in electrical charge across the membrane. The 2 electrons from NADH generate a 6-proton gradient. Thus, oxidation of 1 mole of NADH leads to the availability of a PMF with a free energy of about -31.2 kcal (6 x -5.2 kcal). The energy of the gradient is used to drive ATP synthesis as the protons are transported back down their thermodynamic gradient into the mitochondrion.

Electrons return to the mitochondrion through the integral membrane protein known as ATP synthase (or Complex V). ATP synthase is a multiple subunit complex that binds ADP and inorganic phosphate at its catalytic site inside the mitochondrion, and requires a proton gradient for activity in the forward direction. ATP synthase is composed of 3 fragments: F₀, which is localized in the membrane; F₁, which protrudes from the inside of the inner membrane into the matrix; and oligomycin sensitivity--conferring protein (OSCP), which connects F₀ to F₁. In damaged mitochondria, permeable to protons, the ATP synthase reaction is active in the reverse direction acting as a very efficient ATP hydrolase or ATPase.

Stoichiometry of Oxidative Phosphorylation

For each pair of electrons originating from NADH, 3 equivalents of ATP are synthesized, requiring 22.4 kcal of energy. Thus, with 31.2 kcal of available energy, it is clear that the proton gradient generated by electron transport contains sufficient energy to drive normal ATP synthesis. Electrons from succinate have about 2/3 the energy of NADH electrons: they generate PMFs that are about 2/3 as great as NADH electrons and lead to the synthesis of only 2 moles of ATP per mole of succinate oxidized.

Regulation of Oxidative Phosphorylation

Since electron transport is directly coupled to proton translocation, the flow of electrons through the electron transport system is regulated by the magnitude of the PMF. The higher the PMF, the lower the rate of electron transport, and vice versa. Under resting conditions, with a high cell energy charge, the demand for new synthesis of ATP is limited and, although the PMF is high, flow of protons back into the mitochondria through ATP synthase is minimal. When energy demands are increased, such as during vigorous muscle activity, cytosolic ADP rises and is exchanged with intramitochondrial ATP via the transmembrane adenine nucleotide carrier ADP/ATP translocase. Increased intramitochondrial concentrations of ADP cause the PMF to become discharged as protons pour through ATP synthase, regenerating the ATP pool. Thus, while the rate of electron transport is dependent on the PMF, the magnitude of the PMF at any moment simply reflects the energy charge of the cell. In turn the energy charge, or more precisely ADP concentration, normally determines the rate of electron transport by mass action principles. The rate of electron transport is usually measured by assaying the rate of oxygen consumption and is referred to as the cellular respiratory rate. The respiratory rate is known as the state 4 rate when the energy charge is high, the concentration of ADP is low, and electron transport is limited by ADP. When ADP levels rise and inorganic phosphate is available, the flow of protons through ATP synthase is elevated and higher rates of electron transport are observed; the resultant respiratory rate is known as the state 3 rate. Thus, under physiological conditions mitochondrial respiratory activity cycles between state 3 and state 4 rates.

Inhibitors of Oxidative Phosphorylation

The pathway of electron flow through the electron transport assembly, and the unique properties of the PMF, have been determined through the uses of a number of important antimetabolites. Some of these agents are inhibitors of electron transport at specific sites in the electron transport assembly, while others stimulate electron transport by discharging the proton gradient. For example, antimycin A is a specific inhibitor of cytochrome b. In the presence of antimycin A, cytochrome b can be reduced but not oxidized. As expected, in the presence of cytochrome c remains oxidized in the presence of antimycin A, as do the downstream cytochromes a and a3.

An important class of antimetabolites are the uncoupling agents exemplified by 2,4-dinitrophenol (DNP). Uncoupling agents act as lipophilic weak acids, associating with protons on the exterior of mitochondria, passing through the membrane with the bound proton, and dissociating the proton on the interior of the mitochondrion. These agents cause maximum respiratory rates but the electron transport generates no ATP, since the translocated protons do not return to the interior through ATP synthase.

Inhibitors of Oxidative Phosphorylation

Energy from Cytosolic NADH

In contrast to oxidation of mitochondrial NADH, cytosolic NADH when oxidized via the electron transport system gives rise to 2 equivalents of ATP if it is oxidized by the glycerol phosphate shuttle and 3 ATPs if it proceeds via the malate aspartate shuttle. The glycerol phosphate shuttle is coupled to an inner mitochondrial membrane, FAD-linked dehydrogenase, of low energy potential like that found in Complex II. Thus, cytosolic NADH oxidized by this pathway can generate only 2 equivalents of ATP. The shuttle involves two different glycerol-3-phosphate dehydrogenases: one is cytosolic, acting to produce glycerol-3-phosphate, and one is an integral protein of the inner mitochondrial membrane that acts to oxidize the glycerol-3-phosphate produced by the cytosolic enzyme. The net result of the process is that reducing equivalents from cytosolic NADH are transferred to the mitochondrial electron transport system. The catalytic site of the mitochondrial glycerol phosphate dehydrogenase is on the outer surface of the inner membrane, allowing ready access to the product of the second, or cytosolic, glycerol-3-phosphate dehydrogenase.

In some tissues, such as that of heart and muscle, mitochondrial glycerol-3-phosphate dehydrogenase is present in very low amounts, and the malate aspartate shuttle is the dominant pathway for aerobic oxidation of cytosolic NADH. In contrast to the glycerol phosphate shuttle, the malate aspartate shuttle generates 3 equivalents of ATP for every cytosolic NADH oxidized.

In action, NADH efficiently reduces oxaloacetate (OAA) to malate via cytosolic malate dehydrogenase (MDH) . Malate is transported to the interior of the mitochondrion via the a-ketoglutarate/malate antiporter. Inside the mitochondrion, malate is oxidized by the MDH of the TCA cycle, producing OAA and NADH. In this step the cytosolic, NADH-derived reducing equivalents become available to the NADH dehydrogenase of the inner mitochondrial membrane and are oxidized, giving rise to 3 ATPs as described earlier. The mitochondrial transaminase uses glutamate to convert membrane-impermeable OAA to aspartate and a-ketoglutarate. This provides a pool of a-ketoglutarate for the aforementioned antiporter. The aspartate which is also produced is translocated out of the mitochondrion.

Other Biological Oxidations

Oxidase complexes, like cytochrome oxidase, transfer electrons directly from NADH and other substrates to oxygen, producing water. Oxygenases, widely localized in membranes of the endoplasmic reticulum, catalyze the addition of molecular oxygen to organic molecules. There are 2 kinds of oxygenase complexes, monooxygenases and dioxygenases. Dioxygenases add the 2 atoms of molecular oxygen (O₂) to carbon and nitrogen of organic compounds. Monooxygenase complexes play a key role in detoxifying drugs and other compounds (e.g., PCBs and dioxin) and in the normal metabolism of steroids, fatty acids and fat soluble vitamins. Monooxygenases act by sequentially transferring 2 electrons from NADH or NADPH to 1 of the 2 atoms of oxygen in O₂, generating H₂O from 1 oxygen atom and incorporating the other oxygen atom into an organic compound as a hydroxyl group (R-OH). The hydroxylated products are markedly more water-soluble than their precursors and are much more readily excreted from the body. Widely used synonyms for the monooxygenases are: mixed function oxidases, hydroxylases, and mixed function hydroxylases.

The chief components of monooxygenase complexes include cytochrome b5, cytochrome P450, and cytochrome P450 reductase, which contains FAD plus FMN. There are many P450 isozymes; for example, up to 50 different P450 gene products can be found in liver, where the bulk of drug metabolism occurs. Some of these same gene products are also found in other tissues, where they are responsible for tissue-specific oxygenase activities. P450 reducing equivalents arise either from NADH via cytochrome b5 or from NADPH via cytochrome P450 reductase, both of which are associated with cytochrome P450 in the membrane-localized complexes.

Enzymatic reactions involving molecular oxygen usually produce water or organic oxygen in well regulated reactions having specific products. However, under some metabolic conditions (e.g., reperfusion of anaerobic tissues) unpaired electrons gain access to molecular oxygen in unregulated, non-enzymatic reactions. The products, called free radicals, are quite toxic. These free radicals, especially hydroxy radical, randomly attack all cell components, including proteins, lipids and nucleic acids, potentially causing extensive cellular damage. Tissues are replete with enzymes to protect against the random chemical reactions that these free radicals initiate. Several free radical scavenging enzymes have been identified.

Superoxide dismutases (SODs) in animals contain either zinc (Zn²⁺) and copper (Cu²⁺), known as CuZnSOD, or manganese (Mn²⁺) as in the case of the mitochondrial form. These SODs convert superoxide to peroxide and thereby minimizes production of hydroxy radical, the most potent of the oxygen free radicals. Peroxides produced by SOD are also toxic. They are detoxified by conversion to water via the enzyme peroxidase. The best known mammalian peroxidase is glutathione peroxidase, which contains the modified amino acid selenocysteine in its reactive center.

Glutathione (see the Pentose Phosphate Page) is important in maintaining the normal reduction potential of cells and provides the reducing equivalents for glutathione peroxidase to convert hydrogen peroxide to water. In red blood cells the lack of glutathione leads to extensive peroxide attack on the plasma membrane, producing fragile red blood cells that readily undergo hemolysis.

Catalase (located in peroxisomes) provides a reductant route for the degradation of hydrogen peroxide. Mammalian catalase has the highest turnover number of any documented enzyme.