CHAPTER OUTLINE
Principles of Reduction/Oxidation (Redox) Reactions
Complexes of the Electron Transport Chain
Regulation of Oxidative Phosphorylation
Inhibitors of Oxidative Phosphorylation
Generation of Reactive Oxygen Species
Mitochondrial Dysfunction in Type 2 Diabetes and Obesity
Mitochondrial Encephalomyopathies
Brown Adipose Tissue and Heat Generation
High-Yield Terms
Mitochondrial DNA (mtDNA): the circular genome found within mitochondria, encompasses genes involved in respiratory functions of this organelle
Electrochemical half-cell: consists of an electrode and an electrolyte, 2 half-cells compose an electrochemical cell that is capable of deriving energy from chemical reactions
Ubiquinone (CoQ): a mobile component of the electron transport chain that can undergo either 1- or 2-electron reactions, transfers electrons from complexes I and II to complex III
Chemiosmotic potential: more correctly referred to as proton motive force (PMF), refers to the electrochemical gradient formed across the inner mitochondrial membrane due to the transport of protons, H+, whose energy is used to drive ATP synthesis
Reactive oxygen species: any of a group of chemically reactive molecules containing oxygen, such as hydrogen peroxide and superoxide anion
Encephalomyopathy: a disorder most often resulting from a defect in mitochondrial function affecting both skeletal muscle and central nervous system function
Adaptive thermogenesis: the regulated production of heat in response to environmental changes in temperature and diet; for example, shivering in humans is adaptive thermogenesis
Mitochondria
Oxidative phosphorylation is a critical energy (ATP)–generating metabolic pathway that occurs within the mitochondria. Mitochondria evolved from a symbiotic relationship between aerobic bacteria and primordial eukaryotic cells. Mitochondria contain a 16-kb circular genome (mtDNA) that contains 37 genes critical for the processes oxidative phosphorylation. However, the mtDNA does not encode all of the proteins in the mitochondria. Over 900 mitochondrial proteins are actually encoded by the nuclear genome. Thirteen of the mtDNA genes encode protein subunits of respiratory complexes I, III, IV, and V. Only complex II is solely composed of proteins encoded by nuclear genes. The mtDNA genome also encodes 22 mitochondrial tRNAs and 2 rRNAs that are essential for translation of mtDNA transcripts.
Mammalian cells can have hundreds to thousands of mitochondria, and each mitochondrion contains several mtDNA genomes. This phenomenon is referred to as heteroplasmy. Also, any given mitochondrion is not a discrete, autonomous organelle because it has the capacity to fuse with a neighboring mitochondrion in the near future. Therefore, the entire mitochondrial population within any given cell is in constant flux. The concept of heteroplasmy is significant in the context of inherited disorders in mitochondrial biogenesis (discussed below). It is why the spectrum of symptoms with these types of diseases can be quite broad. In addition, because of the stochastic nature of mitochondrial inheritance during cell division, a daughter cell can occasionally inherit a population of mitochondria whose ratio of mutant to wild-type mtDNA differs significantly from that of the parental cell.
Normal biogenesis of mitochondria is triggered in response to changes in the ATP:ADP ratio and to activation of 5′ adenosine monophosphate–activated protein kinase (AMPK) (Chapter 34) which in turn results in increased expression of peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) and nuclear respiratory factor-1 (NRF1). PGC-1α is a master transcriptional coactivator of numerous genes involved in mitochondrial biogenesis. NRF1 is a transcription factor that regulates the expression of mitochondrial transcription factor A (TFAM, for transcription factor A, mitochondrial; also designated mtTFA), which is a nuclear transcription factor essential for replication, maintenance, and transcription of mitochondrial DNA.
Principles of Reduction/Oxidation (Redox) Reactions
Coupled electrochemical half-cells have the thermodynamic properties of other coupled chemical reactions. An example of a coupled redox reaction is the oxidation of NADH by the electron transport chain:
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, Eo. 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 Eo′. 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 Eo′ by the Nernst equation, 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) (Table 17-1):
High-Yield Concept
Mitochondria are maternally inherited due to the transmission of mitochondria from the egg to the zygote. Paternal mitochondria from the sperm are selectively marked with ubiquitin and degraded.
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.
Energy from Cytosolic NADH
The oxidation of glucose is a major pathway for the generation of cellular energy both aerobically and anaerobically. When carried out aerobically, the NADH derived from cytosolic glycolysis is transferred into the mitochondria where it can be reoxidized and coupled to ATP synthesis. The energy yield from cytosolic NADH depends upon the transport system used to get the electrons into the mitochondria. Two shuttle mechanisms exist: the malate-aspartate shuttle (see Figure 10-2) and the glycerol phosphate shuttle (see Figure 10-3). The glycerol phosphate shuttle involves 2 different glycerol-3-phosphate dehydrogenases: one is cytosolic, acting to produce glycerol 3-phosphate, and the other 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 as FADH2. 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.
Complexes of the Electron Transport Chain
The large quantity of NADH resulting from glycolysis (Chapter 10) and the NADH and FADH2 generated from fatty acid oxidation (Chapter 25) and the TCA cycle (Chapter 16) are used to supply the energy for ATP synthesis via oxidative phosphorylation (Figure 17-1).
FIGURE 17-1: Role of the respiratory chain of mitochondria in the conversion of food energy to ATP. Oxidation of the major foodstuffs leads to the generation of reducing equivalents (2H) that are collected by the respiratory chain for oxidation and coupled generation of ATP. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Oxidation of NADH and FADH2, with phosphorylation of ADP to form ATP, is a process supported by the mitochondrial electron transport assembly and ATP synthase, often referred to as complex V, 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 (Figure 17-2).
FIGURE 17-2: Overview of electron flow through the respiratory chain. (cyt, cytochrome; Q, coenzyme Q or ubiquinone.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
The reduced electron carriers, NADH and FADH2, are oxidized by a series of catalytic redox carriers that are integral proteins of the inner mitochondrial membrane. 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 (Figure 17-3). 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 (Figure 17-3).
FIGURE 17-3: Flow of electrons through the respiratory chain complexes, showing the entry points for reducing equivalents from important substrates. Q and cyt c are mobile components of the system as indicated by the dotted arrows. (ETF, electron transferring flavoprotein; Fe–S, iron–sulfur protein; cyt, cytochrome; Q, coenzyme Q or ubiquinone.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
The mitochondrial electron transport proteins are clustered into multiprotein complexes known as complexes I, II, III, and IV. Complex I, also known as NADH-CoQ oxidoreductase (also NADH-ubiquinone oxidoreductase or NADH dehydrogenase), is composed of NADH dehydrogenase with FMN as cofactor, plus non-heme iron proteins having at least 1 iron sulfur center. There are a total of 45 protein subunits in complex I. Complex I is responsible for transferring electrons from NADH to CoQ. The ΔE°′ for the latter transfer is 0.42 V, corresponding to a ΔG′ 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-CoQ oxidoreductase (also succinate-ubiquinone oxidoreductase or succinate dehydrogenase). Complex II is composed of 4 protein subunits. The ΔE°′ for electron flow through complex II is about 0.05 V, corresponding to a ΔG′ 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 2.5 (more commonly reported as 3) equivalents of ATP, while 2 electrons from succinate (as FADH2) support the production of only 1.5 (more commonly reported as 2) equivalents of 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 ATP equivalents for FADH2.
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. Complex III is known as ubiquinol-cytochrome c oxidoreductase (also CoQ-cytochrome reductase) and is composed of 11 protein subunits. 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 (Fe3+) and reduced (Fe2+) forms. The electron carrier from complex III to complex IV is cytochrome c.
Complex IV, also known as cytochrome c oxidase, contains the heme proteins 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. Complex IV is composed of a total of 13 protein subunits. Oxygen is the final electron acceptor, with water being the final product of oxygen reduction.
In addition to the core protein subunits of each of the complexes of oxidative phosphorylation, there are numerous assembly factors required to ensure correct formation of each complex. The importance of the assembly factors in functional formation of these complexes can be demonstrated by the mitochondrial encephalomyopathies that result due to mutations in several of these genes. For example, GRACILE syndrome (Growth Retardation, Amino aciduria, Cholestasis, Iron overload, Lactic acidosis, and Early death) is caused by mutations in the BCS1L gene which is required for proper assembly of complex III.
Normal oxidation of NADH or FADH2 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 (Figure 17-4). 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 FADH2 with the obligatory 1-electron reactions of the cytochromes.
FIGURE 17-4: The Q cycle. During the oxidation of QH2 to Q, one electron is donated to cyt c via a Rieske Fe–S and cyt c1 and the second to a Q to form the semiquinone via cyt bL and cyt bH, with 2H+ being released into the intermembrane space. A similar process then occurs with a second QH2, but in this case the second electron is donated to the semiquinone, reducing it to QH2, and 2H+ are taken up from the matrix. (cyt, cytochrome; Fe–S, iron-sulfur protein; Q, coenzyme Q or ubiquinone.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
The free energy available as a consequence of transferring 2 electrons from NADH or FADH2 to molecular oxygen is −57 and −36 kcal/mol, respectively. Oxidative phosphorylation traps this energy as the high-energy phosphate of ATP. All of the available energy from electron flow is not captured in the synthesis of ATP and what is not is released as heat which maintains the normal human body temperature.
In order for oxidative phosphorylation to proceed, 2 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 (Figure 17-5), or PMF. The energy of this gradient is used to drive ATP synthesis as the protons are transported back down their thermodynamic gradient into the mitochondrion.
FIGURE 17-5: The chemiosmotic theory of oxidative phosphorylation. Complexes I, III, and IV act as proton pumps creating a proton gradient across the membrane, which is negative on the matrix side. The proton motive force generated drives the synthesis of ATP as the protons flow back into the matrix through the ATP synthase enzyme. Uncouplers increase the permeability of the membrane to ions, collapsing the proton gradient by allowing the H+ to pass across without going through the ATP synthase, and thus uncouple electron flow through the respiratory complexes from ATP synthesis. (cyt, cytochrome; Q, coenzyme Q or ubiquinone.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Electrons return to the mitochondrion through the integral membrane protein known as ATP synthase or complex V (Figure 17-6). 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 (Figure 17-6).
FIGURE 17-6: Mechanism of ATP production by ATP synthase. The enzyme complex consists of an F0 subcomplex which is a disk of “C” protein subunits. Attached is a γ subunit in the form of a “bent axle.” Protons passing through the disk of “C” units cause it and the attached γ subunit to rotate. The γ subunit fits inside the F1 subcomplex of three α and three β subunits, which are fixed to the membrane and do not rotate. ADP and Pi are taken up sequentially by the β subunits to form ATP, which is expelled as the rotating γ subunit squeezes each β subunit in turn and changes its conformation. Thus, three ATP molecules are generated per revolution. For clarity, not all the subunits that have been identified are shown—eg, the “axle” also contains an ε subunit. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
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.
Inhibitors of Oxidative Phosphorylation
The processes of electron flow through the electron transport assembly have been determined through the use 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 (Figure 17-7). 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, cytochrome c remains oxidized in the presence of antimycin A, as do the downstream cytochromes a and a3.
FIGURE 17-7: Sites of inhibition (
) of the respiratory chain by specific drugs, chemicals, and antibiotics. (BAL, dimercaprol; TTFA, an Fe-chelating agent. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
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 transport of electrons generates no ATP, since the translocated protons do not return to the interior through ATP synthase (Table 17-2).
High-Yield Concept
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.
Generation of Reactive Oxygen Species
The mitochondrial electron transport chain (ETC) of oxidative phosphorylation is the major site for the cellular generation of reactive oxygen species (ROS) (Figure 17-8).
FIGURE 17-8: Reactive oxygen species (ROS) are toxic by-products of life in an aerobic environment. (A) Many types of ROS are encountered in living cells. (B) Generation of hydroxyl radical via the Fenton reaction. (C) Generation of hydroxyl radical by the Haber–Weiss reaction. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
As electrons pass through the complexes of the ETC, some of them leak out to molecular oxygen (O2) resulting in the formation of superoxide. This superoxide is rapidly acted upon by copper-zinc-superoxide dismutase (CuZn-SOD; also identified as SOD1) and mitochondrial superoxide dismutase (Mn-SOD; also identified as SOD2) to yield H2O2.
SOD1 is found in the mitochondrial intermembrane space and SOD2 is located within the mitochondrial matrix. Following the generation of H2O2 within the mitochondria, it rapidly diffuses into the cytosol where it is eliminated by several antioxidant enzymes that include the glutathione peroxidases (GPx1-GPx4), catalase, and the peroxiredoxins (PRX1 and PRX2). Within the ETC itself the major site of ROS generation is the flavin mononucleotide (FMN) of complex I. ROS generation also occurs within the plasma membrane and the endoplasmic reticulum (ER) membranes via the action of NADP(H) oxidases.
Mitochondrial and ER production of ROS contributes to the processes of aging as well as progression of numerous disorders such as Type 2 diabetes and Parkinson disease. Dietary constituents can lead to increased ROS production which is evident in obesity and plays a major contributing role in the progression to insulin resistance and diabetes. Consumption of a high-fat diet results in a surplus of NADH and FADH2 that then increases the flux through the ETC with a resultant increase in ROS generation. Indeed, a high-fat diet is known to increase the rate of H2O2 production in skeletal muscle mitochondria. Ultimately the increased rate of ROS production by the mitochondria results in mitochondrial dysfunction.
ER production of ROS is also a major contributor to disease states such as diabetes. Within the ER, proteins undergo folding into their functional conformations as they transit through to the Golgi and finally to the plasma membrane or secretory vesicles. Proper folding requires intra- and interchain disulfide bond formation that involves the oxidation of cysteine residues and the release of electrons. The electrons are passed to protein disulfide isomerase, then to ER oxidoreductin, and finally to the O2-generating superoxide anion. The nutrient excess seen in obesity and diabetes may play a role in overloading the ER protein folding capacity resulting in increased ROS production. An increase in ER ROS production results in ER stress and the induction of the ER stress response pathways (see Chapter 37) that in turn impair insulin receptor signaling and also activate pro-inflammatory pathways.
Mitochondrial Dysfunction in Type 2 Diabetes and Obesity
It is well established that mitochondrial dysfunction, particularly as it relates to the processes of oxidative phosphorylation, is contributory to the development of encephalomyopathy, mitochondrial myopathy, and several age-related disorders that include neurodegenerative diseases, the metabolic syndrome, and diabetes. Indeed, with respect to diabetes, several mitochondrial biogenesis diseases (see below) manifest with diabetic complications such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and maternally inherited diabetes and deafness (MIDD).
As pointed out earlier, normal biogenesis of mitochondria involves the transcription factors PGC-1α and NRF1. Evidence has demonstrated that both PGC-1α and NRF1 expression levels are lower in diabetic patients and in nondiabetic subjects from families with Type 2 diabetes. The expression of NRF1 is highest in skeletal muscle which is also the tissue that accounts for the largest percentage of glucose disposal in the body and, therefore, is the tissue that is most responsible for the hyperglycemia resulting from impaired insulin signaling.
Mitochondrial dysfunction results in increased production of ROS which activates stress responses leading to increased activity of MAPK and JNK. Both of these serine/threonine kinases phosphorylate IRS1 and IRS2 resulting in decreased signaling downstream of the insulin receptor. Decreased insulin receptor signaling results in decreased activation of PI3K, which is responsible for stimulating the translocation of GLUT4 to the plasma membrane allowing for increased glucose uptake. Thus, inhibition of PI3K activation results in reduced glucose uptake in skeletal muscle and adipose tissue.
