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Citric Acid Cycle, Electron Transport Chain, and 7 Oxidative Phosphorylation Citrate to Succinyl–Coenzyme A CONTENTS...

Citric Acid Cycle, Electron Transport Chain, and 7 Oxidative Phosphorylation Citrate to Succinyl–Coenzyme A CONTENTS Citrate synthetase PATHWAY REACTION STEPS Acetyl-CoA condenses with OAA to form citrate and free CoA. Citric Acid Cycle—Acetyl-Coenzyme A to CO2 Electron Transport Chain and Oxidative Phosphorylation— NADH/Hþ/FADH2 and O2 to H2O Aconitase REGULATED REACTIONS Citrate is isomerized to isocitrate. Aconitase forms cis- Regulation of Citric Acid Cycle aconitate as an enzyme-bound intermediate in this reversible Regulation of Electron Transport Chain and Oxidative reaction. Phosphorylation UNIQUE CHARACTERISTICS Citric Acid Cycle Isocitrate dehydrogenase Electron Transport Chain and Oxidative Isocitrate undergoes oxidative decarboxylation, producing the Phosphorylation 5-carbon a-ketoglutarate. Oxidative decarboxylation produces INTERFACE WITH OTHER PATHWAYS free CO2 and NADH. Citric Acid Cycle Electron Transport Chain and Oxidative Phosphorylation a-Ketoglutarate dehydrogenase RELATED DISEASES The 5-carbon a-ketoglutarate undergoes oxidative decarbox- Citric Acid Cycle ylation to succinyl-CoA. This produces the second CO2 and Electron Transport Chain and Oxidative one more NADH. Phosphorylation Succinyl–Coenzyme A to Oxaloacetate Succinate thiokinase CoA is removed from succinyl-CoA, producing free succinate; lll PATHWAY REACTION STEPS this is coupled with substrate-level phosphorylation of guano- sine diphosphate (GDP) to GTP. Citric Acid Cycle—Acetyl–Coenzyme A to CO2 Succinate dehydrogenase The citric acid cycle (CAC) accepts the 2-carbon acetyl- Succinate is oxidized to fumarate, producing FADH2; this en- coenzyme A (CoA) molecule and oxidizes it completely to zyme is part of the succinate-Q reductase (complex II) in the CO2 and H2O. Energy is obtained in three forms: nicotine electron transport chain (ETC). adenine dinucleotide (NADH), flavine adenine dinucleotide (FADH2), and guanosine triphosphate (GTP). Note that in Fumarase comparison with the glycolytic pathway, none of the CAC The fumarate double bond is hydrated to form malate. intermediates are phosphorylated. The CAC is composed of two smaller energy-capturing pathways (Fig. 7-1): (1) four reactions that assimilate acetyl-CoA and then remove Malate dehydrogenase both of its carbon atoms as CO2 to produce succinate, Malate is oxidized to OAA with production of NADH; this and (2) four reactions that convert succinate back to oxalo- returns the cycle to the beginning, with OAA available to con- acetate (OAA). dense with another molecule of acetyl-CoA.  58 Citric Acid Cycle, Electron Transport Chain, and Oxidative Phosphorylation Acetyl-CoA Electron Transport Chain and Oxidative Phosphorylation—NADH/H+/FADH2 OAA Citrate and O2 to H2O The concept of a metabolic pathway for electron transport and oxidative phosphorylation is not very different from that of NADH IC other metabolic pathways except that the products and reac- NAD; tants are almost entirely electrons and protons rather than NAD; metabolites. Instead of an occasional reduction/oxidation Malate NADH CO2 step, this mechanism applies to every step in the ETC. An- other difference regarding the production of protons is that H2O protons in other metabolic pathways are simply buffered. KG However, the protons produced during electron transport Fumarate NAD; are pumped from the mitochondrial matrix to the inner mem- FADH2 NADH brane space, where they form a proton gradient across the FAD inner mitochondrial membrane. CO2 GTP GDP+Pi Succinate S-CoA Electron Transport Chain CoA All enzyme complexes involved in the ETC and oxidative phosphorylation (ATP synthesis) are embedded in the inner Figure 7-1. Steps in the citric acid cycle pathway. IC, isocitrate; mitochondrial membrane (Fig. 7-2). Therefore the ETC and KG, a-ketoglutarate; S-CoA, succinyl-coenzyme A; OAA, oxalo- acetate. See text for expansion of other abbreviations. ATP synthesis are isolated from the cytoplasm but exposed to the metabolites in the matrix, such as adenosine diphos- phate (ADP) and NADH. HISTOLOGY Mitochondria as Symbionts Exchange between the mitochondrial matrix and the IM space Inner membrane Matrix cytoplasm is highly selective and requires specific NADH transporters. This is consistent with the concept of the NADH-Q mitochondrion as a highly specialized derivative of a H+ reductase H+ symbiotic prokaryote. The DNA of mitochondria is circular, Succinate DH and its ribosomes also have prokaryotic characteristics. An increase in the number of mitochondria requires DNA Glycerol replication and fission of the original mitochondrion into two Q FADH2 phosphate DH daughter mitochondria. This process serves the purpose of allowing separate regulation for cytoplasmic and Fatty acyl– mitochondrial metabolism. The mitochondria are not true CoA DH symbionts, however, since most of the mitochondrial proteins Cytochrome are specified by the nuclear DNA. H+ H+ reductase Cytochrome C KEY POINTS ABOUT THE CITRIC ACID CYCLE Cytochrome n The CAC releases both carbons from acetyl-CoA as CO2 and pro- H+ oxidase H+ duces NADH, FADH2, and GTP. O2 n The CAC has three points of regulation—the most important of which is IDH—that are controlled by the supply of adenosine tri- H2O phosphate (ATP) and NADH. ADP+Pi n The CAC serves as a metabolic traffic circle that receives carbon ATP H+ synthase H+ skeletons from amino acids and fatty acids and donates carbon skeletons to amino acids and porphyrins. F0 ATP n An increase in flow of acetyl-CoA into the CAC is made possible F1 by pyruvate carboxylase conversion of pyruvate to OAA, thus providing substrate to combine with the increased amount of Figure 7-2. Steps in the electron transport chain. The entire acetyl-CoA. pathway is a sequence of oxidation and reduction steps. IM, intermembrane. See text for expansion of other abbreviations.  Pathway reaction steps 59 NADH-Q reductase (also known as NADH O dehydrogenase or complex I) K This multisubunit complex transfers electrons from NADH in 1 2 3 4 5 6 7 8 9 10 Q the mitochondrial matrix (and not from NADH in the cyto- Ip Ip Ip Ip Ip Ip Ip Ip Ip Ip K plasm) to coenzyme Q through its riboflavin coenzyme, flavin O mononucleotide (FMN). Figure 7-3. Structure of coenzyme Q. This quinone (Q) is made very hydrophobic by adding 10 isoprene units (Ip) as a “tail.” Succinate-Q reductase (complex II) Isoprene is formed in the pathway for cholesterol synthesis. Similar to complex I, this multisubunit complex donates elec- trons from a riboflavin coenzyme, FADH2, to coenzyme Q. Proton Pumping and Adenosine This complex contains three enzymes, all of which have Triphosphate Synthesis FAD as a prosthetic group: Complex I, complex III, and complex IV pump several protons l Succinate dehydrogenase, from the CAC into the intermembrane space for every pair of electrons that l Glycerol-3-phosphate dehydrogenase, from the glycerol they transport to O2. A sufficient number of protons are phosphate shuttle pumped to maintain a 10:1 concentration gradient (one pH l Fatty acyl–CoA dehydrogenase, from the first step in unit) between the intermembrane space and the matrix. b-oxidation of fatty acids Adenosine triphosphate synthase complex Coenzyme Q The ATP synthase complex (FoF1-ATP synthase) allows protons Coenzyme Q, a lipid-soluble quinone (Fig. 7-3), also known as to flow back into the matrix and uses the free energy change ubiquinone, accepts electrons from FMNH2 in complex I and from this process to synthesize ATP from ADP and inorganic FADH2 in complex II and carries them rapidly by diffusion phosphate Pi. It is located in knob-shaped structures embedded through the inner mitochondrial membrane to cytochrome c in the cristae (invaginations of the inner mitochondrial mem- reductase (complex III). brane) and extending into the matrix. l The Fo protein (the “o” in Fo refers to its sensitivity to Cytochrome c reductase (complex III) oligomycin, a poison that blocks the flow of protons) This multisubunit complex accepts electrons from coenzyme extends through the inner mitochondrial membrane and Q and donates them to cytochrome c. Two of the protein com- serves as the proton channel between the intermembrane ponents of complex III are cytochrome b and cytochrome c1. space and the matrix. The ATP synthase (F1-ATPase) is attached to the Fo protein on the inside of the matrix. F1-ATPase uses the protons flow- Cytochrome c ing into the matrix to bind ADP and Pi and release ATP. The This water-soluble protein diffuses along the surface of the F1-ATPase is named by the reverse reaction it catalyzes when inner membrane facing the intermembrane space (between it is isolated from mitochondria and thus uncoupled from the the outer and inner mitochondrial membranes) to transfer proton gradient. electrons from complex III to complex IV. Cytochrome oxidase (complex IV) KEY POINTS ABOUT THE ELECTRON This multisubunit protein transfers electrons from cyto- TRANSPORT CHAIN chrome c to O2. Two of the protein components of complex IV are cytochromes a and a3. This complex is unique in the n The ETC is located in the mitochondrial inner membrane and con- tains several different kinds of electron carriers: FMN, iron-sulfur ETC in having copper as a component. However, copper is proteins, coenzyme Q, heme-containing cytochromes, and cop- a common component in other oxidase enzymes that also per ions. react with O2. The product of O2 reduction by the ETC is a water molecule. One molecule of water is produced for each n Three large multiprotein complexes serve as proton pumps by harnessing the energy from electron flow through the ETC to ox- molecule of NADH or FADH2 oxidized in the ETC. ygen; in turn, the chemiosmotic energy in the proton gradient that is created by the pumps is coupled to the synthesis of ATP by the PHARMACOLOGY (F1-ATPase) complex. n ATP regulates its own synthesis and the flow of electrons through Zidovudine and Fat Metabolism respiratory control; if ATP synthesis slows down, electron trans- Mitochondrial DNA replication enzymes are sensitive to port slows down and vice versa. nucleoside analogs, such as zidovudine (AZT), that are used in n Cytosolic NADH cannot pass through the mitochondrial mem- HIV therapy. This causes a reduction in the mitochondrial brane, so it shuttles its electrons through the glycerol phosphate population and a reduced ability to metabolize fats. The CAC, shuttle and the malate-aspartate shuttle. which is contained only in the mitochondria, is an absolute necessity for fat metabolism, since the main product of fat n ATP and ADP are transported in exchange for each other by the oxidation is acetyl-CoA. ATP/ADP translocase.  60 Citric Acid Cycle, Electron Transport Chain, and Oxidative Phosphorylation lll REGULATED REACTIONS a regulatory effect on the flow of electrons. Tight coupling pre- vents unnecessary O2 consumption when the ATP supply is Regulation of Citric Acid Cycle adequate. This is termed respiratory control. Oxygen is not There are three main regulatory points for the CAC (Fig. 7-4). consumed unless energy is needed, and the rate of oxygen con- More than one site of regulation is needed to allow shunting of sumption increases with energy needs (e.g., during exercise). carbons into gluconeogenesis (OAA) during fasting or into fat l Electron transport and O2 consumption increase when (citrate) after feeding. Note that acetyl-CoA input into the cy- ADP becomes plentiful. cle is substantial in both fasting (from b-oxidation) and feeding l Electron transport and O2 consumption decrease when (from glycolysis). ADP becomes limiting. Isocitrate dehydrogenase (IDH) is the primary regulation l Likewise, any condition that slows or blocks electron trans- point, the “pacemaker,” for the CAC. It is the only allosteric port will slow the synthesis of ATP (see Related Diseases enzyme in the cycle and is stimulated by ADP; ATP and section). NADH allosterically inhibit this enzyme. Thus, when energy needs are met, isocitrate levels increase and shift the equilib- lll UNIQUE CHARACTERISTICS rium to increase citrate. Citrate can then be transported out Citric Acid Cycle of the mitochondrion as an acetyl carrier for fat synthesis, or it can inhibit citrate synthase (CS), to redirect OAA into Anaplerosis gluconeogenesis. As the concentration of acetyl-CoA entering the CAC increases, CS is inhibited by an increase in its product, citrate, or a de- a proportional increase in OAA is required for the formation of crease in the substrate, OAA. Thus an increase in citrate will citrate. To provide the additional OAA, pyruvate is converted prevent the entry of acetyl-CoA into the CAC, causing acetyl- directly to OAA by pyruvate carboxylase (Fig. 7-5). This process CoA to be shunted toward the pathway that forms ketone of replenishment is referred to as anaplerosis. Pyruvate carbox- bodies (see Chapter 10). ylase is allosterically stimulated by acetyl-CoA, ensuring that a-Ketoglutarate dehydrogenase complex (KGDC) is inhib- increased formation of acetyl-CoA stimulates increased forma- ited by its products, NADH and succinyl-CoA. tion of OAA by pyruvate carboxylase. Energy Production Regulation of Electron Transport Chain Each molecule of acetyl-CoA that enters the CAC produces and Oxidative Phosphorylation the equivalent of 12 ATP. Although substrate-level phosphor- ylation produces GTP, it is readily converted to ATP. The An isolated ETC that is uncoupled from ATP synthesis will total energy produced by oxidation of one mole of glucose transport electrons and pump protons as fast as O2 can diffuse through the CAC is 36 to 38 moles of ATP. to the cytochrome oxidase and be reduced to water. However, in the cell, the ETC is tightly coupled to ATP synthesis, exerting Pyruvate − ATP Acetyl-CoA CO2 + Acetyl-CoA ADP OAA − Citrate OAA Citrate Acetyl-CoA IC stimulates formation of extra OAA for Malate − Malate increased citrate NADH synthesis ATP + IC ADP Fumarate Fumarate KG NADH − KG Succinate Succinate S-CoA S-CoA Figure 7-4. Regulated reactions in the citric acid cycle. Each Figure 7-5. Anaplerosis: pyruvate carboxylase catalysis of regulated step is irreversible. See text for expansion of all pyruvate conversion to oxaloacetate. See text for expansion of abbreviations. all abbreviations.  Interface with other pathways 61 Multienzyme Complex free energy state is used to pump protons and create the pro- Both pyruvate and a-ketoglutarate are keto acids. Thus, the ton gradient, thus transforming electrochemical energy into KGDC is a multienzyme complex with striking similarities chemiosmotic energy. to the pyruvate dehydrogenase complex. Both complexes There are three sites where the free energy change is suffi- bind an a-keto acid to a thiamine pyrophosphate coenzyme, cient to do work in the form of proton pumping—complexes followed by decarboxylation. The shortened carbon skeleton I, III, and IV: is then transferred to lipoic acid and then to CoA, leaving l 3 ATP are generated for every electron pair donated by behind a reduced lipoic acid. Subsequent oxidation of the NADH. lipoic acid produces NADH. While the first two enzyme com- l 2 ATP are generated for every electron pair donated by ponents of these complexes are similar in their structure and FADH2. function, the third component, lipoamide dehydrogenase, is l Complete oxidation of glucose to CO2 yields between 36 and identical for both complexes. 38 ATP. The difference is determined by the shuttle mecha- nism used to transport NADH-reducing equivalents from the Electron Transport Chain and Oxidative cytoplasm (see Interface with Other Pathways section). Phosphorylation P/O Ratio Iron-Sulfur Proteins The P/O ratio is a calculation of the moles of ATP synthesized Iron-sulfur proteins, a unique form of nonheme iron (Fe-S) per mole of O2 consumed. (Fig. 7-6), are characteristic components of the ETC. Iron is l NADH produces 3 ATP for each pair of electrons and there- bound to sulfur either in the elemental form or to the thiol fore has a P/O ratio of 3. in the cysteine side chain. This iron participates in electron l FADH2 produces 2 ATP for each pair of electrons and transport by the same mechanism as heme iron through reduc- therefore has a P/O ratio of 2. tion and oxidation. l Leaky membranes (i.e., those in which electron transport and phosphorylation of ATP are uncoupled) have a low Heme Prosthetic Groups P/O ratio because many of the protons reenter the mito- The cytochrome proteins in the ETC contain heme groups that chondrial matrix by pathways independent of the ATPase. participate in electron transport. The prosthetic group of cytochromes b, c, and c1 is heme C, the same heme found in myoglobin and hemoglobin. How- lll INTERFACE WITH OTHER ever, unlike the heme groups in the oxygen-binding proteins, PATHWAYS the heme iron of cytochromes is reversibly reduced and oxi- Citric Acid Cycle dized during ETC activity. The prosthetic group for heme A in cytochrome a differs The CAC interfaces with several other pathways (Fig. 7-7). It slightly from that of heme C in having a formyl group and a serves not only as a destination for the oxidation of carbon long hydrophobic isoprene side chain. skeletons from amino acids but also as a source of precursors for biosynthesis pathways. Chemiosmotic Theory If the citrate concentration increases beyond that needed The energy needed to synthesize the high-energy bond of for energy generation by the CAC, then it is transported to ATP is not found in a chemical bond but in another form of the cytoplasm, where it is converted to acetyl-CoA and chemical energy, a proton gradient. The energy obtained OAA by citrate lyase (see Chapter 10). when electrons pass from a high free energy state to a lower Carbon skeletons from deamination of amino acids enter at acetyl-CoA, a-ketoglutarate, succinyl-CoA, fumarate, or OAA. Carbons entering the CAC at succinyl-CoA, fumarate, Protein or OAA can contribute their carbon skeletons to gluconeogen- J J J J J esis; they are termed glucogenic (see Chapter 12). Cys Cys a-Ketoglutarate and OAA can leave the cycle, also through S S transamination, to be used for the synthesis of the carbon skel- J Fe etons of the nonessential amino acids. J J Succinyl-CoA can leave the cycle to serve as a precursor in S S the synthesis of porphyrins (see Chapter 12). It can also con- J J Fe tribute to the use of ketone bodies in peripheral tissues by J J S S donating its CoA group to acetoacetate. Succinyl-CoA is J J J J formed from propionyl-CoA, a product of odd-chain fatty Cys Cys acid oxidation and the catabolism of several amino acids. Acetyl-CoA carbons are always oxidized to CO2 and en- Protein ergy and never contribute carbon skeletons to gluconeogene- Figure 7-6. Iron-sulfur bonding in iron-sulfur proteins. Both sis. Therefore, fatty acid carbons cannot be used for glucose elemental and cysteine (Cys) sulfur (S) are involved in bonding synthesis even though the fatty acids can be used to energize with iron (Fe). this pathway.  62 Citric Acid Cycle, Electron Transport Chain, and Oxidative Phosphorylation Acetyl-CoA AA FFA Asp OAA Citrate Acetyl-CoA Malate IC AA Fumarate KG Glu Succinate S-CoA Heme Propionyl-CoA Odd-chain AA FFA Figure 7-7. Intersection of the citric acid cycle with other metabolic pathways. See text for expansion of abbreviations. Electron Transport Chain and Oxidative HISTOLOGY Phosphorylation Mitochondrial Composition The ETC has three intermediates that interface with other The mitochondrial inner membrane is structurally and pathways of metabolism: NADH, FADH2, and ADP. functionally more complex than the outer membrane. NADH Interfaces It is composed of about 80% protein and is highly selective in its permeability. The ETC is located NADH input to the ETC is primarily derived in the mitochon- entirely within foldings of the inner membrane called drial matrix from the CAC, the PDC, and b-oxidation. A sec- cristae, structures that are more prominent in metabolically ond source of NADH is the cytoplasm, but it has to be supplied active cells. While NADH-Q reductase is specified by the indirectly by a shuttle mechanism because the mitochondrial mitochondrial DNA, the remainder of the enzymatic inner membrane is impermeable to NADH. The shuttle that composition of the inner membrane is specified by transports NADH into the mitochondrion is termed the the nuclear DNA. malate-aspartate shuttle (Fig. 7-8), since it relies on specific transporters for malate and aspartate in the mitochondrial inner membrane. l OAA in the cytosol is reduced to malate, regenerating Flavine Adenine Dinucleotide Interface NADþ from NADH. FADH2 input to the ETC is primarily derived from the citric l Malate is transported to the mitochondrial matrix and oxi- acid cycle and b-oxidation. However, another source of dized back to OAA, producing NADH in the mitochondrial FADH2 is from the cytoplasm, and it is supplied by a second matrix. shuttle mechanism that is designed to transport electrons from l OAA is then transaminated to aspartate, which is trans- cytoplasmically generated NADH. This is termed the glycerol ported to the cytoplasm by exchange with glutamate. phosphate shuttle (see Fig. 7-8), since it relies on both a cyto- l The shuttle cycle is completed by transamination of aspar- plasmic and a mitochondrial form of glycerol phosphate dehy- tate back to OAA, which can then be reduced again by drogenase (GPDH). cytoplasmic NADH. l NADH is used by the cytoplasmic form of GPDH to l All reactions in the malate-aspartate shuttle are reversible reduce dihydroxyacetone phosphate (DHAP) to glycerol and can reverse to increase cytoplasmic NADH under ab- 3-phosphate. normal conditions that increase the matrix concentration l Glycerol 3-phosphate then diffuses into the intermembrane of NADH (e.g., hypoxia). space.  Related diseases 63 Outer IM space Inner Matrix membrane membrane Malate Malate NAD+ NAD+ NADH NADH Tr Tr OAA Asp Asp OAA NADH NADH-Q Malate-aspartate reductase shuttle enters at NADH-Q reductase DHAP DHAP NADH FADH2 Glycerol phosphate cGl-3PD mGl-3PD Q shuttle enters at NAD+ FAD coenzyme Q Gl-3P Gl-3P Cytochrome reductase Cytochrome c O2 Cytochrome oxidase H2O Figure 7-8. Shuttle mechanisms for cytoplasmic nicotine adenine dinucleotide (NADH). The malate-aspartate shuttle (top) produces NADH in the matrix for entry into the electron transport chain at NADH-Q reductase. The glycerol phosphate shuttle (bottom) produces flavine adenine dinucleotide (FADH2) in the mitochondrial inner membrane so that it enters the electron transport chain at complex II by reducing coenzyme Q. Gl-3P, glycerol 3-phosphate; cGl-3PD, cytoplasmic glycerol-3-phosphate dehydrogenase; mGl-3PD, mitochondrial glycerol-3-phosphate dehydrogenase; IM, intermembrane; Tr, transaminase; DHAP, dihydroxyacetone phosphate. l The mitochondrial GPDH localized in the inner mitochon- It is considered to be a suicide substrate, since it is activated drial membrane oxidizes the glycerol 3-phosphate to DHAP to fluoroacetyl-CoA, which then undergoes condensation with a transfer of electrons to FADH2. This FADH2 is localized with OAA to produce fluorocitrate, a potent inhibitor of within the membrane and donates its electrons directly to aconitase. This inhibition blocks any conversion of citrate to coenzyme Q through complex II. isocitrate, thus preventing any CAC activity. Fluoroacetate is formed in some plants after uptake of fluoride from water, Adenosine Diphosphate/Adenosine air, or soil. This has resulted in the poisoning of field workers Triphosphate Translocation and livestock. Fluoroacetate also enters aquatic ecosystems ADP has access to the F1-ATPase only from the matrix side of by way of atmospheric degradation of hydrofluorocarbons the inner membrane. Therefore, the cytoplasmic ADP has to to fluoroacetate. Originally used in purified form as a roden- be transported into the matrix by ATP-ADP translocase. This ticide, this compound has been banned because of its extreme membrane transporter operates by facilitated exchange diffu- toxicity. sion (antiport). It is specific for ADP and ATP; thus the ex- change of ATP and ADP is tightly coupled. Electron Transport Chain and Oxidative Phosphorylation lll RELATED DISEASES Abnormalities associated with the ETC and oxidative phos- Citric Acid Cycle phorylation are caused by inherited enzyme deficiencies, drugs, or poisons (Table 7-1). The crucial and central role of the CAC in metabolism is underscored by the fact that there are few identified enzyme Inherited Defects deficiencies in this complex pathway. Thus a deficiency in any of the CAC enzymes will either be incompatible with life or Leber Hereditary Optic Neuropathy will produce a mitochondrial myopathy that impairs energy A mutation in mitochondrial DNA reduces the activity of metabolism. complex I (NADH-Q reductase). It is characterized by a loss The importance of the CAC in metabolism is under- of central vision and eventual blindness due to degeneration scored by a potent environmental poison, fluoroacetate. of the optic nerve.  64 Citric Acid Cycle, Electron Transport Chain, and Oxidative Phosphorylation TABLE 7-1. Action of Various Inhibitors of ATP Synthesis INHIBITOR MODE OF ACTION SITE OF INHIBITION Rotenone, amobarbital (Amytal) Blocks electron transport NADH-Q reductase (barbiturate) Antimycin A (antibiotic) Blocks electron transport Cytochrome reductase Cyanide, azide, carbon monoxide Blocks electron transport Cytochrome oxidase Oligomycin Blocks proton flow through ATP synthase ATP synthase Dinitrophenol Uncouples ATP synthesis from electron Nonspecific site transport Atractyloside Inhibits ATP-ADP exchange ATP-ADP translocase ATP, adenosine triphosphate; NADH-Q, nicotine adenine dinucleotide; ADP, adenosine diphosphate. Uncouplers Electron Transport Blockers Lipophilic organic acids, such as dinitrophenol and pentachlo- Several drugs and poisons block the ETC at various sites. rophenol, can carry protons across the mitochondrial mem- Rotenone, an insecticide, and amobarbital (Amytal), a barbi- brane effectively, short-circuiting the proton gradient across turate, inhibit complex I. The inhibition can be bypassed by the membrane at sites away from the F1-ATPase complex. the addition of succinate, since its electrons enter the ETC Since respiratory control is dependent on the integrity of at coenzyme Q after the block (Fig. 7-9). the proton flow through the F1-ATPase complex, the tight Antimycin A, an antibiotic, inhibits complex III. This inhi- coupling between ATP synthesis and electron flow is lost. bition cannot be bypassed by succinate, since it is downstream Uncouplers allow unregulated flow of electrons through the ETC to O2. Because the flow of protons through the F1-ATPase synthase complex is reduced, the P/O ratio is reduced. The en- IM space Inner membrane Matrix ergy that would have been captured in the ATP high-energy bond is lost as heat, resulting in hyperthermia. NADH NADH-Q reductase – Rotenone PHARMACOLOGY Amytal Q Cyanide Antidotes – Antimycin A The inhibitory action of cyanide on electron transport is due to Cytochrome its tight binding to the copper ions in cytochrome oxidase. reductase Since this poison blocks at the last step in the ETC, there is no effective antidote that will bypass the block. The only effective Cytochrome c CN – antidotes aim to remove the cyanide with nitrates (inducing Azide methemoglobin formation to bind the cyanide) or thiosulfate Cytochrome CO (which hastens the conversion of cyanide to the less toxic oxidase thiosulfate). In general, treatment involves the use of both O2 compounds. Atractyloside – H2O ATP ATP/ADP ATP translocase PHARMACOLOGY ADP ADP Pentachlorophenol Poisoning H+ H+ DNP DNP Pentachlorophenol is a volatile lipophilic wood preservative that is readily absorbed through the lungs. Since it ADP+Pi uncouples oxidative phosphorylation from the ETC, the ATP transfer of electrons to O2 proceeds unregulated, greatly H+ synthase H+ increasing the O2 demand of the tissues. Any of the – ATP energy from the proton gradient that would have been Oligomycin captured in ATP is released as heat, creating a potentially fatal hyperthermia. There is no specific antidote for pentachlorophenol poisoning. Figure 7-9. Inhibitors of adenosine triphosphate synthesis. IM, intermembrane. See text for expansion of other abbreviations.  Related diseases 65 from coenzyme Q, but the inhibition can be bypassed by deoxyribonucleoprotein will allow electron transport and ascorbate, which can reduce cytochrome c directly. O2 consumption to resume. All carriers upstream of the block become highly reduced, and all carriers downstream from the block become oxidized. Adenosine Triphosphate Synthase Owing to the tight coupling of respiratory control, ETC Complex Inhibition blockers reduce the synthesis of ATP. Proton flow through the F1-ATPase complex is blocked by the antibiotic oligomycin. This blocks ATP synthesis and the flow Adenosine Triphosphate/Adenosine of electrons through the ETC. As in the case of atractyloside Diphosphate Translocase Inhibition inhibition, addition of deoxyribonucleoprotein uncouples Inhibition of the ATP/ADP translocase by atractyloside, a respiratory control and allows electron transport and O2 con- plant toxin, depletes the supply of ADP in the matrix as it sumption to resume. is converted to ATP. As ATP synthesis slows owing to the lack of ADP, respiratory control also slows the flow of elec- Self-assessment questions can be accessed at www. trons through the ETC. Addition of an uncoupler such as StudentConsult.com.