ll = CHAPTER 20 Biological Oxidation and Electron Transport Chain Chapter at a Glance ‘The reader willbe able to answer questions on the following topics: Redox potentials Biological oxidation Enzymes and co-enzymes High energy compounds (Organization of electron transport chain vvvvy > Chemiosmotic theory > Proton pump > ATP synthase. Inhibitors of ATP synthesis Stages of Oxidation of Foodstuffs First Stage Digestion in the gastrointestinal tract converts the macromolecules into small units. For example, proteins are digested to amino acids. This is called primary ‘metabolism (Fig, 20.1), Second Stage ‘The products of digestion are absorbed, catabolizedto smaller components, and ultimately oxidized to CO, The reducing ‘equivalents are mainly generated in the mitochondria by the final common oxidative pathway, citric acid cycle. In this process, NADH and FADH, are generated. This is called secondary or intermediary metabolism, Third Stage ‘These reduced equi the electron transport chai ents (NADH and FADH,) enter into (ETC), of Respiratory chai where energy is released. This is the tertiary metabolism or Internal respiration or cellular respiration (Fig. 20.1). ‘The energy production by complete oxidation of one molecule of glucose is 2850 k3/mol and that of palmitate is ‘9781 kJ/mol. This energy is then used for synthetic purpose in the body (Fig. 20.2) Fig,20.1: Oxidation of foodstuffs in three stagesTextbook ofBlochemistry Phototrophs harvest the eneray of light (plants). Chemotrophs harvest energy from oxidation of fel molecules. Principles of bio- ‘energetics and thermodynamics are described ia Chapter 1 | REDOX POTENTIALS Redox potential of a system is the electron transfer potential E,. Oxidation is defined as the loss of electrons, and reduction as the gain in electrons. When a substance ‘exists both in the reduced state and in the oxidized state, the pair is called a redox couple. The redox potential of this couple is estimated by measuring the electromotive force (EMF) of a sample half cell connected to a standard half cell. The sample half cell contains one molar solution each of the reductant and oxidant, The reference standard half cell has 1 M Hl solution in equilibrium with hy drogen gasat one atmosphere pressure. The reference half cell has a reduction potential of zero mV, Negative and Positive Redox Potential When a substance has lower affinity for electrons than hydrogen, it has a negative redox potential, Ifthe substance has a positive redox potential, it has a higher affinity for clectrons than hydrogen. Thus NADH, a strong reducing agent, has a negative redox potential (~0.32 V ), whereas a strong oxidant like oxy gen has a positive redox potential (+082 V). Table 20.1 gives the redox potentials of some of the important redox couples of the biological system. A summary is shown in Box 20.1 Meabotam of C0,7H,0 carbohydrates, bee af fat and amino oats NAD NADHH™ 0 0, ae Apr +P Energy is used for: Muscle contraction Active vaneport Biosynthesis Fig. 202: ATP generation, Food is catabollzed: energy from food Is trapped as ATP; ts then used for anabolic reactions Substrate Level Phosphorylation Here energy from a high energy compound is directly transferred to nucleoside diphosphate to form a triphosphate ‘without the help of electron transport chain, e.g a. Bisphosphoglycerate kinase (see Fig. 9.11); '. Pyruvate kinase (see Fig, 9.13) ©. Succinate thiokinase (see Fig, 19.2) ATP generation is coupled with a more exergonic metabolic reaction, BIOLOGICAL OXIDATION The transfer of electrons from the reduced co-enzymes through the respiratory chain to oxygen is known as biological oxidation, Energy released during this process is trapped as ATP. This coupling of oxidation with phosphorylation is called oxidative phosph the body, this oxidation is carried out by successive steps of dehydrogenations Electron Transport Chain ‘The electron flow occurs through successive dehydrogenase enzymes, together known as electron transport chain (ETC), The electrons flow from electronegative potential (-0.32) to electropositive potential (+ 0.82). A summary is shown in Figure 20.13. Oxidant NADY ‘Cytochrome br Reductant NADH+ ‘Cytochrome bY Coenzyme OH, Cytochrome Cytochrom Ho Co-enzymeQ ‘Cytochrome c* ‘Cytochrome ar 0,+2H 1. Free energy is 2 measure ofthe energy available to perform wseful work 2. AG can predict the direction ofa chemical reaction 3. Chemical reactions can be coupled, which allows an ‘energetically unfavorable reaction to conclusion, 4G measured under physiological conditions may be different from thata a standard state.Energetics of Oxidative Phosphorylation ‘The E, and G® of biological oxidation may be calculated as follows: %40,+2H' — + H,O(E, = +082) NAD’ +H’ + 2e - NADH (E, =~ 0.32) ‘When these two equations are computed, % 0, + NADH +H’ H,0 +NAD*(E, = 1.14V) AG” = -nF E, = -2 * 23.06 « 1.14 = -52.6 kcal/mol ‘The free energy change between NAD and water is equal to $3 kcal/mol. This is so great that, if this much energy is released at one stretch, body cannot utilize it Hence, with the help of ETC assembly, the total energy change is released in small increments so that energy can be trapped as chemical bond energy, ATP (Fig. 20.2). ENZYMES AND CO-ENZYMES Al the enzymes involved in this process of biological oxidation belong to the major class of oxidoreductases. ‘They can be classified into the following 5 headings: Oxidases ‘These enzymes catalyze the removal of hydrogen from substrates, but only oxygen can act as acceptor of hydrogen, so that water is formed, AH, + 40, > A+H,0 This group includes Cytochrome oxidase (terminal component of ETC), tyrosinase, polyphenol oxidase, catechol oxidase and monoamine oxidase Aerobic Dehydrogenases ‘These enzymes catalyze the removal of hydrogen from a substrate, but oxygen can act as the acceptor. These enzymes are flavoproteins and the product is usually hydrogen peroxide. AH, +0, A+HO, These flayoproteins contain cither FMN or FAD as prosthetic group. Examples are L-amino acid oxidase which catalyzes the oxidative deamination of L-amino acids (see Chapter 15) and Xanthine oxidase (see Chapter 43) Anaerobic Dehydrogenases ‘These enzymes catalyze the removal of hydrogen from a substrate but oxygen cannot act as the hy drogen acceptor. ‘They therefore require co-enzymes as acceptors of the Chapter 20: Biological Oxidation and Electron Transport Chain hhydrogen atoms. When the substrate is oxidized, the co- enzyme is reduced a. NAD* linked dehydrogenases: NAD" is derived from nicotinic acid, a member of the vitamin B ‘complex (see Chapter 37). When the NAD” accepts the two hydrogen atoms, one of the hydrogen atoms is removed from the substrate as such. The other Ihydrogen atom is split into one hydrogen ion and one electron. The electron is also accepted by the NAD’ so as to neutralize the positive charge on the co-enzyme molecule, The remaining hydrogen ion is released into the surrounding medium (Fig. 20.3). H, + H+W'te ‘AH,+NAD* A+ NADH +H" ‘The NAD* linked dehydrogenases are (Fig. 20.13) i. Glyceraldchyde-3-phosphate dehydrogenase ii, Isocitrate dehydrogenase iii, Malate dehydrogenase iv. Glutamate dehydrogenase v. Beta hydroxyacy! CoA dehydrogenase vi. Pyruvate dehydrogenase Vii. Alpha ketoglutarate dehydrogenase. b. NADP* linked dehydrogenases: NADPH cannot be oxidized with concomitant production of energy NADPH is used in reductive biosynthetic reactions like fatty acid synthesis and cholesterol synthesis. ‘An example of NADPH linked dehydrogenase is the slucose-6-phosphate dehydrogenase (see Fig. 10.11) ©. FAD-linked dehydrogenases: When FAD is the co- ‘enzyme, (unlike NAD"), both the hydrogen atoms are attached to the flavin ring. Examples: i. Succinate dehydrogenase (see step 6, Fig. 19.2) ii, Fatty acyl CoA dehydrogenase (sce Fig. 12.9) iii, Glycerolphosphate dehydrogenase (Fig. 20.6). 4. Cytochromes: All the cytochromes, except eytochrome oxidase, are anaerobic dehydrogenases. (Cytochrome oxidase is an oxidase, see above). rar R Fig.20.3: NAD" accepts H NADH-_ ce ee All cytochromes are hemoproteins having iron atom Cytochrome b, cytochrome cl, and cytochrome ¢ are in mitochondria while cytochrome P-450 and cytochrome bS are in endoplasmic reticulum. Hydroperoxidases 44 Peroxidase: Examples of peroxidases ate glutathione peroxidase in RBCs (a selenium containing enzyme), leukoeyte peroxidase and horse radish peroxidase, Peroxidases remove free radicals like hydrogen peroxide. (see Chapter 33) HO, + AH, (peroxidase) —m 21,0 + A Catalase: Catalases are hemoproteins Peroxisome are subcellular ‘organelles having both aerobic dehydrogenases and catalase 2H.0, —{eatalase) > 2 H,0~0, Oxygenases 4 Mono-onygenases: ‘These are otherwise called mixed function ‘oxidase Here, one oxygen atom incorporated int the substrate ‘and te other oxygen atom i reduced to water These enzymes are also called hydroxylases because OH group ts ineorporated into the subst Ad+ 0, ydoxylase)-+ A-OH 10% B. 1. Phenvlalanne hydroxylase fi, Tyrosine hydroxylase Tryptophan hydroxylase (see Chapter 18) i: Microsemal cytochrome P-450 mono-oxygenise is concerned ‘with drug metabotism Mitochondrial cytochrome P-180 mono-oxygenase. ‘The figure “450” denotes that it absorbs light at 450 nm, when the heme combines with carbon monoxide Its equied for steroid bydroxylation in adrenal cortex, testis and ovary ¥. Nitric oxide synthase (see Chapter 17) b. Dioxygenases: They are enzymes which incorporate both atoms of « molecule of oxygen imo the substrate, eg tryptophan pyttlase and homogeatisic acd oxidase (see Chapter 18) A+0,——> 00, HIGH ENERGY COMPOUNDS ‘These compounds when hydrolyzed will release a large quantity of energy, that is, they have a large AG*". The = ies a i\ ‘| Kart Fitz Lipmann ‘Alexander Todd Lohmann, NP 1953 NP 1957 1896-1978 1890-1986 1907-1997 high energy bond in compounds is usually indicated by a squiggle bond (~). The free energy of hydrolysis AG® of an ‘ordinary bond varies from —1 10-6 kcal/mol. For example. slucose-6-phosphate has a free energy of only 13.8 kJ/mol (33 kcal/mol), On the other hand, the free energy of high ‘energy bonds varies from >25 kJ/mol (~7 to-15 kcal/mol), High energy compounds are listed in Table 20.2, Adenosine Triphosphate (ATP) i. ATP is the universal currency of energy within the living cells, Structure of ATP is shown in Fig. 53. fi, The hydrolysis of ATP to ADP (under standard conditions) releases ~30.5 kJ/mol or —7.3 kcal/mol (AG =~7.3), The energy in the ATP is used to drive all endergonic (biosynthetic) reactions. The energy efficiency of the cell is comparable to any machine so far invented. ATP captures the chemical energy Energy ch compound Phosphate Compounds 1. Nucleotides: (ATR. GT, UTR, UDP-glucose) [ATP to AMP + PPA [ATP ADP +P Creatine phosphate Arginine phosphate | Larbisphosphoghycerte | - 494 Phosphocnolpyrwvate 619 Inorganic pyrophosphate “107 keal 73. ~105 4566) 305 4 =101 48 ES -514 “123 Cerbemoyl phosphate Aminoacy| adenylate (aminoacyl AMP) Sulfur Compounds 9. CoA derivatives: ‘cetyl CoA Succinyl COA Fatty acyl CoA HMG.CoA S-adenosy! methionine aM) Adenosine phosphosulfate (active sulfate)released by the combustion of nutrients and transfers it to synthetic reactions that require energy iii, At rest, Na’-K" -ATPase (see Chapter 2) uses up one-third of all ATP formed. Other energy requiring processes are, biosynthesis of macromolecules, muscle contractions, cellular motion using kinesin, dyenin etc iv, ATP is continually being hydrolyzed and regenerated ‘An average person at rest consumes and regenerates, ATP at a rate of approximately 3 molecules per second, i.c. about 1.5 kg/day! AL this juncture, itis interesting to review different types reactions wndergone by ATP ose ~ ATP ~ Glucose-G-phosphate + ADP ere ATPistiydolyzed to ADPlevel and phosphate isincorporated in the product. 2. Pyruvate + CO, “ATP —+ Oxaloacetate + ADP + Pi ere ATP is hydrolyzed to ADP level, but phosphate is released 3. Fatty acid + CoA + ATP Fatty seyl Co ~ AMP ~ PP: The ATP is hydrolyzed to AMP level, but pyrophosphate is eased 4. Ribose--P + ATP-— Phosphoribosy! pyrophosphate + AMP Although ATP is hydrolyzed to AMP level, the pyrophosphate is ‘ald to the substrate 5. Amino acid > ATP— Aminoaeyl adenylate + PPA Here AMP group is incorporated into the privet ‘6. Methionine » ATP S-adenossl methionine » PP Pi Here adenoss! group is incorporated ito the pret (Cyrus Fiske and Yellapragada Subbarao discovered ATP in 192 ‘Karl Lohmann showed its importance in muscle contraction in 1929. In 1941, Fritz Lipmana (Nobel prize, 1953) showed that ATPisthe universal caurier of chemical energy in the cell and coined the expression “energy ‘ich phosphate bonds, Alexander Todd (Nobel Prize 1957) elucidated its sseucture Creatine Phosphate Phosphocreatine (Creatine phosphate or CP) provides a high energy reservoir of ATP to regenerate ATP rapidly by the Lohmann’s reaction, catalyzed by creatine kinase, ATP + Creatine —> Phosphocreatine + ADP + AG” 48.1 kmol (-10.5 kCal/mo)), The reaction is mitochondrial and of special significance in the myocardium which has a high energy £2 hl ‘Yellapragada Subbarao (1895-1048). His article 's the 4th most ced paper inthe word iterature || Bom in Andie Pradesh, he studied medicine In Madras, and conducted research at USA. He discovered ATP, assayed phosphates and isolated tetracycins and many other drugs Chapter 20: Biological Oxidation and Electron Transport Chain = requirement, about 6 kg of ATP per day. Energy transfer 10 the heart's myofibrils is by creatine kinase energy shuttle, since being a smaller molecule than ATP, CP can rapidly diffuse from the myocardium to the myofibrils. Structure of Mitochondrion ‘The electron transport chain is functioning inside the mitochondria. The mitochondrion is a subcellular organelle hhaving the outer and inner membranes enclosing the matrix (Fig. 20.4). The inner membrane is highly selective in its permeability, containing specific transport proteins. Certain enzymes are specifically localized in mitochondria (Table 20.3), The inner membrane contains the respiratory chain and translocating systems, The knob like protrusions represent the ATP synthase system (Fig, 20.4). High W’ concetraion —_Cristae Acyl CoA synthetase Phospholipase AZ In between outer and inner membrane: ‘Adenylate kinase Creatine kinase Inner membrane, outer surface: Giycerol3-phosphate dehydrogenase Inner membrane, inner surface: ‘Succinate dehydrogenase Enzymes of respiratory chain Soluble matrix: Enzymes of cite acid cycle Enzymes of beta oxidation of fatty acidTextbook of Biochemistry Inner and outer mitochondrial membrane differs greatly in their composition. Inner membrane is 22% cardiolipin and contains no cholesterol, whereas outer membrane is similar to cell membrane, with less than 3% cardiolipin and 45% cholesterol. | ORGANIZATION OF ELECTRON TRANSPORT CHAIN i. In the Electron transport chain, or respiratory chain, the electrons are transferred from NADH 10 a chain of electron carriers. The electrons flow from the more electronegative components to the more electropositive components, ii, All the components of electron transport chain (ETC) are located in the inner membrane of mitochondria ili, There are four distinct multi-protein complexes; these are named as complex-I, I, HL and IV, These are connected by two mobile carriers, co-enzyme Q and cytochrome € iv. The arrangement is schematically represented in Figure 20.7. The sequence of reaction is depicted in Box 20.2, NADH Generation ‘The NADH is generated during intermediary metabolism (Fig. 20.13), A detailed list of the reactions using NADH is given in Box 37.3 (Malate Aspartate Shuttle Mitochondrial membrane is impermeable to NADH. The NADH. ‘equivalents generated in glycolysis ar therefore to be transporte from ‘ytoplasm to mitoehonda for oxidation This is achieved by malate- ‘aspartate shutle oF malate shuttle, which operates mainly in liver, Kidney and heart. The eyele is operated withthe help of enzymes malate dehydrogenase (MDH) and aspartate aminotransferase (Fig. 205). From one molecule of NADH in the mitochondria, 2's ATP molectles are generated Giycerol-3-phosphate Shuttle In skeletal muscle and brain. the reducing equivalents fiom eytoplasmie NADH are transported to mitochondria as FADH, through glycerol} ‘Compler: NADH + FAN» FeS-+CoQ—> Complex Succinate» FAD -» Fe-S -» CoQ—> Complec i: CoQ ->Fe-S—+ cytb eytel cyte Complex W:Cyt.c>eyta-a3 +0, ‘phosphate shuttle (Fig. 20.6) Hence only 1% ATP are generated when this system is operating ETC Complex-I i. Itis also called NADH-CoQ reductase or NADH dehydrogenase complex, It is tightly bound to the inner membrane of mitochondria. ii, It contains a flavoprotein (Fp). consisting of FMN as prosthetic group and an iron-sulfur protein (Fe-S) NADH is the donor of electrons, FMN accepts them and gets reduced to FMNH, (Fig. 20.8). Two electrons and one hydrogen ion are transferred from NADH to the flavin prosthetic group of the enzyme NADH +H’ + FMN > FMNH, + NAD iii, The electrons from FMNH, are then transferred 10 Fe-S. The electrons are then transferred to co-enzyme Q (ubiquinone) (CoQ), iv. Overall function of this complex is to collect the pair of electrons from NADH and pass them to CoQ, The reactions are shown in Figure 20.8, Fig, 205: Mitochondrial wansport of NADH by malate-aspartate Shuttle MOH = malate dehydrogenase, AST = aspartate amino transferase: Gu= glutamic acid: AKG = alpha ketoglutaic ai 1= malate transporter; 2=glutamate aspartate transporter Cytopiasm ‘Mitochondria Giycorot- Giycoro S-phosphate S-phosphate NADY FAD NADH Ht FADH, Disdydroxy Diydroxy acetone phosphate acetone phosphate Fig.20.6:Glycerol-3-phosphate shuttle in muscle and brain\. There is a large negative free energy change: the energy released is 12 kcal/mol. This is utilized to drive 4 protons out of the mitochondria, Complex I! or Succinate-Q-Reductase The reaction in Complex-II is represented in Fig. 20.9, The electrons from FADH, enter the ETC at the level of coenzyme Q. This step does not liberate enough energy to act as a proton pump. In other words, substrates oxidized by FAD-linked enzymes bypass complex-I ‘The three major enzyme systems that transfer their clectrons directly to ubiquinone from the FAD prosthetic ‘group are: i, Succinate dehydrogenase, (see step 6, Fig. 19.2) fi, Fatty acyl CoA dehydrogenase (see step 1, Fig. 12.9) iii, Mitochondrial glycerol phosphate dehydrogenase (Fig, 206). Co-enzyme Q i, The ubiquinone (Q) is reduced successively to semi- ‘quinone (QH) and finally to quinol (QH,), Fig. 20.8: Complex | or NADH-CoO reductase (NADH dehydrogenase complex) Chapter 20: Biological Oxidation and Electron Transport Chain fi, It accepts a pair of electrons from NADH ot FADH, through complex-I or complex-II respectively (Figs 20.7 and 20.13). Co-enzyme Q is a quinone derivative having a long isoprenoid tail. The chain length of the tail is different in various species, mammals have 10 isoprene units (Fig. 20.10), Two molecules of cytochrome ¢ are reduced, iv. The Q eyele thus facilitates the switching from the two electron carrier ubiquinol to the single electron carrier eytochrome ¢. Complex III or Cytochrome Reductase i. This is a cluster of iron-sulfur proteins, cytochrome 1b and cytochrome €1, both contain heme prosthetic ‘group. The sequence of reaction inside the Complex lis shown in Figure 20.11 ii, During this process of transfer of electron, the iron in hheme group shuttles between Fe” and Fe forms, ‘The free energy change is—10 keal/mol; and 4 protons are pumped out, Cytochrome c It is a peripheral membrane protein containing one heme prosthetic group. The term cytochrome is derived from Greck, ili ‘Summary: Succinate —> FAD—* Fe-S—* CoQ = Fig.208: Complex i; Succinate O reductase ° RY RR R2_ RY Ra RY RO RI RO RI Ra g g @ (any (a) (2 quinone (oncized sat); OH = sem-quinore: OH, = quiol ‘or hydroquinone (reduced state). Ri = CH,O-gr0up; R2 = CH, group; R3 = 10 isoprene units (one isoprene unit contains 5 carbon atoms) J Fig. 20.10: Addition of # to co-enzymme QTextbook of Biochemistry ‘meaning cellular colors. It is one of the highly conserved proteins among different species. Axel Theorell (Nobel prize, 1955) isolated it. Cytochrome c collects electrons from ‘Complex Ill and delivers them to Complex IV. Complex IV or Cytochrome Oxidase i, It contains different proteins, including cytochrome 4 and cytochrome a3. The Complex IV is tightly bound to the mitochondrial membrane ii, ‘The reaction is depicted in Figure 20.12, Four electrons are accepted from cytochrome ¢, and passed on to molecular oxygen. 4H +0,+4Cyt.c-Fe" +2 H,0+4 Cyt 0 li, 2 protons are pumped out to the inter-membrane space. iv. Cytochrome oxidase has 4 redox centers, namely, a, a3, CuA and CuB. The electron transfer in this complex is as shown Cyto e CUA Cyto a Cyto a3 CUB Cytochrome oxidase contains two heme groups and two copper ions. The two heme groups are denoted as cytochrome-a and cytochrome a-3. The functional unit of the enzyme is a single protein, and is referred 10 as cytochrome a~a3, ‘The sequential arrangement of members of electron ‘transport chain is shown in Box 20.2 and Fig. 20.13, P: 0 Ratio ‘The P:O ratio is defined as the number of inorganic phosphate molecules incorporated into ATP for every atom of oxygen consumed, When a pair of electrons from NADH reduces an atom of oxygen (¥ 0,), 2.5 mol of ATP are formed per 0.5 mol of O, consumed. In other words, the P:O ratio of NADH oxidation is 2.5; The P:O value of FADH, is 1. OH, cyte (Rea) (Fe) 2 one (ond) Fe) [TU Summeycoa— Fes baton | Fig 2.11: Complex il or cytochrome reductase (cytochrome b-<1 complex) of respiratory chain oytelFe™) ~| tot Fig, 20.12: Complex v(cytoctome oxidase) of respiratory chain ‘Site 1 ‘4 protons pumped out 6. Pyruvate. 7. Alpha ketoglutarate ‘sito2 4 protons pumped out Site 3 2 protons pumped out Fig, 20.13: Components and sequence of reactions of electron transport chainCurrent Concept, Energetics of ATP Synthesis The free energy released by electron transport through complex I to IV must be conserved in a form that ATP synthase can perform energy coupling. The energy of electron transfer is used to drive protons out of the matrix, by the complexes I, III and IV that are proton pumps. The proton gradient thus created is maintained across the inner mitochondrial membrane till electrons are transferred 10 oxygen to form water. The electrochemical potential ofthis, gradient is used to synthesize ATP. ‘According to the estimated free energy of synthesis, it was presumed that around 3 protons are required per ATP synthesized. Hence, when one NADH transfers its, electrons to oxygen, 10 protons are pumped out. This, ‘would account for the synthesis of approximately 3 ATP. Similarly the oxidation of | FADH, is accompanied by the pumping of 6 protons. accounting for 2 molecules of ATP. However, Peter Hinkle recently proved that the actual energy production is less, because there is always leakage of protons, This results in hamessing of energy required for the production of 2.5 ATP from NADH and 1.5 ATP from FADH, The synthesis of one ATP molecule is driven by the flow of 3 protons through the ATP synthase (see below). ‘When NADH is oxidized, 10 hydrogen ions (protons) are pumped out (Fig. 20.13), According to recent findings, one NADH may generate only 2.5 ATP; and one FADH, may generate only 1.5 ATP. So, one molecule of glucose will generate only 32.ATPs. The traditional values and the new values are compared in Table 20.4. (Please note that there is no change in the values of ATP generation by substrate level phosphorylation. Energy efficiency of glucose oxidation giving 32 molecules of ATP and palmitate giving 106 molecules of ATP is given as 34% and 33 % respectively Chaptes 20: Biological Oxidation and Electron Transport Chain Sites of ATP Synthesis ‘Traditionally, the sites of ATP synthesis are marked, as site 1,2 and 3, as shown in Figure 20.13. But now itis known that ATP synthesis actually occurs when the proton gradient is dissipated, and not when the protons are pumped out (Fig. 20.15), (| CHEMIOSMOTIC THEORY ‘The coupling of oxidation with phosphorylation is termed ‘oxidative phosphorylation, Peter Mitchell in 1961 (Nobel prize, 1978) proposed this theory to explain the oxidative phosphorylation, The transport of protons from inside to ‘outside of inner mitochondrial membrane is accompanied by the generation of a proton gradient across the membrane. Protons (HH° ions) accumulate outside the membrane, creating an electrochemical potential difference Fig. 20.15). This proton motive force drives the synthesis of ATP by ATP synthase complex (Fig. 20.14). Proton Pump and ATP Synthesis ‘The proton pumps (complexes I, If and IV) expel H’ from inside to outside of the inner membrane. So, there is high H concentration outside the inner membrane. This causes Hi" to enter into mitochondria through the channels (Fo); this proton influx causes ATP synthesis by ATP synthase. A summary is shown in Figure 20.15. Fig. 20.14 ATP synthase. Protons from outside pass through the pore (off into the matrix, when ATP is synthesizedTentbook of Biochemistry The pH outside the mitochondrial inner membrane is 1.4 units lower than inside, Further, outside is positive relative to the inside (+0.14 V) (Fig. 20.15). The proton ‘motive force (PMF) is 0.224 V corresponding to a free ‘nergy change of 5.2 kcal/mol of protons. ATP Synthase (Complex V) Itisa proteinassembly inthe inner mitochondrial membrane is sometimes referred to as the Sth Complex (Figs 20.14 and 20.15). Proton pumping ATP synthase (otherwise called F1-Fo ATPase) is a multisubunit transmembrane protcin. It has two functional units, named as Fl and Fo. It looks like a lollipop since the membrane embedded Fo component and FI are connected by a protein stalk Peter 0 Theorel Mitchel NP 1955 NP 1978 1903-1982 1970-1902 Fo Unit: The “o" is pronounced as “oh”; and not a8 “zero” The “0° stands fr oligomycin, as Fo is inhibited by oligomycin, Fo unit spans inner mitochondial membrane. It serves as a proton channel ‘rough which protons enter into mitochondria (Fig. 20.14). Fo eit has 4 polypeptide chains and is connected to FI. Fo is water insoluble where 48 L isa water soluble penpheral membrane protein 1 Unit: It projects into the mat I catalyzes the ATP synthesis (Fig 2014) FY unithas9 polypeptide chains, (3 alpha, 3 beta, 1 gamma, 1 sigma, 1 epsilon). The alpha chains have binding sites for ATPand ADP and eta chains have caalytic sits, ATP symhess requires Me ions, Mechanism of ATP synthesis: Translocation of protons carried out by the Fo catalyzes the formation of phospho-anhydride bond of ATP by FI. Coupling of the dissipation of proton gradient with ATP synthesis (oxidative phosphorylation) is through the interaction of Fl and Fo. Binding Change Mechanism ‘The binding change mechanism proposed by Paul Boyer (Nobel prize, 1997) explains the synthesis of ATP by the proton gradient. The ATP syathase is a “molecular machine”. comparable to a “water-diven hammer, minting coins”. Foi the wheel ow of protons is the waterfall and the structural changes in FI lead to ATP coin being minted for each tum ofthe wheel. The FI has 3 conformation states forthe alpha-beta fanetional unit (0 state—Does nt bind substrate or products L state—Loose binding of substrate and products ‘Tstate—Tight binding of substrate and pro termeribranes ETC completes pu ycogen 1:8 Summaryof AP yess. One mocha sdepiced waver ander menbeanes EC comple ip Fe pst maven the marta space, 5 termed spaces more gh cde than mata $0 hygtooen ns teed ‘tomate tough. Then Aste seed (1 Mv = components ET‘According to this theory, the thee beta subunits (catalytic sites) are in thiee functional states © form is open and has 10 affinity for subsirates. 1 form binds substrate with sluggish affity: T form binds substrate AAs protons translocate othe matnx, the free energy i released and hiss harnessed to ne the T state and ATP is release inthe O sate The sequence of events is as fallow: 1. ADPand Pi bind tL binding site 2. Lio T conversion is by energy daven conform: ‘atalyzes the formation of ATP 3. Tstate reverts to O state when ATP s released 4. L states regenerated for funer ADP binding. For the complete rotation of F1 head though the 3 states, 10 protons located Protons entering the system, cause conformational changes in the FI particle. Initially the ADP and Pi are loosely bound tothe catalytic site on FI. As the Fo aceepts protons the affinity for ADP is increased (step 1, Fig. 20.16), Further conformational change induces catalytic sctivity, and ATPis synthesized (step 2, Fig. 20.16), This moves protons to the matrix side As the ATPs are released, the orginal conformation ofthe enzyme is assumed (step 3, Fig 20.16) Then ADP again hound and the eycle repeats (step 4, Fig. 20.16) The energy surplus produced by the proton gradient is stored as chemical energy in ATP. The energy ‘requiring step isnot atthe syathesis of AE, but energy is required forthe conformational changes. Regulation of ATP Synthesis ‘The availability of ADP regulates the process. When ATP level is low and ADP level is high, oxidative phosphorylation proceeds at a rapid rate. This is called respiratory control or acceptor control. The major source of NADH and FADH, is the citric acid cycle, the rate of which is regulated by the energy charge of the cell, Since electron transports directly coupled to proton translocation, the flow of electrons through the electron transport system is rep by the magnitude ofthe PMF The higher the PME, the lower the rat of ily and catalyzes ATP synthesis. ert these 3 sates. The bond is synthesize in foal change that Chapter 20: Biological Oxidation and Electron Transport Chain cfectrom 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 PME is high. flow of protons back into the mitochondria ‘through ATP synthase is minimal, When energy demands ar increase, such as during vigorous muscle activity, eylosoic ADP rises and is ‘exchanged with intramitochondial ATP via the transmembrane adenine auclootide carrer ADP/ATP transloease. Increased itramitochondsial concentrations of ADP cause the PMF to become discharged as protons pour through ATP synthase, regenerating the ATP pool. Thus, while the tale of electron transport is dependent on the PMF, the magnitude of the PMF at any moment simply reflects the encrgy charge oft tum the energy charge, oF more precisely ADP concentation, normally < ¢ toe ~< ap abe 13 ee ig, 20.19: Creatine phosphate shuttle 1 = Chm (muscle creatine kinase; 2 = CKmt (mitochondrial creatine phosphate) 3 = ANT (adenine nucleotide transporter (C= creatine, CP = creatine phosphatel Textbook of Blochemistry Diseases Associated with Mitochondria Mitochondtial DNA is inberited eytoplasmically and is therefore transmitted maternally (see Table 46.6). OXPHOS (oxadative phosphorylation) diseases are described in Chapter 46. Mutations in mitochondrial DNA are responsible forthe following diseases. 1. Lethal inane mitochondrial ophthalmoplegia 2 Leber’ herelitary optic neuropathy (HON) 3. Myoctonic epilepsy 4. Mitochondrial encephalomyopathy lactic acidosis stroke like episodes (MELAS) Unlike nuclear DNA, there are hundreds of copies of mitochon dial genes per cell (heteroplasmy). Leber’ hereditary optic myopathy i characterized by blindness in young males. Ii caused by a single ‘se mutation in NADH Coenzyme Q reductase. Stepomycin- induced deafness is also found to be due w 4 mutation in the sitochondhial RNA. (Mitochondrial Permeability Transition Pore (MPTP) Cytochrome © is also the mediator of apoptonis (programmed cll death), Since estochrome ¢ is a peripheral membrane protein, itis loosely bound to mitochondria, So it is released from the mitochondria when the mitochondrial membrane permeabilization (MMP) occurs “This can happen in response to an oxidant stress due to ROS, increase in caleium concentration in mitochondria ‘As the membrane permeability increase, there is transient opening of 4 mitochondrial permeability wanston pore (MPTP). However, if the injury i ony transient, the pote closes ‘Butifthe pore remains open, it resulsin dissipation of mitochondrial proton gradient, ATP depletion and release of eytochrome ©. This Cytochrome ¢ acts asa tigger for apoptosis by forming an apoptosome ‘complex with other pro-apoptotic factors. The initiator caspase is then ‘setvated leading to stivation of effector caspases, and finally the eel ‘eath (Fig. 2020) any other form of sites, Fig. 20.20: Role of mitochondria in apoptosis Repesfuson injury can also result from generation of ROS which leads to activation (opening ) of MPTP and resultant events can lead necrosis. In myocardial and cerebral ischemia, the core of the damaged risa undergoes necrosis, but the surrounding tissue which sot intally damaged can undergo delayed apoptosis MPTP is located at the contact site between the inner and outer ‘mitochondeial membranes. Its mode up of Voltage Dependent Anion ‘Channel (VDAC) located inthe outer membrane, Adenine Nudeotide Translocase (ANT) located inthe inner membrane and Cyelophilin-D Clinical Case Study 20.1 A 68-year-old female in a hypertensive crisis is being treated in the intensive care unit (ICU) with intravenous nitroprusside for 48 hours. The patient's blood pressure ‘was brought back down to normal levels; however, she was complaining of a burning sensation in her throat and mouth followed by nausea and vomiting, diaphoresis, agitation, and dyspnea. An arterial blood gas revealed a significant ‘metabolic acidosis. A serum test suggests a metabolite of nitroprusside, thiocyanate, is at toxic levels, 1. Whats the likely cause of her symptoms? 2. What is the biochemical mechanism of this problem? 3. What is the treatment for this condition? Clinical Case Study 20.2 [A'55-year-old man was treated in the ICU with intravenous nitroprusside for hypertensive crisis for 48 hours. BP ‘was restored, but he had a burning sensation in his throat ‘and mouth, followed by nausea and vomiting, excessive ‘sweating, agitation and dyspnea, There was a sweet almond, smell in his breath and arterial blood gas analysis revealed severe metabolic acidosis. What is the likely condition? How is it treated” What is the pathogenesis? Clinical Case Study 20.1 Answer Diagnosis: Cyanide poisoning from toxic dose of nitro- prusside, Biochemical mechanism: Cyanide inhibits mitochondrial cytochrome oxidase, blocking electron transport and pre- venting oxygen utilization, Lactic acidosis results second- ary to anaerobic metabolism. Treatment: Supportive therapy, oxygen, and antidotal therapy with sodium nitrite, and sodium thiosulfate. Clinical correlation: Malignant hypertension is diagnosed when there is elevated blood pressure (systolic levels of 220 mm Hg and/or diastolic blood pressures exceeding,120 mm Hg). The symptoms may include severe headache, neurological deficits, chest pain, or heart failure. Hyperten- sive emergencies require immediate lowering of the blood pressure to lower levels. One hazard of abruptly lowering the blood pressure is causing hypotension and subsequent ischemia to the brain ‘or heart. Sodium nitroprusside induces a smooth fall in blood pressure, One side effect of sodium nitroprusside is that its metabolite is thiocyanate, and with prolonged use, cyanide poisoning may result, which inhibits the electron transport chain. Thus, in clinical practice, short-term nitroprusside is used. Clinical Case Study 20.2 Answer Patient is most probably suffering from cyanide poisoning. Cyanide inhibits mitochondrial cytochrome oxidase, blocks the electron transport chain and prevents oxygen utilization. Lactic acidosis is secondary to anaerobic metabolism. Cellular oxygen metabolism is impaired and ccan produce death within minutes. NItroprusside therapy which is the drug of choice for hypertensive emergency, on prolonged usage can produce cyanide poisoning. Hence, in clinical practice, nitroprusside is used only for short term. Causes for cyanide poisoning include smoke inhalation from residential or industrial fires, metal trades, mining, electroplating, jewelry manufacture and X-ray film recovery It can occur during fumigation of ships, warchouses, etc and are also used commonly as suicidal agents, especially by terrorists and healthcare and laboratory workers, Cyanide affectsall body tissues and attaches to many metalloenzymes, rendering them inactive Treatment includes administration of amy! nitrite, sodium nitrite and sodium thiosulfate, increasing oxygen concentration in inspired air and sodium bicarbonate therapy. Amyl and sodium nitrites induce methemoglobin formation, it combines with cyanide and reduces its toxicity Sodium thiosulfate converts cyanide 19 thiocyanate and which is excreted in urine, Hydroxocobalamin combines Chapter 20: Biological Oxidation and Becton Transport Chain with cyanide to form cyanocobalamin which is excreted through urine. Sodium bicarbonate reduces lactic acidosis, QUICK LOOK OF CHAPTER 20 1. Oxidation of food stuff occurs in 3 stages—primary metabolism where macromolecules are converted to smaller units, secondary metabolism where reducing ‘equivalents are formed and tertiary metabolism where energy is released 2. Oxidation is loss of electrons and reduction is gain of electrons. A pair that exists in both oxidized and reduced state is a redox couple. 3. In substrate level phosphorylation, energy from high- ‘energy compound is directly transferred to NDP to form NTP without the help of electron transport chain 4. Transfer of electrons from reduced co-enzymes through respiratory chain to O, is known as Biological ‘Oxidation 5. The energy released is trapped as ATP. This coupling of oxidation with phosphory ation is called Oxidative phosphorylation. All enzymes of biological oxidation are oxidoreductases. 6, Electron flow occurs through successive dehy drogenase ‘enzymes (located in the inner mitochondrial membrane) together known as Electron Transport Chain; the electrons are transferred from higher to lower potential 7. NADH is impermeable to mitochondrial membrane. Hence it is transferred via malate-aspartate shuttle in liver, kidney and heart as NADH reducing equivalents and in skeletal muscles as FADH, through glycerol 3-phosphate shuitle 8. The ETC has 4 distinct multiprotein complexes —viz: complex I, Il, III and IV connected by two mobile carriers to Co Q and cytochrome ¢ 9. Inhibitors of oxidative phosphorylation include atracty loside and oligomyein. Cyanide inhibits terminal cytochrome and brings cellular respiration to stand stl.