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1 Plant Physiology and Biochemistry Respiration Dr. M.S. Rawat Department of Botany, HansRaj College Malkaganj Delhi-110007 Contents: Historical Aspects Aerobic Respiration Glycolysis Transition Reaction Krebs Cycle Oxidative Phosphorylation Anaerobic Respiration Obligate anaerobiosis Facultative anaerobiasis Factors Availability of Respiratory Fuels Temperature Oxygen Hydration Kind of Plant Material Light CO2 Concentration Wounding / Injury Age Inhibitors/ Respiratory Poisons Key words: Aerobic Respiration, Glycolysis, Transition Reaction, Krebs Cycle Oxidative Phosphorylation, Anaerobic Respiration,Obligate anaerobiosis, Facultative anaerobiasis 2 RESPIRATION Respiration, the most important process in all living beings, is the oxidation of food to produce energy. Continuous production of energy is necessary for occurrence of various physiological processes, such as growth, transport, movements, activation and synthetic pathways. Study of respiration has a vast scope, as it comprises three different aspects : breathing, gaseous exchange and cellular respiration. 1. Breathing involves the act of inhalation (inspiration) and exhalation (expiration). Plants do not have any respiratory organs. Monera, Protista and Fungi also do not possess organ systems for breathing. However, different groups of animals have well defined respiratory systems for uptake of oxygen and release of carbondioxide. 2. Gaseous exchange is the other essential aspect of respiration; the process in which oxygen enters the tissues/cells and carbondioxide leaves the cells. In unicellular organisms, the plasmamembrane is permeable to both oxygen and carbondioxide. In higher plants, diffusion of these gases occurs mainly through stomata, the minute apertures on the surface of leaves or through epidermal cells, which are thin walled and not much cuticularised. These gases may be stored in the intercellular spaces or air cavities in the leaves for the gaseous exchange to take place continuously. In land animals the gaseous exchange occurs in the alveoli of lungs where the gases are in contact with epithelial cells of the walls of blood capillaries. The driving force for diffusion of oxygen or carbondioxide is its concentration gradient or gradient of its partial pressure. Thus, in the lungs, oxygen enters the blood capillaries because partial pressure of oxygen (i.e. concentration of oxygen) in the alveoli is higher than the partial pressure of oxygen in the blood capillaries. Similarly, CO2 diffuses out of blood capillaries into the alveoli since the partial pressure of CO2 is higher in the blood (as CO2 is produced during respiration by cells and tissues) than that in the alveoli. Exchange of CO2 and O2 through the stomata follows the same principle. During daytime oxygen is given out of stomata whereas, carbondioxide diffuses in, even if O2 concentration is about 21% in the atmosphere and CO2 concentration is just 0.033%. Molecules of a gas move from its higher concentration to lower concentration. In plants, during daytime, the leaf cells take CO2 and release O2 in photosynthesis. At the same time in respiration, O2 is utilized and CO2 is given out. However, since photosynthesis is approximately 10 times respiration, the net gaseous exchange causes high partial pressure of oxygen and a very low partial pressure of carbondioxide inside the leaf. As a result oxygen diffuses out of stomata from its higher partial pressure inside to lower partial pressure outside. CO2 enters through the stomata as its partial pressure outside is higher than its partial pressure inside the leaf. During night the gaseous exchange reverses as there is no photosynthesis and only respiration occurs. Understandably, CO2 is given out and oxygen is taken up through stomata. 3. Cellular respiration pertains to the process of energy production in the cells of all organisms, plants or animals. The process of cellular respiration is common to all organisms. This chapter deals exclusively with cellular respiration and concentrates on the oxidation of food and production of energy. Historical Aspects Although the importance of oxygen to life is known from the time immemorial, the term respiration came into existence somewhere in 15th century. Crooke (1615) emphasized that life is not possible without air. Only animals were expected to respire. Malpighi (1679) observed that seeds failed to germinate in the absence of air. IngenHousz (1779) demonstrated that green plants also make the air impure in dark just like a breathing mouse or a burning candle. Thus, implying that in dark, green plants respire by taking oxygen from the atmosphere and release carbondioxide. They also respire in daytime but do not make the air impure (rather purify the air) because of domination of photosynthesis in which CO2 is taken in and O2 is given out. De Saussure (1804) was the first to demonstrate that the volume of O2 consumed by germinating seeds was equal to the volume of CO2 evolved. 3 Louis Pasteur (1870) studied fermentation by yeast cells, the process in which sugar is converted into ethyl alcohol and carbondioxide. That the process of fermentation is connected to anaerobic respiration was proposed by the Russian scientist Kostychev (1927). Significant breakthrough in the mechanism of respiration came when Embden, Meyerhof and Parnas worked out the process of glycolysis i.e. breakdown of glucose into pyruvic acid (a 3-C compound). Sir Hans Kreb subsequently worked out the process of oxidation of pyruvic acid (known as Krebs Cycle) and received the Nobel Prize for his outstanding work in 1937. Davis and Majelis Young later established the occurrence of enzymes of the Krebs Cycle in the mitochondria. Discovery of Acetyl Coenzyme A by Fritz Lipmann is another piece of Nobel Prize winning work in this field. A revolutionary idea regarding the mechanism of biosynthesis of ATP in respiration came from Peter Mitchell, known popularly as Chemiosmotic theory of ATP synthesis. Although he proposed this hypothesis in 1961, he was awarded Nobel Prize in 1978 after the importance of his work was realized J. N. Siedow and A. Berthold described alternate oxidase — a cyanide resistant pathway, unique to higher plants. Kinds of Respiration Respiration is an intricate process where complex compounds are oxidised into simpler compounds and energy is released. Depending upon the utilization or non-utilization of oxygen, there may be complete oxidation or incomplete oxidation. Accordingly, respiration may be of two types. A. Aerobic Respiration / Aerobiosis / Complete Oxidation B. Anaerobic Respiration / Anaerobiosis / Incomplete Oxidation A. Aerobic Respiration Respiration occurring in the presence of oxygen is called aerobic respiration. Here, complete oxidation of food occurs to produce water, carbon dioxide and large amount of energy. It used to be called intermolecular respiration as oxygen is also consumed along with glucose. C6H12O6 + 6O2 6H2O + 6CO2 + 686 KCal / 2871 kJ Glucose Substantial amount of energy is released in the form of ATP, which is synthesized from ADP and H3PO4. In short, the features of aerobic respiration are : Oxygen is absorbed / consumed. Carbondioxide is given out. Energy currency, ATP is synthesized. Heat energy is also released in addition to ATP synthesis. Dry weight of plant organs / tissues decreases. Dehydrogenation i.e. release of hydrogens (protons and electrons) is a regular feature of aerobic respiration. Such dehydrogenation reactions are the source of energy for electron transport chain, which ends up in ATP synthesis. In eukaryotes, mitochondria are the sub-cellular organelles besides cytoplasm to carry aerobic respiration. In prokaryotes (bacteria and blue green algae), aerobic respiration can occur without mitochondria. Demonstration of Aerobic Respiration Aerobic respiration can be demonstrated and its rate can be calculated by using Ganong’s respiroscope or a simple respiroscope as shown in the given figure. 4 In a conical flask, sufficient amount of plant material (preferably germinating seeds / flowers / roots) and a glass vial containing KOH solution are taken. A one-holed cork is fitted to the flask and a delivery tube bent at two right angles is then fitted to the cork. The other end of the delivery tube is dipped in coloured water / mercury taken in a beaker. All the connections are made air tight. As respiration occurs, the level of the fluid rises in the delivery tube. The volume of fluid displaced is equal to the volume of oxygen used in aerobic respiration. In the respiroscope, oxygen is used by the plant material and CO2 which is released is absorbed by KOH. So, a partial vacuum or suction is created, resulting in the upliftment of fluid in the delivery tube. The maximum level to which fluid rises is about one-fifth of the total volume of respiroscope. Mechanism of Aerobic Respiration Although aerobic respiration is visualised as intermolecular respiration, direct combination between glucose and oxygen is neither possible nor feasible. The process involves a complex series of reactions, so that energy is gradually released, several intermediates are formed, which may function as precursors for the synthesis of other metabolites and there are several control points. Respiration is a continuous process, nevertheless, for the sake of convenient study it is divided into following major steps : 1. Glycolysis 2. Transition reaction 3. Krebs cycle 4. Oxidative phosphorylation Glycolysis In this process, hexose sugars, such as glucose is oxidised into pyruvic acid (pyruvate) to release some energy. Besides several enzymes and cofactors, NAD+, ADP and Phosphate are needed for glycolysis. Glycolysis is common to both aerobic and anaerobic respiration. Thus, glycolysis is known to occur universally in all living organisms. In aerobic respiration, oxygen is not directly used in any of the reaction of glycolysis. However, oxygen is necessary to oxidise NADH into NAD+ and H2O, so that NAD+ is available to carry one of the reactions of glycolysis in which NADH is formed. Cytoplasm (cytosol) is the site for glycolysis, although oxidation of NADH in aerobic respiration occurs on the cristae of mitochondria. 5 Availability of Hexose Glucose is available to cells for respiration due to the hydrolysis of reserve carbohydrates. In animals, glycogen is hydrolysed by glycogen phosphorylase into glucose-1-phosphate. In plants, glucose is formed by the hydrolysis of starch, maltose, sucrose and fructans etc. Starch + water Amylase Maltose Maltose + H2O Maltase 2 Glucose Sucrose + H2O Inulin + water Invertase Inulase Glucose + Fructose Fructose Phosphorylation of Sugars This part is also known as activation of sugars since it involves utilization of ATP. This is comparable to ignition of fuels. Hexose is a respiratory fuel, so initially some energy is supplied to activate it. During this process glucose is converted into fructose-1,6-diphosphate, utilizing 2 ATP. Reaction 1 This is the first step of glycolysis and therefore, quite important. Hexokinase is an allosteric enzyme and is inhibited by a higher concentration of the product, glucose-6-phosphate. Magnesium ions are needed by hexokinase and all other kinases to link ATP to the active site of the enzyme. Reaction 2 Glucose-6-phosphate is isomerised into fructose-6-phosphate. 6 Reaction 3 This reaction is considered to be the committed reaction of glycolysis as this conversion is irreversible. Conversion of fructose-1,6-diphosphate into fructose-6-phosphate requires a different enzyme, phosphatase. Moreover, once fructose-1,6-diphosphate is formed, it is destined to be used further in respiratory breakdown, whereas glucose-6-phosphate and fructose-6-phosphate may be used in synthetic pathways. The enzyme Phosphofructokinase (PFK) is also an allosteric enzyme, which is inhibited at a higher concentration of ATP. The enzyme is rightly referred to as “pace maker of respiration” as it acts as a valve controlling the rate of glycolysis. Thus, when the level of ATP increases, the process of glycolysis slows down. As ATP concentration decreases, the pace of the process increases. AMP (adenosine monophosphate) reverses the inhibition due to ATP. The enzyme adenylate kinase combines ATP and AMP to form two moles of ADP. Availability of ADP promotes glycolysis. During the formative phase of glycolysis (Reactions 1 to 3) there is no gain of ATP/energy; rather two moles ATP are utilized in phosphorylation of sugars. Breakdown of Fructose-1,6-diphosphate into 3-C compounds This part of glycolysis is designated as pay off phase, as energy (ATP) is released here. Reaction 4 The enzyme aldolase catalysing this reaction belongs to the class desmolases or lyases, which break a bond without adding water. The two trioses formed are isomers. Reaction 5 CHO Phosphotrioisomerase CH2OH CHOH C=O CH2OPO32- CH2OPO32- 3-phosphoglyceraldehyde or Glyceraldehyde 3-phosphate Dihydroxyacetone phosphate 7 The two isomers, PGAL and DHAP are readily interconvertible, therefore when phosphoglyceraldehyde is used in further reaction, equilibrium is shifted towards left. Practically, two moles of PGAL are generated from one mole fructose-1,6-diphosphate. Dihydroxyacetone phosphate is the precursor in the synthesis of glycerol. Reaction 6 COOPO32- CHO CHOH + NAD+ + H3PO4 PGAL dehydrogenase CHOH CH2OPO32- + NADH + H+ CH2OPO32- 3-phosphoglyceraldehyde 1,3-diphosphoglycerate Oxidation of PGAL is another crucial step of glycolysis. Here, one phosphate is added to C-1 coupled with dehydrogenation. The enzyme PGAL dehydrogenase needs a coenzyme NAD+ (nicotinamide adenine dinucleotide), which accepts hydrogens released in this step. NAD+ bears a positive charge, therefore accepts two electrons and one proton. NAD+ + 2H (2H+ + 2 e–) NADH + H+ However, it is for the sake of convenience that NADH + H+ is often written as NADH2. In aerobic respiration, NADH is oxidised on the cristae of mitochondria. During this process called oxidative phosphorylation, ATP is synthesized and NAD+ is regenerated to be used in reaction no. 6. Reaction 7 COOPO32- COOPhosphoglycerate kinase CHOH + ADP CHOH CH2OPO32- + ATP CH2OPO32- 1,3-diphosphoglycerate 3-phosphoglycerate In this step, phosphoglycerate kinase transfers a phosphoryl group along with its energy to ADP to form ATP. Such a process, in which ATP is synthesized without involving electron transfer is called substrate level phosphorylation. Here, ATP is synthesized at the level of the substrate when energy rich phosphate bond from substrate is directly added to ADP. Reaction 8 COO- Phosphoglycerate mutase COO- CHOH CHOPO32- CH2OPO32- CH2OH 3-phosphoglycerate 2-phosphoglycerate The enzyme phosphoglycerate mutase transfers phosphoryl group from C-3 to C-2. Reaction 9 COOCHOPO32 CH2OH 2-phosphoglycerate COOPhosphoglycerate mutase COPO32CH2 Phosphoenol pyruvate (PEP) 8 This reaction yields a substrate (PEP) having a high energy phosphate for the synthesis of ATP later on. Reaction 10 COOCOPO32- COO+ ADP Pyruvate kinase C=O + ATP CH3 CH2 Phosphoenolpyruvate Pyruvate This last step of glycolysis is another example of substrate-level phosphorylation, where there is direct synthesis of ATP due to the transfer of energy rich phosphate from PEP to ADP. This is also a good example of “coupled reactions” where an exergonic reaction and an endergonic reaction are coupled to synthesize ATP i.e. a reaction which is not thermodynamically possible, occurs with the help of a reaction which is thermodynamically possible. ADP + H3PO4 ATP + H2O ; ∆Go = + 30.5 J kJ (endergonic) PEP + H2O Pyruvate + H3PO4 ; ∆Go = – 62.2 kJ (exergonic) Adding the above two PEP + ADP Pyruvate + ATP ; ∆Go = – 31.7 kJ Due to coupling, overall reaction becomes exergonic (exothermic), so ATP is readily synthesized. From the breakdown of 1 mole glucose, 2 moles pyruvate, 2 moles ATP and 2 moles of NADH are formed. In aerobic respiration pyruvate is further oxidised into H2O and CO2 in the presence of oxygen. For glycolysis to take place continuously and consistently it is necessary that * Glucose supply is maintained continuously. * NAD+ is adequately available i.e. NADH produced in oxidation of PGAL must be oxidised to regenerate NAD+. * Regular supply of ADP and Pi (inorganic phosphate) should be maintained. * Pyruvate must be oxidised further in Krebs cycle or under anaerobic conditions it must be reduced to ethanol or lactic acid. * ATP level should be maintained low i.e. ATP produced in the overall process must be utilized. 9 Glucose ATP ADP Glucose-6-phosphate Fructose-6-phosphate ATP ADP Fructose-1,6-diphosphate Dihydroxyacetone phosphate 3-phosphoglyceraldehyde (DHAP) (PGAL) Pi NAD+ NADH2 1,3-diphosphoglycerate (DPGA) ATP ADP 3-phophoglycerate (PGA) 2- phosphoglycerate H2O Phosphoenol Pyruvate ATP ADP Pyruvate Summary Of Glycolysis 10 Transition Reaction Under aerobic conditions, pyruvate is transported to the mitochondrial matrix where it is fully oxidised into H2O and CO2. The major pathway of oxidation is Krebs cycle. Before the cycle begins there is a bridging step between glycolysis and Krebs cycle called transition reaction. Here, 3-C compound pyruvate is oxidised and decarboxylated to form a 2-C compound acetyl Coenzyme A. Thus, the reaction is called oxidative decarboxylation. O O CH3 – C – COO– + CoA – SH + NAD+ Pyruvate CH3 – C – S – CoA + CO2 + NADH + H+ Coenzyme A Acetyl Coenzyme A This reaction is catalysed by pyruvate dehydrogenase a multi enzyme-complex. It comprises three different enzymes involving five different coenzymes — Thymine pyrophosphate (TPP), Lipoic acid, Coenzyme A (CoASH), FAD and NAD+. Besides these, Mg++ is also necessary. Coenzyme A is chemically a conjugate of adenosine monophosphate (a nucleotide) and pantothenic acid (a vitamin B). This molecule has a phosphate group at one end and a highly reactive thiol (–SH) group at the other end. When its chemical nature was poorly known, it was called coenzyme A because of its high reactivity with an acid. The conversion of pyruvate into acetyl coenzyme A comprises following steps : 1. Pyruvate combines with the cofactor TPP (thymine pyrophosphate) and loses CO2. The reaction is catalysed by pyruvate dehydrogenase component (called E1) of the multi-enzyme complex. E1 Pyruvate + TPP Hydroxyethyl – TPP complex+ CO2 2. Hydroxyethyl group is oxidised by lipoamide, a derivative of lipoic acid by the same enzyme component, E1. E1 Hydroxyethyl-TPP Complex + Lipoamide Acetyl lipoamide + TPP 3. Coenzyme A removes the acetyl group from acetyl lipoamide with the help of the 2nd component of enzyme complex called dihydrolipoyl transacetylase (called E2). Acetyl lipoamide complex + CoA–SH E2 Acetyl CoA + Dihydrolipoamide 4. The third enzyme component called dihydrolipoyl dehydrogenase (E3) regenerates lipoic acid. The hydrogen acceptor is FAD. Dihydrolipoamide + FAD E3 Lipoamide + FADH2 5. Protons and electrons are then transferred from FADH2 to NAD+, which is unusual but is possible here because of flavoproteins tightly associated with FAD. FADH2 + NAD+ E3 FAD + NADH + H+ 11 TCA Cycle Acetyl coenzyme A combines with oxaloacetate to form citrate, which, then undergoes various reactions in a cyclic process to regenerate oxaloacetate. This cycle is called Krebs cycle or Citric acid cycle or Tricarboxylic acid (TCA) cycle. 1. Entry of Acetyl Coenzyme A into TCA cycle COO– O CH3 – C – S – CoA + H2C – COO– HO – C – COO– C = O + H2O + CoA–SH H2C – COO– CH2 COO– Acetyl Coenzyme A Oxaloacetate Citrate Acetyl coenzyme A combines with oxaloacetate to form citrate with the help of citrate synthase (also called condensing enzyme). This is the first step of Krebs cycle and has a large negative ∆G. This enzyme experiences feed back inhibition as it is inhibited by NADH or succinyl coenzyme A. 2. Isomerisation of Citrate into Isocitrate The enzyme aconitase catalysing this reaction contains an iron-sulphur cluster having three iron and four sulphur atoms. The reaction occurs in two steps. H2C – COO– H2C – COO– HO – C – COO– H–C–H H2O H2C – COO– C – COO– H – C – COO– H – C – COO– H – C – COO– COO– Citrate H2O OH Cis-aconitate Isocitrate The significance of this step is that isocitrate (2o alcohol) is readily oxidised as compared to citrate (3o alcohol). Fluoroacetate, a rodent poison indirectly inhibits TCA cycle. Fluoroacetate is metabolised by citrate synthase into fluorocitrate which inhibits aconitase. 12 3. Oxidation of Isocitrate This reaction occurs in two steps catalysed by one enzyme isocitrate dehydrogenase, which is known to be activated by ADP. H2C – COO– H – C – COO- H2C – COO– + NAD+ H – C – COO– H – C – COO- + NAD + H+ C=O COO– OH Isocitrate Oxalosuccinate H2C – COO– H2C – COO– H – C – COO– H 2C C=O C=O – COO– COO + CO2 α-Ketoglutarate Oxalosuccinate It is for the first time that in this reaction the TCA cycle is connected to electron transport chain and oxidative phosphorylation, as NADH is further oxidised in the presence of oxygen to produce ATP and regenerate NAD+. NADH + H+ + ½ O2 NAD+ + H2O + 3ATP This step highlights the role of oxygen in the operation of TCA cycle. The cycle does not operate in the absence of oxygen, although there is no direct combination of oxygen with any metabolite of TCA cycle. Oxygen is necessary for oxidative phosphorylation so that NAD+ is available for some reactions in the TCA cycle. This reaction also connects TCA cycle to nitrogen metabolism, as α-Ketoglutarate is precursor for the synthesis of amino acids. In protoplasmic respiration (when proteins are used as respiratory fuels), glutamate (an amino acid) enters TCA cycle at this juncture by first changing into α-ketoglutarate. 4. Oxidative decarboxylation of α-Ketoglutarate H2C – COO– H2C – COO– H2C H 2C + CoA–SH + NAD+ C=O + CO2 + NADH + H+ O = C – S – CoA COO– α-Ketoglutarate Succinyl coenzyme A 13 The mechanism of this reaction catalysed by multi-enzyme complex α-Ketoglutarate dehydrogenase is same as oxidative decarboxylation of pyruvate (discussed earlier under transition reaction). This enzyme complex also requires the same five cofactors viz. CoA-SH, TPP, Lipoic acid, FAD and NAD+. Succinyl coenzyme A and NADH produced in this reaction are both energy rich metabolites. 5. Substrate-level phosphorylation H2C – COO– H2C H2C – COO– + GDP + Pi H2C – COO + GTP + CoA–SH – O = C – S – CoA Succinyl Coenzyme A Succinate This reaction, catalysed by succinate thiokinase (also called succinyl coenzyme drives the phosphorylation of GDP (guanosine diphosphate) into GTP (guanosine triphosphate). A synthetase), This step provides example of substrate-level phosphorylation in mitochondria in which the substrate (succinyl CoA) directly provides energy for phosphorylation (synthesis of GTP) without an electron transport chain or proton gradient. Generation of one mole GTP in this reaction is equivalent to generation of one mole ATP because of following reasons : a. Free energies of hydrolysis of ATP or GTP are similar. GDP + H3PO4 ; ∆Go = – 30 kJ GTP + H2O b. GTP produced mostly in mammals, is readily changed into ATP by donating its terminal phosphoryl group to ADP. GTP + ADP ====== ATP + GDP c. In bacteria and plants, substrate-level phosphorylation in the mitochondria yields ATP instead of GTP. However, GTP is an important source of energy in the translational process of protein synthesis. 6. Oxidation of Succinate H2C – COO– H2C – COO HC – COO– + FAD – HC – COO Succinate + FADH2 – Fumarate Dehydrogenation of succinate to form fumarate is caused by Succinate dehydrogenase (SDH), using the coenzyme FAD. This enzyme is unique in that it is bound to inner-membrane of mitochondria as part of Succinate-Coenzyme Q reductase complex, whereas, other enzymes of TCA cycle are present in the matrix. This reaction is not sufficiently exergonic to reduce NAD+ but FAD is readily reduced to FADH2. Subsequently on oxidative phosphorylation, FADH2 produces less energy as compared to NADH. FADH2 + ½ O2 FAD + H2O + 2 ATP The enzyme SDH is inhibited by malonate as a competitive inhibitor. 14 7. Hydration of Fumarate OH HC – COO– H – C – COO– + H2O HC – COO– H2C – COO– Fumarate Malate This reaction involves the addition of H and OH from water across the double bond by fumarase, a kind of lyase also referred to as hydrolyase. 8. Regeneration of Oxaloacetate OH HC – COO– H2C – COO– Malate O + NAD+ C – COO– + NADH + H+ H2C – COO– Oxaloacetate This is the last reaction of TCA cycle in which oxaloacetate, the starting metabolite is regenerated. If we sum up all the above reactions of TCA cycle (1 to 8), it is obvious that only one molecule of acetyl coenzyme A is used up and has produced 2 CO2, 3 NADH, FADH2 and GTP (ATP). For the oxidation of one molecule of glucose two turns of TCA cycle occur and products would be 6 NADH, 2 FADH2, 4 CO2 and 2 GTP (2 ATP). SUMMARY OF TCA CYCLE 15 Significance of TCA Cycle 1. Bulk of ATP generated in respiration is derived from TCA cycle. Out of total 38 ATP produced from glucose, 24 ATP owe their production to TCA cycle. If conversion of pyruvate to acetyl CoA is also considered as part of TCA cycle, then the figure comes to 30 ATP. 2. NADH and FADH2 may directly act as reducing power (source of hydrogen) in nitrogen frixation, nitrate reduction and fatty acid biosynthesis. 3. TCA cycle provides precursors for various biosynthetic pathways. Acetyl coenzyme A is the starting substance for synthesis of fatty acids, terpenes, gibberellins, rubber and many other substances. α-Ketoglutarate, oxaloacetate and pyruvate produce the amino acids glutamate, aspartate and alanine, respectively, by reductive amination. Succinyl CoA is a precursor of synthesis of porphyrins (part of chlorophylls) and heme of hemoglobin. 4. Since TCA cycle involves several steps, it is well regulated, thus, the overall process of respiration is better controlled and energy is not wasted. Principal regulatory signals are concentration of acetyl CoA, ATP, NAD+ and NADH. The TCA cycle is turned on when NAD+/NADH and ADP/ATP ratios are higher. ATP is an inhibitor of isocitrate dehydrogenase. NADH is an inhibitor of citrate α-ketoglutarate dehydrogenase. synthase, isocitrate dehydrogenase and 16 Summary of ATP production in respiration CYTOSOL MITOCHONDRION GLYCOLYSIS MATRIX TRANSITION CRISTAE TCA CYCLE REACTION OXIDATIVE PHOSPHORYLATION Glucose 2NADH 2 Pyruvate 6 ATP 2 Pyruvate 2 ATP 2 NADH 2 Acetyl Co A 6 ATP 2 Acetyl CoA 2 CO2 6 NADH 18 ATP 2 FADH2 4 ATP 2 GTP or 2 ATP 4 CO2 The above scheme shows that * Gross ATP production = 38 per glucose. * Net ATP production = 38 – 2 = 36. Expenditure of 2 ATP is accounted for the transport of 2 NADH from cytosol to the mitochondrial cristae. * Out of 38 ATP, 36 ATP are produced in mitochondrion and 2 ATP in the cytosol. * Out of 38 ATP, 4 ATP are produced in substrate-level phosphorylation and 34 ATP in oxidative phosphorylation / electron transport chain. * Out of 38 ATP, 34 ATP are produced on cristae, 2 ATP in the matrix of mitochondrion and 2 ATP in the cytosol. * Complete oxidation of one acetyl coenzyme A produces 12 ATP or one turn of TCA cycle produces 12 ATP. * Complete oxidation of one pyruvate produces 15 ATP. 17 Electron Transport & Oxidative Phosphorylation Synthesis of ATP from ADP and inorganic phosphate (Pi) during the oxidation of NADH or FADH2 is called oxidative phosphorylation. This process is accompanied with electron transport from NADH/FADH2 to oxygen. NADH/FADH2 is the source of protons plus electrons and oxygen is the ultimate acceptor. NADH + H+ NAD+ + 2 H+ + 2e– 2H+ + 2e– + ½O2 H 2O 3 ADP + 3 Pi 3 ATP NADH+ + ½ O2 + 3ADP + 3 Pi NAD+ + H2O + 3 ATP The process is too complicated as it involves a series of redox systems (electron carriers) through which electrons are transferred from NADH to oxygen. These redox systems are bound to inner membrane of mitochondria. The basis of transport of electrons from one substance to another is the difference between their redox potentials. It occurs from low reduction potential to higher i.e. more -ve to less -ve values. Redox Potential It is also called reduction potential or oxidation-reduction potential, expressed as Eo, (volts). Reduction potential is a measure of ability of a substance to give electron or its ability to accept electron. In studying energy changes in oxidation reduction reactions, data are often obtained by looking at the electrode potential generated when a half cell containing unit concentrations of oxidised and reduced forms of a substance is compared to a standard hydrogen-cell. Suppose a half cell containing 1 M oxidised form of a substance (X+) and 1 M reduced form of the same substance (X–) is connected to a standard half cell i.e. H-half cell through a voltmeter and an agar bridge, then i. If electrons move from X+/X– cell to H2/H+ cell, the substance X is said to have -ve redox potential (Eo volts). More -ve the value of Eo more is the tendency of substance X to be oxidised i.e. to transfer electrons to other species. Such a substance is said to have low reduction potential but high oxidation potential. ii. If elecrons move from H2/H+ cell to the X+/X– cell, then the substance X is said to have +ve redox potential. Such a substance has higher tendency to get reduced i.e. to accept electrons from other species. For example E’o for NAD+/NADH is –0.32 V, and E’o for ½ O2/H2O is +0.82 volts. This indicates that electrons readily flow from NADH to oxygen, which is designated as downhill electron transport. No energy is gained in downhill electron transport, rather energy is released. 18 Electron Transport Chain The scheme for electron transport from NADH/FADH2 to oxygen in oxidative phosphorylation is given as follows : FeS : Iron-sulphur Centres CoQ : Coenzyme Q or Ubiquinone (UQ) Cyt : Cytochrome, a protein containing a heme group (Fe) The standard free energy change (∆Go) during electron transfer from one electron carrier to the next may be calculated as, ∆Go = – n F ∆E’o where, n = no. of electrons transferred F = Faraday’s number (96500 Coloumbs) ∆E’o = Difference in redox potentials of two electron carriers. In the given ETC, three steps are shown to produce ATP. ATP is not synthesized directly. This scheme only indicates the steps where free energy change is sufficient to synthesize ATP i.e. at each of the step given above the energy released is more than 32 kJ. Various components of electron transport chain are arranged into 4 complexes. I. NADP - Coenzyme Q reductase This complex comprises NADH dehydrogenase, FMN and FeS centre. Two protons and two electrons from NADH + H+ are transferred to FMN, which gets reduced to FMNH2. Electrons are then transferred from FMNH2 to coenzyme Q (CoQ), also called ubiquinone (UQ) which gets reduced to CoQH2. During this process 2H+ are transferred from the matrix side of inner membrane of mitochondrion to the intermembrane space. II. Succinate - Coenzyme Q reductase It includes the membrane bound enzyme of TCA cycle, Succinate dehydrogenase, FAD and FeS centre. Two hydrogens removed from succinate reduce FAD to FADH2. From FADH2 the electrons are transferred to CoQ via FeS centre and CoQ is reduced toCoQH2. 19 III. Cytochrome c reductase It consists of Cytochrome b, Rieske FeS centre and Cytochrome c1. Electrons donated by CoQH2 are transferred through cyt b, FeS centre and cyt c1 to cytochrome c, which gets reduced. 4 H+ are transferred from matrix to intermembrane space. Like CoQ, cytochrome c is a mobile electron carrier and is not the part of any complex mentioned here. IV. Cytochrome c oxidase Simply called cytochrome oxidase, it includes cytochrome a, cytochrome a3 and two copper ions named α and β. Electrons are received from cytochrome c (which gets oxidised). Electrons are ultimately transferred to oxygen to form water. This step is called terminal oxidation. 2 Reduced Cyt a3 + 2 H+ + ½ O2 2 Oxidised Cyt a3 + H2O Water synthesized in respiration is called metabolic water. During oxidative phosphorylation involving NADH2, electrons are transferred as : NADH2 Complex I CoQ Complex III Cyt C Complex IV Oxygen During oxidative phosphorylation involving FADH2, electrons are transferred as : Succinate Complex II CoQ Complex III Cyt C Complex IV Oxygen Following is the route of electron transport during oxidation of NADH and FADH2. Succinate Complex II Malate NADH + H+ Complex I CoQ Complex III Cyt C Complex IV Oxygen Synthesis of ATP In aerobic respiration, bulk of ATP (34 ATP out of 38 ATP per mole glucose) is synthesized as a result of electron transport chain. The exact mechanism of ATP synthesis is very complex. Engelhardt in 1930 visualised that ATP is synthesized as a result of electron transport. By 1940, Severo Ochoa deduced that the number of ATP molecules produced per atom of oxygen or per pair of electrons transported is 3 for NADH and 2 for FADH2. The exact mechanism in this regard is chemiosmotic hypothesis given by Peter Mitchell. According to this hypothesis, electron transport is coupled with proton transport from matrix to intermembrane space of mitochondria building up a proton gradient, which is then used to synthesize ATP. Figure given below is structure of Mitochondria. 20 F0 - F1 ATPase Complex Electron microscopic studies of mitochondria revealed that small particles (~ 8 nm diameter) present on the cristae and projected in the matrix, are the sites of ATP synthesis. These particles are called F0-F1 ATPase or ATP synthase complexes. These Fo-F1 (named after Fernandez-Moran) particles were earlier called oxysomes or elementary particles. Now the term couplers is also used to these particles. Inner membranes of mitochondria from which these particles have been removed, can carry electron transport but ATP is not synthesized. ATP synthase consists of two parts F0 and F1. F0 is embedded in the inner membrane and consists of three hydrophobic subunits a, b and c. It forms a channel through which protons move from intermembrane space to matrix. The F1 consists of five polypeptide chains α, β, γ, δ, and ε. F1 has a short stalk joined to Fo and a headpiece protruded into the matrix. Catalytic site for the synthesis of ATP lies in the β subunit. Proton Gradient and Synthesis of ATP The mitochondrion has two membrane bound spaces. One, the intermembrane space between outer and inner membranes, and second, the matrix enclosed by the inner membrane. Thus, the inner membrane has two faces — one facing the matrix called n-side (n for negative) and the other facing outer membrane is called p-side (p for positive). The four redox complexes of ETC along with F0-F1 ATPase are embedded in the inner membrane. Chemiosmotic synthesis of ATP occurs as follows : 1. There is uphill proton transport form matrix of mitochondria to the intermembrane space accompanied with downhill electron transport. In other words, active transport of protons occurs across the membrane which is otherwise impermeable to protons. Energy for this active transport of protons is derived from the coupling with electron transport. Thus, the flow of electrons in ETC is not used to directly synthesize ATP, but used to transport protons uphill i.e. from their low to higher concentration. Figure given below is the electron transport scheme accompanied with uphill proton transport given by William G. Hopkins (1999). 21 2. As a result of above mentioned coupling of downhill electron transport and uphill proton transport, a proton gradient is established across the membrane i.e. there is high concentration of protons in the intermembrane space as compared to the matrix. Since the membrane is impermeable to protons, their diffusion back into matrix is prevented. The proton gradient established is also referred to as proton motive force (PMF). This force is due to membrane potential i.e. potential difference on two sides of the inner membrane. 3. Next, the PMF is used to drive the synthesis of ATP. As the protons move from the intermembrane space back to the matrix through F0-F1 ATPase complex, ATP is synthesized from ATP and H3PO4, with the help of ATP synthase. How does the flow of H+ drive synthesis of ATP ? It is proposed that ADP and Pi bind to ATP synthase to form ATP, but ATP does not leave the site unless protons flow through the ATP synthase. According to Paul Boyer, proton flow through Fo drives the rotation of γ subunit of F1 head. This facilitates the release of ATP and its synthesis. The figure below shows Chemiosmotic synthesis of ATP. Some Inhibitors of Oxidative Phosphorylation OUTER MEMBRANE INTERMEMBRANE SPACE INNER MEMBRANE MATRIX Rotenone (an insecticide), several barbiturates such as amytal and pain-killers like demerol inhibit NADH-CoQ reductase to block electron transport. Cyanide, azide and carbon monoxide inhibit cytochrome c oxidase and block electron transport. Oligomycin (an antibiotic) inhibits ATP synthesis by blocking the flow of protons through F0. Some inhibitors of oxidative phosphorylation such as 2,4-Dinitrophenol and dicumarol affect ATP synthesis but do not affect electron transport. Such inhibitors are called uncouplers because they disrupt the tight coupling 22 between electron transport and development of proton gradient. Uncouplers destroy proton gradient by allowing protons to diffuse through inner membrane. In the presence of uncouplers, since electron transport continues without ATP synthesis, lot of heat is generated. Transport of ATP ATP synthesized in mitochondria has to be transported to the cytosol. An adenine nucleotide transporter located on the inner membrane exchanges ADP and ATP. Outer membrane of mitochondrion is permeable to most of the substances.. There is also an inorganic phosphate translocator, which exchanges H2PO4– with OH– ions. Shuttle Systems The inner membrane of mitochondrion is impermeable to NADH. Shuttle systems are transport mechanisms to transfer cytoplasmic NADH to the electron transport chain inside mitochondria. A shuttle system will carry NADH from cytosol to matrix with a very little or no expenditure of energy. There are two shuttle systems for this purpose. 1. Glycerophosphate shuttle NADH produced during glycolysis in the cytosol is used to reduce dihydroxyacetone phosphate to glycerol phosphate. Glycerophosphate is oxidised back to DHAP in the mitochondrial membrane, reducing FAD to FADH2 which enters ETC yielding 2 ATP. So, there is a net use of one ATP to transport NADH from cytosol to matrix. 2. Malate-Aspartate shuttle This shuttle is reversible i.e. NADH can be transported from cytosol to matrix and from matrix to cytosol without using any ATP. This shuttle is based on the fact that malate and aspartate are readily permeable through the inner membrane of mitochondria. In the cytosol NADH is used to convert oxaloacetate into malate. Malate enters the matrix where it changes into oxaloacetate and NADH is produced. 23 Cyanide Resistant Respiration Cyanide (CN–) is a potent inhibitor of Cytochrome c oxidase. In animals, cyanide completely inhibits respiratory O2 uptake. However, many plants such as spinach, pea, arums, lilies and others show considerable resistance to cyanide ions. This cyanide resistant respiration is attributed to an alternate oxidase and the pathway is commonly called alternate respiratory pathway. The alternate oxidase is also called Coenzyme Q : O2 Oxidoreductase. Electrons from CoQ (ubiquinone) are transferred to cytochrome oxidase and then to oxygen bypassing complex III and complex IV. Here, less ATP is synthesized and a large amount of energy is released as heat. Thus, electrons transferred from NADH would produce only one ATP whereas, electrons transferred from FADH2 produce none. Complex II Cyanide ions 2e– NADH 2e– Complex I 2e– CoQ Complex III Cyt c Complex IV No O2 reduction 2 e– Alternate oxidase 2e– + 2 H+ + ½ O2 H 2O Carbon monoxide and azide also do not affect O2 uptake in plants. One possible role of cyanide resistant respiration is thermogenesis i.e. heat generation. In Symplocarpus foetidus (skunk cabbage) the inflorescence produces heat to the extent that its temperature rises as much as 10oC above the ambient. High temperature volatilises some amines so that an odour is produced to attract pollinators. Another advantage of this pathway is to burn off the excess of carbohydrates. 24 Pentose Phosphate Pathway (PPP) It is an alternate mechanism of oxidation of glucose-6-phosphate bypassing glycolysis and TCA cycle. That is why the process is also called Hexose Monophosphate Shunt (HMS). The entire process is completed in the cytoplasm. It begins with dehydrogenation of glucose-6-phospphate by the enzyme glucose-6-phosphate dehydrogenase needing NADP+ as hydrogen acceptor instead of NAD+. The product. 6-phosphoglucono-lactone is then hydrolysed by the lactonase into 6-phosphogluconate. Next, oxidative decarboxylation of 6phopshogluconate by 6-phosphogluconate dehydrogenase yields ribulose-5-phosphate. NADP+ is again the hydrogen acceptor. 6-phoshogluconate COOH─C─OH CH2OH HO─CH H─C─OH C=O + NADP+ H─C─OH H─C─OH H─C─OH CH2─OPO326-phosphogluconate + NADPH + H+ + CO2 CH2─OPO32Ribulose-5-phosphate This process is regulated by the concentration of NADP+ as it has to compete with NAD+ as H-acceptor. The enzymes have a relatively low Km for NADP+ implying that NADP+ is the H-acceptor rather than NAD+. Ribulose-5-phosphate is converted into its isomers, ribose-5-phosphate and xylulose-5-phosphate. Various reactions among these sugars lead to the regeneration of glucose-6-phosphate, with a net release of 12 NADPH and 6CO2 at the cost of one molecule of glucose-6-phosphate. 25 Summary of PPP Following are the various steps of Pentose Phosphate Pathway : 6 Glucose-6-P + 6 NADP 6 Phosphogluconate + 6 NADPH2 6 Phosphogluconate + 6 NADP 6 Ribulose-5-P + 6 NADPH2 + 6 CO2 6 Ribulose-5-P 2 Ribose-5-P + 4 Xylulose-5-P 2 Ribose-5-P + 2 Xylulose-5-P 2 Sedoheptulose-7-P + 2 PGAL 2 PGAL + 2 Sedoheptulose-7-P 2 Erythrose-4-P + 2 Fructose-6-P 2 Xylulose-5-P + 2 Erythrose-4-P 2 Fructose-6-P + 2 PGAL 2 PGAL (PGAL + DHAP) Fructose-6-P 5 Fructose-6-P 5 Glucose-6-P Glucose-6-P 12 NADPH2 + 6 CO2 26 Significance of PPP . This pathway occurs both in plants and animals. Its significant features are as follows : 1. This is an alternative to TCA cycle. NADPH can undergo oxidative phosphorylation to generate 3ATP. 2. The pathway generates reducing power in the form of NADPH, which serves as electron and proton donor for N2-fixation, fatty acid biosynthesis, nitrate reduction and other processes. 3. Pentose phosphates, such as ribose-5-phosphate produced in this pathway, serve as precursors for the synthesis of nucleotides (needed for the synthesis of ADP, ATP, DNA, RNA etc.). 4. In plants, erythrose-4-phosphate, an intermediate of PPP is a precursor for the biosynthesis of lignin, flavonoids and aromatic amino acids. Following is the cyclic representation of PPP 27 Fats as Respiratory Fuels Oils & fats are the reserve foods in plants and animals. Fat or oil is an ideal reserve food because it produces large amount of energy in oxidation. Thus, more energy can be stored in a small space. A 10-C fatty acid (decanoic acid) having equivalent molecular mass as that of glucose yields 78 ATP on complete oxidation, as compared to 36 ATP generated by glucose. Fat + 3 H2O Lipase Glycerol + 3 Fatty acids. Glycerol enters glycolysis as it is converted into DHAP (dihydroxyacetone phosphate). Fatty acids enter into mitochondria for further oxidation. Breakdown of Fatty acids The most common pathway for fatty acid breakdown is β-oxidation. It is a complex process, at the end of which the fatty acid is shortened by two carbons and one molecule of acetyl coenzyme A, one molecule of FADH2 and one molecule of NADH are produced. Ultimately a fatty acid with 2n carbons produces n Acetyl CoA, (n-1) FADH2 and (n-1) NADH2 since a total (n-1) β-oxidations take place. However, as usual, activation of a fatty acid is necessary before β-oxidation occurs. Activation of Fatty acid O ATP CH3 – (CH2)n – CH2 – CH2 – C – OH + HS – CoA Fatty acid O AMP + PP Coenzyme A CH3 – (CH2)n CH2 – CH2 – C – S – CoA Fatty acyl Coenzyme A This reaction is catalysed by Fattyacyl coA synthetase which needs hydrolysis of ATP. Since ATP is hydrolysed into AMP & Pyrophosphate rather than ADP & P1, energy worth 2 ATP is considered to be used in the activation process. To complete the ATP cycle AMP has to change into ADP by using ATP, before ATP can be synthesized from ADP. ATP AMP + PP AMP + ATP 2 ADP 2 ATP 2 ADP + 2Pi 28 β-Oxidation of Fattyacyl coenzyme A The process involves oxidation, hydration, oxidation and thiolysis. Following is the summary of one turn of β-oxidation : O CH3 – (CH2)n – CH2 – CH2 – C – S – CoA Fattyacyl coenzyme A FAD OXIDATION FADH2 O CH3 – (CH2)n – CH = CH – C – S – CoA Enoyl coenzyme A H2O HYDRATION HO O CH3 – (CH2)n – CH – CH2 – C – S – CoA NAD+ OXIDATION O Hydroxyacyl coenzyme A O NADH+H+ CH3 – (CH2)n – C – CH2 – C – S – CoA THIOLYSIS CoA–SH O CH3 – (CH2)n – C – S – CoA Fattyacyl coenzyme A Ketoacyl coenzyme A O + CH3 – C – S – CoA Acetyl coenzyme A (shortened by 2-C) NADH & FADH2 enter ETC to produce respectively, 3 & 2 ATP. Acetyl CoA enters TCA cycle producing 12 ATP. The fattyacyl CoA shortened by 2-C undergoes further turn of β-oxidation, and so on. ATP generated by Palmitic acid (a 16-C fatty acid) * No. of β-oxidations = 7 * Products of one β-oxidation =Acetyl Coenzyme A+ FADH2+ NADH + 14–C Fattyacyl coenzyme A * Products of 7 β-oxidations = 8 Acetyl CoA + 7 FADH2 + 7 NADH * ATP produced = (8 × 12) + (7 × 2) + (7 × 3) = 131 * Energy consumed in activation of palmitic acid = 2 ATP * Energy produced by oxidation of palmitic acid = 129 ATP 29 Thus, fatty acid with 2n carbons will yield ATP molecules, which are equal to 12n + 5 (n-1) – 2. For example, ATP produced by decanoic acid would be, (12 × 5) + 5 (4) – 2 = 78 ATP : The energy currency of cells In cells flow of energy is mediated by some reduced coenzymes (NADH and FADH2) and high energy phosphate molecules with unhydride phosphoryl groups,such as ATP, ADP, Phosphoenol pyruvate, Acetyl phosphate etc. However, ATP is most ideal molecule for energy transduction. Energy is harvested during ATP synthesis and released when ATP is hydrolysed. ADP + Pi ATP + H2O Energy Energy ATP + H2O ; ∆Go = 30.5 kJ/mole ADP + H2PO4– + H+ ; ∆Go = – 30.5 kJ/mole Structure of ATP ATP is a conjugate of ribose sugar, adenine (linked C-1 of ribose) and triphosphate (linked to C-5 of ribose). It is a higher nucleotide with two energy rich phosphate bonds, indicated as “wriggle bonds”. Phosphate linkage with adenosine (ribose + adenine) is not considered energy rich because energy released in hydrolysis is less. AMP Adenosine + Pi ; ∆Go = – 9.2 kJ/mole Another important aspect of ATP hydrolysis is its irreversibility, because * The hydrolysis reaction is exergonic (∆Go = – 30.5 kJ/mole) * Entropy of hydrolysis products increases, as kinds of products are more than kinds of reactants. * Sites of synthesis of ATP and utilization of ATP are generally different. 30 ATP is most suitable molecule to serve an energy currency for three reasons. * ATP is relatively stable for short term energy storage. * Energy either needed for ATP formation or energy obtained on hydrolysis to ADP is neither too high nor too low but within the range of energies needed for most of the biosynthetic steps. * AMP and ADP needed for the synthesis of ATP are available in plenty in all cells as these are the metabolites for the synthesis of nucleic acids. There are three ways of ATP synthesis 1. Photophosphorylation Synthesis of ATP in the photochemical reaction of photosynthesis when electrons are transported from H2O to NADP+ via several electron carriers, is called photophosphorylation. Light H2O + ADP + Pi + NADP+ NADPH + H+ + ATP + ½ O2 Chloroplast 2. Oxidative phosphorylation Synthesis of ATP during oxidation of NADH/FADH2 in the presence of oxygen is called oxidative phosphorylation. Here, electrons are transported from NADH/FADH2 to oxygen, resulting in the synthesis of ATP. NADH + H+ + ½ O2 + ADP + Pi Mitochondria NAD+ + H2O + ATP 3. Substrate-level phosphorylation Synthesis of ATP without involving electron transport is called substrate-level phosphorylation. In this case an energy rich phosphoryl group is shifted from a metabolite (substrate) to ADP to form ATP. Phosphoenol pyruvate (PEP) + ADP Pyruvate + ATP The average adult human produces / consumes about 65 kilogram ATP per day. However, the body weight does not increase because of ATP cycle. ATP is only transient energy carrier. The body has only about 50g ADP and ATP. The ratio of ATP production to O2 (P/O ratio) For reach molecule of NADH or FADH2 that is oxidised, one atom of molecular oxygen is reduced to water. P/O ratio expresses efficiency of oxidative phosphorylation. It is the number of ATPs produced per oxygen atom used. Experiments showed that for every NADH oxidised to NAD, 2 to 3 ATP are synthesized. For every FADH2 oxidised to FAD, 1.5 to 2 ATP are synthesized. The free energy release in oxidation of NADH is 224.5 kJ/mole, which is sufficient to synthesize 3 ATP. 31 However, the latest agreement is that 10 H+ are transported to intermembrane space every 2e– passed from NADH to oxygen, and 4 H+ are transported into the matrix for every ATP synthesized, so that P/O ratio is 10/4 or 2.5. Similarly P/O ratio when FADH2 is oxidised is 6/4 or 1.5. Thus, a total of 2.5 ATP per NADH and 1.5 ATP per FADH2 may be produced through the process of electron transport chain. The gross ATP production from glucose is therefore, 32 ATP instead of 38 ATP. Glucose on complete oxidation produces, 2 ATP, 10 NADH, 2 FADH2 and 2 GTP which sums as 2 + (10 × 2.5) + (2 × 1.5) + 2 i.e. 32 ATP. Net gain of ATP is 30 ATP if glycerophosphate shuttle operates or 32 ATP if malate-aspartate shuttle operates for transport of NADH from cytosol to mitochondria. Respiratory Quotient (R.Q.) It is the ratio of volume of CO2 evolved to volume of O2 absorbed in respiration. Volume of CO2 evolved RQ = Volume of CO2 consumed or Moles of CO2 evolved Moles of CO2 consumed RQ values indicate the nature of respiratory substrate being respired. Ratio of carbon to oxygen Respiratory Substrate RQ Volume of CO2 & O2 Carbohydrates 1 Vol. of CO2 = Vol. of O2 C to 0 = 1 Fats & Proteins <1 Vol. of CO2 < Vol. of O2 C to O > 1 Organic acids >1 Vol. of CO2 > Vol. of O2 in the substrate C to O < 1 Experimentally, RQ value obtained for a certain plant part is an average value as more than one type of substrates be respired at one time. Sometimes, even if the substrate is carbohydrate, observed RQ value may be less than one, as oxygen may be used elsewhere (like, oxidation of phenols). Similarly, despite carbohydrate a respiratory substrate, RQ value may be more than one if fermentation is also taking place, where oxygen is not used up but CO2 is given out. In succulent plants (CAM plants), RQ value (normally calculated in dark) may be zero or very less as in these plants CO2 is assimilated in dark and changes into malic acid. RQ values are often less than one in starving plants or plant tissues. Normally, carbohydrates are the first choice as respiratory fuels, but as these are exhausted, fats and then proteins are used in respiration. B. Anaerobic Respiration This process, also called anaerobiosis is the incomplete oxidation of food without using oxygen. Anaerobic respiration is equivalent to fermentation in relation to the product formed. For example, alcoholic fermentation is the mode of anaerobic respiration in which alcohol is the end product. Two well known processes of fermentation are : 32 i. Alcoholic fermentation It occurs in plants and some microbes like yeast. Here, glucose is converted into ethanol and CO2. C6H12O6 2 C2H5OH + 2 CO2 + 2ATP ii.Lactic acid fermentation In animal tissues and several microbes like Lactobacter, glucose is changed into lactic acid. C6H12O6 CH3CH – COOH + 2 ATP OH glucose lactic acid Following are the features of anaerobic respiration in contrast to aerobic respiration : * * * * * * Oxygen is not used. Anaerobic respiration is considered intramolecular respiration. Oxidation of food is incomplete i.e. the product is ethanol/lactate/butyrate/acetate instead of H2O & CO2. Less CO2 or no CO2 is produced. In alcoholic fermentation, 2 moles CO2 are produced per glucose as compared to aerobic respiration, where 6 moles CO2 are produced per mole of glucose. No CO2 is produced in lactic acid fermentation. Less energy is produced. In anaerobic respiration 2 ATP per glucose molecule are produced as compared to 36 ATP per glucose molecule in aerobic respiration. Pyruvate produced at the end of glycolysis is not oxidised into H2O & CO2 but reduced to certain substances, which are not further oxidised. Oxidative phosphorylation and electron transport do not take place; consequently, mitochondria are not needed. Anaerobic respiration has two forms : 1. Obligate anaerobiosis This refers to life without oxygen. Here, oxygen is of no use to the organism. Fermentation occurs whether oxygen is available or not. Examples ─ Clostridium, Lactobacter. 2. Facultative anaerobiosis In this kind of respiration, organisms or tissues respire aerobically in the presence of oxygen but when there is oxygen stress or absence of oxygen, anaerobic respiration occurs. Example, alcoholic fermentation in plants and yeast and lactic acid fermentation in skeletal muscles. Germinating seeds or yeast respire aerobically in the presence of oxygen but if oxygen is eliminated, alcoholic fermentation occurs. Following figure demonstrates anaerobic respiration in germinating seeds 33 In the left side tube, as there is no oxygen, anaerobic respiration occurs. No oxygen is used but CO2 is released. Due to the development of gas pressure, level of mercury falls, as shown in the right side tube. That the space in this inverted tube contains CO2 can be tested by inserting a pellet of KOH into the tube. As KOH absorbs CO2 the level of mercury goes up in the tube In human muscles, so far oxygen is present adequately, aerobic respiration occurs to produce enough energy. As the oxygen availability declines anaerobic respiration occurs to produce lactic acid. White muscles can do work vigorously for a short time for which aerobic respiration occurs. After some time anaerobic respiration sets resulting in fatigue and pain. However, in red muscles, myoglobin prolongs availability of oxygen so the muscles are capable of doing strenuous work for a long time. In practice, athletes with well-developed red muscles are suited for long term events like marathon. Those with well-developed white muscles are only fit to do short term events such as 100 meter race, discus throw. Yeast carries alcoholic fermentation only when oxygen is not available. If oxygen is available, anaerobic respiration is shifted to aerobic breakdown, although the carbohydrates decrease. This effect of inhibition of alcoholic fermentation by oxygen is called Pasteur effect. Mechanism of Anaerobic Respiration (Alcoholic & Lactic acid fermentation) Glycolysis is common to aerobic and anaerobic respiration. Fate of pyruvate and also the fate of NADH is different in anaerobic respiration. In alcoholic fermentation, pyruvate is reduced by NADH into ethanol to regenerates NAD+ (needed in glycolysis). In lactic acid fermentation, pyruvate is reduced to lactate. The entire process occurs in cytosol. First, glycolytic breakdown of glucose occurs as follows. C6H12O6 2ATP Fructose-1, 6-diphosphate 2 Phosphoglyceraldehyde 2NAD+ +2H3PO4 2 Diphosphoglyceric acid 2NADH2 1,3-Diphosphoglycerate 4ATP 2 Pyruvic acid / Pyruvate Reduction of Pyruvic acid In alcoholic fermentation, as in yeast, pyruvate is converted into ethanol and CO2. NADH+H+ NAD+ O CH3 – C – COOH Pyruvic acid CO2 CH3CHO Acetaldehyde CH3CH2OH Ethanol In the above scheme, the first step is catalysed by pyruvate decarboxylase and the second step by alcoholic dehydrogenase. 34 In lactic acid fermentation, as in animal tissues, pyruvate is converted into lactate in a single step and no carbondioxide is released. O NADH+H+ OH NAD+ CH3 – C – COOH Pyruvic acid CH3 – CH – COOH Lactic acid In animal tissues experiencing anaerobic condition, when all the stored oxygen in used up then cramps and muscle fatigue occur which are associated with the build up of lactic acid in muscles. Most of this acid is transported by blood to the liver where it is synthesized into glucose. In anaerobic respiration, the two ATP produced during glycolysis account for the sole production of ATP as no ATP is produced during the breakdown of pyruvate, rather, an energy rich molecule, NADH is consumed. Factors affecting respiration in plants Rate of respiration changes very often due to so many factors, some of these are briefly mentioned below with special reference to plants. 1. Availability of respiratory fuels Supply of monosaccharides such as glucose, increases rate of respiration. Glucose is referred to as instant energy food. Starvation certainly lowers respiration and even leads to protoplasmic respiration, where proteins and amino acids are used in respiration. 2. Temperature With increase in temperature from 5oC to 30oC, the rate of respiration rises exponentially with Q10 value approximately equal to two. rate at (t+10)o C Q10 (temperature coefficient) = rate at to C Q10 is the measure the number of times the rate increases when the temperature is increased by 10oC. Generally, when temperature is increased from 15oC to 25oC, the rate of respiration doubles. Change in temperature basically affects the activity of enzymes. With increase in temperature kinetic energy of various metabolites increases but the stability of enzymes decreases. Also, with an increase in temperature, solubility of oxygen in the cell sap decreases. 3 Oxygen Oxygen is the terminal acceptor of electrons in oxidative phosphorylation. Since cytochrome c oxidase has a high affinity for oxygen (with very low Km), it is rarely a limiting factor. Plants experience oxygen-deficit when flooded with water. With decrease in concentration of oxygen more fermentation occurs. At higher concentration of oxygen rate of fermentation decreases. In facultative anaerobes, anaerobic respiration is shifted to aerobic in the presence of oxygen (Pasteur effect). This spoils the process of wine or beer production. The concentration of oxygen at which respiration may not be observed is called extinction point. 4. Hydration Since enzymes form a colloidal system, the protoplasm must be well hydrated for respiration to take place. Rate of respiration decreases when there is water stress. In dry seeds, the rate of respiration is negligible. When seeds are stored in moist conditions, they may lose their viability. This is due to respiration by microbes, insects and seeds to generate enough heat to inactivate the seeds. 35 5. Kind of plant material Rate of respiration is high in growing systems but very low in storage organs and dormant systems. Rate of respiration is high in shoot apices, flowers, leaves and germinating seeds. In tubers, corms and tuberous roots rate of respiration is low due to poor penetration of oxygen. 6 Light Although, respiration is not affected by light, in green plants the rate of the process is more in light than in darkness. This effect in due to heat rays or due to production of sugars in photosynthesis. The light intensity at which the rate of photosynthesis is just equal to the rate of respiration is called light compensation point. In C3 plants, the rate of overall respiration in light may be affected by photorespiration. In photorespiration, CO2 is evolved by chlorophyllous cells in the presence of light and oxygen. When CO2 level is low or oxygen level is high, the enzyme ribulose biphosphate carboxylase combines ribulose biphosphate with oxygen (instead of CO2) to form glycolate, which later releases CO2. Thus, rapid photorespiration apparently increases respiration in terms of consumption of O2. 7. CO2 concentration Normally CO2 does not affect respiration but a relatively higher level of CO2 depresses respiration. At higher concentration of CO2 stomata close in plants even in the presence of light. Sugars accumulate in potato tissue if stored at high concentration of CO2. This principle of retardation of respiration by increasing CO2 level has been utilized for prolonged storage of fruits. 8. Wounding / Injury Wounding of plant parts accelerates respiration. This so-called fever respiration has been remarkably observed in potato, carrot, beet, onion, radish and leaves of Liriodendron. The mechanism of stimulation of respiration by wounding is not well understood. This is attributed to hormonal production during injury and healing process that follows. The plant hormone, indole acetic acid, is known to accelerate rate of respiration. 9. Age Normally rate of respiration decreases with age. However, in ripening fruits rate of respiration increases tremendously. This high rate of respiration when it is at its peak is called respiratory climacteric. This rapid increase in rate of respiration during ripening is attributed to the production of ethylene. The respiratory climacteric may be due to ethylene-stimulated hydrolysis of starch into simple sugars which fuel respiration. Exogenously applied ethylene can enhance respiratory climacteric in Climacteric fruits - those fruits which experience respiratory climacteric during ripening, such as mango, banana and others. Ethylene can induce respiratory climacteric in Non-climacteric fruits - those fruits which do not show respiratory climacteric, such as orange, grapes and some others. Respiratory Climactericate Rate of respiration Ripeneing phase Time//Age of 36 10. Inhibitors / Respiratory poisons There are numerous substances, which inhibit respiration. Enzyme inhibitors such as heavy metals (Ag, Hg and Pb) and acetoamide inhibit respiration. Cyanide, azides, carbon-monoxide, 2,4-dinitrophenol, rotenone, fluoroacetate and many others are potent inhibitors of respiratory oxidative phosphorylation. Several fungal toxins and antibiotics also inhibit respiration. References 1. Berg, J. M., Tymoczko, J. K. and Stryer L., 2002. Biochemistry. W. H. Freeman and Company. 2. Cross, R. L., 1994. Our primary source of ATP. Nature. 370 : 594 - 595. 3. Darnell, J. Lodish H. and Baltimore, D., 1986. Molecular Cell Biology. Scientific American Books. 4. Garret, R.H. and Grisham, C.M., 1995. Biochemistry. Saunders College Publishing. 5. Hopkins, William G., 1999. Introduction to Plant Physiology. John Wiley and Sons, Inc. 6. Karpe, G., 2002. Cell and Molecular Biology : Concepts and Experiments. John Wiley and Sons, Inc. 7. Kilgour, G. L., 1981. Fundamentals of Biochemistry. D. Van Nostrand Company. 8. Lea, P. J. and Leegood, R.C., 1993 (Edited). Plant Biochemistry and Molecular Biology. John Wiley and Sons. 9. Sparks, S., 1997. The purpose of glycolysis. Science 277 : 459 - 460.