Fatty Acid Catabolism PDF

FATTY ACID CATABOLISM The oxidation of long-chain fatty acids to acetyl-CoA is a central energy-yielding pathway in many organisms and tissues. In mammalian heart and liver, for example, it provides as much as 80% of the energetic needs under all physiological circumstances. The electrons rem...

FATTY ACID CATABOLISM The oxidation of long-chain fatty acids to acetyl-CoA is a central energy-yielding pathway in many organisms and tissues. In mammalian heart and liver, for example, it provides as much as 80% of the energetic needs under all physiological circumstances. The electrons removed from fatty acids during oxidation pass through the respiratory chain, driving ATP synthesis; the acetyl-CoA produced from the fatty acids may be completely oxidized to CO2 in the citric acid cycle, resulting in further energy conservation. In some species and in some tissues, the acetyl-CoA has alternative fates. In liver, acetyl-CoA may be converted to ketone bodies—water-soluble fuels exported to the brain and other tissues when glucose is not available. In higher plants, acetyl-CoA serves primarily as a biosynthetic precursor, only secondarily as fuel. Although the biological role of fatty acid oxidation differs from organism to organism, the mechanism is essentially the same. The repetitive four-step process, called β oxidation, by which fatty acids are converted into acetyl-CoA. As hormone-sensitive lipase hydrolyzes triacylglycerol in adipocytes, the fatty acids thus released (free fatty acids, FFA) pass from the adipocyte into the blood, where they bind to the blood protein serum albumin. This protein (Mr 66,000), which makes up about half of the total serum protein, noncovalently binds as many as 10 fatty acids per protein monomer. Bound to this soluble protein, the otherwise insoluble fatty acids are carried to tissues such as skeletal muscle, heart, and renal cortex. In these target tissues, fatty acids dissociate from albumin and are moved by plasma membrane transporters into cells to serve as fuel. About 95% of the biologically available energy of triacylglycerols resides in their three long-chain fatty acids; only 5% is contributed by the glycerol moiety. The glycerol released by lipase action is phosphorylated by glycerol kinase (Fig. 17–4), and the resulting glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate. The glycolytic enzyme triose phosphate isomerase converts this compound to glyceraldehyde 3-phosphate, which is oxidized via glycolysis. Fatty Acids are Activated and Transported into Mitochondria The enzymes of fatty acid oxidation in animal cells are located in the mitochondrial matrix, as demonstrated in 1948 by Eugene P. Kennedy and Albert Lehninger. The fatty acids with chain lengths of 12 or fewer carbons enter mitochondria without the help of membrane transporters. Those with 14 or more carbons, which constitute the majority of the FFA obtained in the diet or released from adipose tissue, cannot pass directly through the mitochondrial membranes— they must first undergo the three enzymatic reactions of the carnitine shuttle. The first reaction is catalysed by a family of isozymes (different isozymes specific for fatty acids having short, intermediate, or long carbon chains) present in the outer mitochondrial membrane, the acyl-CoA synthetases, which promote the general reaction. Fatty acid + CoA + ATP fatty acyl–CoA + AMP + PPi Thus, acyl-CoA synthetases catalyse the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl–CoA, coupled to the cleavage of ATP to AMP and PPi. The reaction occurs in two steps and involves a fatty acyl–adenylate intermediate. Fatty acyl–CoAs, like acetyl-CoA, are high-energy compounds; Entry of glycerol into the glycolytic pathway. their hydrolysis to FFA and CoA has a large, negative standard free- energy change (ΔG° ≈ 31 kJ/mol). The formation of a fatty acyl–CoA is made more favourable by the hydrolysis of two high-energy bonds in ATP; the pyrophosphate formed in the activation reaction is immediately hydrolysed by inorganic pyrophosphatase, which pulls the preceding activation reaction in the direction of fatty acyl–CoA formation. The overall reaction is – Fatty acid + CoA + ATP fatty acyl–CoA + AMP 2Pi ΔG° = –34 kJ/mol  Fatty acyl–CoA esters formed at the cytosolic side of the outer mitochondrial membrane can be transported into the mitochondrion and oxidized to produce ATP, or they can be used in the cytosol to synthesize membrane lipids. Fatty acids destined for mitochondrial oxidation is transiently attached to the hydroxyl group of carnitines to form fatty acyl–carnitine— the second reaction of the shuttle. This transesterification is catalysed by carnitine acyltransferase I (Mr 88,000), in the outer membrane. Either the acyl-CoA passes through the outer membrane and is converted to the carnitine ester in the intermembrane space, or the carnitine ester is formed on the cytosolic face of the outer membrane, then moved across the MECHANISM FIGURE outer membrane to the intermembrane Conversion of a fatty acid to a fatty acyl–CoA. The conversion is catalysed by space—the current evidence does not fatty acyl–CoA synthetase and inorganic pyrophosphatase. Fatty acid activation by formation of the fatty acyl–CoA derivative occurs in two steps. In step 1, the carboxylate ion displaces the outer two (and) phosphates of ATP to form a fatty acyl–adenylate, the mixed anhydride of a carboxylic acid and a phosphoric acid. The other product is PPi, an excellent leaving group that is immediately hydrolysed to two Pi, pulling the reaction in the forward reveal which. In either case, passage into direction. In step 2, the thiol group of coenzyme A carries out nucleophilic the intermembrane space (the space attack on the enzyme-bound mixed anhydride, displacing AMP and forming between the outer and inner membranes) the thioester fatty acyl–CoA. The overall reaction is highly exergonic. occurs through large pores (formed by the protein porin) in the outer membrane. The fatty acyl–carnitine ester then enters the matrix by facilitated diffusion through the acyl-carnitine/carnitine transporter of the inner mitochondrial membrane. In the third and final step of the carnitine shuttle, the fatty acyl group is enzymatically transferred from carnitine to intramitochondrial coenzyme A by carnitine acyltransferase II. This isozyme, located on the inner face of the inner mitochondrial membrane, regenerates fatty acyl–CoA and releases it, along with free carnitine, into the matrix. Carnitine reenters the intermembrane space via the acyl-carnitine/carnitine transporter. Fatty acid entry into mitochondria via the acyl-carnitine/ carnitine transporter. After fatty acyl–carnitine is formed at the outer membrane or in the intermembrane space, it moves into the matrix by facilitated diffusion through the transporter in the inner membrane. In the matrix, the acyl group is transferred to mitochondrial coenzyme A, freeing carnitine to return to the intermembrane space through the same transporter. Acyltransferase I is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis. This inhibition prevents the simultaneous synthesis and degradation of fatty acids. Oxidation of Fatty Acids As noted earlier, mitochondrial oxidation of fatty acids takes place in three stages. In the first stage— β oxidation—fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-CoA, starting from the carboxyl end of the fatty acyl chain. For example, the 16-carbon palmitic acid (palmitate at pH 7) undergoes seven passes through the oxidative sequence, in each pass losing two carbons as acetyl-CoA. At the end of seven cycles the last two carbons of palmitate (originally C-15 and C-16) remain as acetyl-CoA. The overall result is the conversion of the 16-carbon chain of palmitate to eight two-carbon acetyl groups of acetyl-CoA molecules. Formation of each acetyl-CoA requires removal of four hydrogen atoms (two pairs of electrons and four H) from the fatty acyl moiety by dehydrogenases. In the second stage of fatty acid oxidation, the acetyl groups of acetyl-CoA are oxidized to CO2 in the citric acid cycle, which also takes place in the mitochondrial matrix. Acetyl-CoA derived from fatty acids thus enters a final common pathway of oxidation with the acetyl-CoA derived from glucose via glycolysis and pyruvate oxidation. The first two stages of fatty acid oxidation produce the reduced electron carriers NADH and FADH2, which in the third stage donate electrons to the mitochondrial respiratory chain, through Stages of fatty acid oxidation. Stage 1: A long- which the electrons pass to oxygen with the concomitant chain fatty acid is oxidized to yield acetyl residues phosphorylation of ADP to ATP. The energy released by fatty acid in the form of acetyl-CoA. This process is called oxidation is thus conserved as ATP. oxidation. Stage 2: The acetyl groups are oxidized to CO2 via the citric acid cycle. Stage 3: Electrons We now take a closer look at the first stage of fatty acid derived from the oxidations of stages 1 and 2 pass to O2 via the mitochondrial respiratory chain, oxidation, beginning with the simple case of a saturated fatty acyl providing the energy for ATP synthesis by chain with an even number of carbons, then turning to the slightly oxidative phosphorylation. more complicated cases of unsaturated and odd-number chains. We also consider the regulation of fatty acid oxidation, the β -oxidative processes as they occur in organelles other than mitochondria, and, finally, two less-general modes of fatty acid catabolism, α oxidation and ω oxidation. The β Oxidation of Saturated Fatty Acids Has Four Basic Steps Four enzyme-catalysed reactions make up the first stage of fatty acid oxidation. First, dehydrogenation of fatty acyl–CoA produces a double bond between the and carbon atoms (C-2 and C-3), yielding a tra ns Δ2 -enoyl Co A (the symbol Δ2 designates the position of the double bond). Note that the new double bond has the trans configuration, whereas the double bonds in naturally occurring unsaturated fatty acids are normally in the cis configuration. This first step is catalysed by three isozymes of acyl-CoA dehydrogenase, each specific for a range of fatty-acyl chain lengths: very-long-chain acyl-CoA dehydrogenase (VLCAD), acting on fatty acids of 12 to 18 carbons; medium- chain (MCAD), acting on fatty acids of 4 to 14 carbons; and short-chain (SCAD), acting on fatty acids of 4 to 8 carbons. All three isozymes are flavoproteins with FAD as a prosthetic group. The electrons removed from the fatty acyl-CoA are transferred to FAD, and the reduced form of the dehydrogenase immediately donates its electrons to an electron carrier of the mitochondrial respiratory chain, the electron-transferring flavoprotein (ETF). The oxidation catalysed by an acyl-CoA dehydrogenase is analogous to succinate dehydrogenation in the citric acid cycle; in both reactions the enzyme is bound to the inner membrane, a double bond is introduced into a carboxylic acid between the and carbons, FAD is the electron acceptor, and electrons from the reaction ultimately enter the respiratory chain and pass to O2, with the concomitant synthesis of about 1.5 ATP molecules per electron pair.  In the second step of the β-oxidation cycle, water is added to the double bond of the trans- Δ2 -enoyl-CoA to form the L stereoisomer of β-hydroxyacyl-CoA (3-hydroxyacyl-CoA). This reaction, catalysed by enoyl-CoA hydratase, is formally analogous to the fumarase reaction in the citric acid cycle, in which H2O adds across an α – β double bond. In the third step, L-β -hydroxyacyl-CoA is dehydrogenated to form β-ketoacyl-CoA, by the action of β-hydroxyacyl-CoA dehydrogenase; NAD+ is the electron acceptor. This enzyme is absolutely specific for the L stereoisomer of hydroxyacyl-CoA. The NADH formed in the reaction donates its electrons to NADH dehydrogenase, an electron carrier of the respiratory chain, and ATP is formed from ADP as the electrons pass to O2. The reaction catalyzed by β-hydroxyacyl-CoA dehydrogenase is closely analogous to the malate dehydrogenase reaction of the citric acid cycle. The fourth and last step of the β -oxidation cycle is catalysed by acyl-CoA acetyltransferase, more commonly called thiolase, which promotes reaction of β-ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA. The other product is the coenzyme A thioester of the fatty acid, now shortened by two carbon atoms. This reaction is called thiolysis, by analogy with the process of hydrolysis, because the β-ketoacyl-CoA is cleaved by reaction with the thiol group of coenzyme A. The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain. For fatty acyl chains The β -oxidation pathway. In each pass of 12 or more carbons, the reactions are catalyzed by a multienzyme through this four-step sequence, one acetyl complex associated with the inner mitochondrial membrane, the residue (shaded in pink) is removed in the trifunctional protein (TFP). TFP is a heterooctamer of α4β4 subunits. form of acetyl-CoA from the carboxyl end of Each subunit contains two activities, the enoyl-CoA hydratase and the fatty acyl chain— in this example the β-hydroxyacyl-CoA dehydrogenase; the subunits contain the palmitate (C16), which enters as palmitoyl- thiolase activity. This tight association of three enzymes may allow CoA. efficient substrate channeling from one active site to the Oxidation of a monounsaturated fatty acid. Oleic acid, as Oxidation of a polyunsaturated fatty acid. The example here is oleoyl-CoA (Δ9), is the example used here. Oxidation requires linoleic acid, as linoleoyl-CoA (Δ9,12). Oxidation requires a an additional enzyme, enoyl-CoA isomerase, to reposition second auxiliary enzyme in addition to enoyl-CoA isomerase: the double bond, converting the cis isomer to a trans isomer, NADPH-dependent 2,4-dienoyl-CoA reductase. The combined a normal intermediate in β-oxidation. action of these two enzymes converts a trans-Δ2, cis-Δ4- dienoyl-CoA intermediate to the trans-Δ2-enoyl-CoA substrate necessary for β-oxidation.  GLYCOLYSIS : A CYTOSOLIC PROCESS Glycolysis converts carbohydrates into pyruvate, producing NADH & ATP. Glycolytic & fermentative pathway in plants are identical with those of animal cells. In animals the substrate of glycolysis is glucose & the end product pyruvate. Because sucrose is the major translocated sugar in most plants & is therefore the form of carbon that most non-photosynthetic tissue import, sucrose (not glucose) is the true sugar substrate for plant respiration. The end product of plant glycolysis includes another organic acid, malate. In most plant tissues sucrose synthase, localized in the cytosol, is used to degrade sucrose by combining sucrose with UDP to produce fructose & UDP-glucose. UDP-glucose pyro phosphorylase then converts UDP-glucose & pyrophosphate (PPi) into UTP & glucose-6-phosphate. In some tissues, invertases present in the cell wall, vacuole or cytosol hydrolyze sucrose to its two component hexoses (glucose & fructose). Plastids such as chloroplasts or amyloplasts also supply substrate for glycolysis. Starch is synthesized & categorized only in plastids & carbon obtained from starch degradation enters the glycolytic pathway in the cytosol primarily as hexose phosphate (which is translocated out of amyloplasts) or triose phosphate (which is translocated out of chloroplasts.) Photosynthetic products can also directly enter the glycolytic pathway as triose phosphate. This series of reactions consumers two to 4 molecules of ATP per sucrose unit, depending on whether the sucrose is split by sucrose synthesis or invertase. These reactions also include 2 of the 3 essentially hexokinases & phosphofructokinase. The phosphofructokinase reaction is the control points of glycolysis in both plants and animals. The Energy Conserving Phase of Glycolysis - The standard free energy change ADP ATP of hydrolysis is -49.3 K J mol-1 or - 11.8 Kcal mol-1 for sucrose as I. 1,3-bisphosphoglycerate 3 phosphoglycerate substrate. On the other hand, - phosphoglycerate kinase 18.9 K J mol-1 or -4.5 Kcal mol-1 for glucose Mixed acid anhydride Phosphate on 3-phosphoglycerate is transferred to carbon2 & a molecule of water is removed, yielding the compound PEP. The phosphate group on PEP also has a high tree energy of hydrolysis (-30.5 K J mol-1 or -703 Kcal mol-1 for glucose, -61.9 K J mol-1 or -14.8 Kcal mol -1 for sucrose). Pyruvate into an energetically unfavored enol (– C=C – OH) configuration & this high level of free energy makes PEP an extremely good phosphate donor for ATP formation as well. In the final step of glycolysis, the enzyme pyruvate kinase catalyzes a second substrate level phosphorylation because it involves the direct transfer of a phosphate moiety from a substrate molecule to ADP to form ATP. It is distinct from the mechanism of ATP synthesis during oxidative phosphorylation that is utilized by the electron transport chain in the final stage of respiration & from phosphorylation that is used to synthesize ATP photosynthetic electron transfer. In the absence of O2, fermentation regenerates the NAD+ needed for glycolysis, In the absence of O2, citric acid & oxidative phosphorylation cannot function. Glycolysis thus cannot continue to operate because the cell supply of NAD+ is limited, & once all the NAD+ becomes tied up in the reduced state (NADH), the reaction catalyzed by glyceraldelyde-3-phosphate dehydrogenase cannot take place. To overcome this problem, plants & other organisms can further metabolize pyruvate by carrying out one or more forms of fermentative metabolism. Plant tissues may be subjected to low (hypoxic) or zero(anoxic) concentrations of ambient O2, forcing them to carry out fermentative metabolism. The best & died ex-flooded or submerged waterlogged soil in which the diffusion of O2 is sufficiently reduced to cause root tissues to become hypoxic. In corn the initial response to low O2 is lactic acid fermentation. But the subsequent response is alcoholic fermentation. Ethanol is thought to be a less toxic end product of fermentation because it can diffusion out of the cell, whereas lactate accumulates & promotes acidification of the cytosol. Pyruvate dehydrogenase complex contains, Pyruvate dehydrogenase (containing TPP as prosthetic group). Dihydrolipoyl transacetylase containing lipomide as prosthetic group. Dihydrolipoyl dehydrogenase containing FAD as prosthetic group. 2 Regulatory enzymes, Pyruvate dehydrogenase kinase (kinase helps in phosphorylation) which phosphorylate pyruvate dehydrogenate component forms inactive enzyme (pyruvate dehydrogenase phosphate). Pyruvate dehydrogenase phosphate phosphatase, which by dephosphorylation converted pyruvate dehydrogenase complex in its active form. 5 coenzymes are, TPP- Thiamine pyrophosphate Pyrimidine Thiazole Pyrophosphate Lipomide (protein) ; Lipomic acid forms peptide bond with an amino acid forming protein. The overall process occurs in a 3-step process – Decarboxylation, oxidation &conjugation to FAD CoA NAD+ CoASH Fate of NADH H ( –) H H CONH2 CONH2 NAD+ N kR R + +  The NADH produce in glycolysis when glyceraldehydes-3-phosphate is converted 1,3-bisphosphoglycerate, may undergo reoxidation to NAD+ by different ways depending upon the conditions – I. Under aerobic conditions :- NADH is oxidized to NAD+ under aerobic condition when pyruvate is converted to ethanol or lactate. Glyceraldehyde- 3- P NAD+ Ethanol or Lactate 1,3- bis phosphoglycerate NADH Pyruvate II. Under aerobic conditions :- The cytosolic NADH ultimately transfers into the electrons to molecular O2 under aerobic condition through electron transport chain in mitochondria. But mitochondrial membrane is impermeable to NADH and NAD+. It is actually the electron from NADH rather than NADH itself are carried across the mitochondrial membrane through two different shuttles. a. Glycerol phosphate shuttle b. Malate aspartate shuttle (liver, kidney & heart mitochondria) c. Glycerol phosphate shuttle- (only skeleton muscle & brain) CH2OH CH2OH Glycerol phosphate dehydrogenase C=O CH OH CH2OP CH2OP (Dihydroxy acetone NADH+ + H+ NAD+ (Glycerol –3– phosphate) phosphate) CYTOSOL MITOCHONDRIA CH2OH CH2OH Glycerol phosphate dehydrogenase C=O CH OH CH2OP CH2OP (Dihydroxy acetone FADH2 FAD+ (Glycerol –3– phosphate) phosphate) NADH + H+ NADH + H+ + FAD FADH2 + NAD+ The cytosolic glycerol phosphate dehydrogenase is quite different from mitochondrial glycerol phosphate dehydrogenase which contain FAD as prosthetic group. ETC NADH+ + H+ 2.5 ATP  ETC FADH2 1.5 ATP The reduced flavin transfer its elections to the respiratory chain at co-enzyme a level inside mitochondria & produce 2 ATP. CITRIC ACID CYCLE The citric acid cycle of plants has unique features – The step catalysed by Succinyl-CoA synthetase produces ATP in plants GTP in in animals. A feature of the plant citric acid cycle that is absent in many other organisms is the significant activity of NAD + malic enzyme, which has been found in the matrix of all plant mitochondria. Malate + NAD+ Pyruvate + CO2 + NADH Cls –aconitate normally does not dissolute from the active site. Aconitase contains an iron-sulfur centre which acts both in the binding of the substrate at the active site 2 in the catalytic addition or removal of H2O. Oxidation of succinate to fumarate – In eukaryotes, succinate dehydrogenase is tightly bound to the inner mitochondrial membrane; in prokaryotes, to the plasma-membrane. It is the only enzyme of the citric acid cycle that is membrane bound. The enzyme from beet heart mitochondria contains three different iron-sulfur clusters 2 one molecule of covalently bound FAD. Electron flow from succinate through these carriers to the final electron acceptor, O2 is coupled to the synthesis of about 1.5 ATP molecule per pair of electrons (respiration- linked phosphorylation). Malonate, an analogue of succinate, is a strong competitive inhibitor of succinate dehydrogenase 2 therefore blocks the activity of the citric acid cycle. In aerobic organisms, the citric acid is an amphibolic pathway means both catabolic & anabolic pathway. Anaplerotic Reaction – An enzyme catalysed reaction that can replenish the supply of intermediates in the citric acid cycle. An intermediate of the citric acid cycle are removed to serve as biosynthetic precursors, they are replenished by anaplerotic reactions. Reactions & their occurring places (tissue / organism) Pyruvate Carboxylase Pyruvate + HCO3– + ATP Oxaloacetate + ADP + Pi (Liver, kidney) PEP Carboxykinase Phosphoenolpyruvate + CO2 + GDP Oxaloacetate + GTP (Heart, Skeletal muscle) PEP Carboxylase PEP + HCO3– Oxaloacetate +Pi (Higher plants, Yeast, Bacteria)  Malic enzyme Pyruvate + HCO3– + NAD(P)H Malate + NAD(P)+ (Widely distributed in eukaryotes & prokaryotes) ❖ Pyruvate enters the mitochondria & is oxidized via the citric acid cycle – Pyruvate generated in the cytosol during glycolysis be transported the impermeable inner mitochondrial membrane via a specific transport protein. The products are NADH (from NAD+), CO2 & acetic acid in the form of acetyl- CoA, in which a thioester bond links the acetic acid to a sulphur containing cofactor, coenzyme A (CoA) ATP One molecule of ATP is synthesized by a substrate level phosphorylation during the reaction catalyzed by Succinyl-Co A Synthetase.  Malic enzyme decarboxylates malate to pyruvate and enables NAD+ NADH plant mitochondria to oxidize malate. Malate Pyruvate + CO2 Malic enzyme GLYOXYLATE CYCLE In plants, certain invertebrates, some microorganisms such as E. coli & yeast, acetate can serve both as energy rich fuel & as a source of PEP for carbohydrate synthesis. In these organisms, enzymes of the glyoxylate cycle catalyze the net conversion of acetate to succinate or other 4-C intermediates of the citric acid cycle. 2 Acetyl-CoA + NAD+ + 2H2O Succinate + 2CoA + NADH + H+ In plants, the enzyme of the glyoxylate cycle are sequestered in membrane bounded organelles called glyoxysomes. Those enzymes common to the citric acid & glyoxylate cycle have two isozymes, one specific to mitochondria, the other to glyoxysomes. Glyoxysomes are not present in all plant tissues at all times. They develop in lipid-rich seeds during germination, before the developing plant acquire the ability to make glucose by photosynthesis. A cytosolic isozyme of malate dehydrogenase oxidizes malate to oxaloacetate, a precursor for gluconeogenesis, germinating seeds can therefore convert the carbon of stored lipids into glucose. Vertebrate animals do not have the enzymes specific to the glyoxylate cycle (isocitrate lyase & malate synthase) & therefore cannot bring about the net synthesis of glucose from lipids. Malate is transformed into the cytosol & oxidized to oxaloacetate, which is converted into phosphoenolpyruvate by the enzyme PEP carboxy kinase. The resulting PEP is then metabolized to produce sucrose via the gluconeogenic pathway. ELECTRON TRANSPORT AND ATP SYNTHESIS AT THE INNER MITOCHONDRIAL MEMBRANE – OXIDATIVE PHOSPHORYLATION The electron transport chain of plants (and fungi) contains multiple NAD(P)H dehydrogenases and the alternative oxidase not found in mammalian mitochondria ATP: the F0F1- ATP Synthases For each molecule of sucrose oxidized through glycolysis and the citric acid cycle pathways – 4 molecules of NADH generated in the cytosol 16 molecules of NADH + 4 molecules of FADH2 generated in the mitochondrial matrix. (associated with succinate dehydrogenase) The role of the electron transport chain is to bring about the oxidation of NADH (& FADH2) & in the process, utilize some of the free energy released to generate an electrochemical proton gradient, UH across the inner mitochondrial membrane. Complex I (NADH dehydrogenase): Electrons from NADH generated in the mitochondrial matrix during the citric acid cycle are oxidized by complex I (an NADH dehydrogenase). The electron carriers in complex I include a tightly bound cofactor (FMN) & several iron-sulfur centers. Complex I then transfer these electrons to ubiquinone. Four protons are pumped from the matrix to the intermembrane space for every electron pair passing through the complex. Ubiquinone, a small lipid- soluble electron and proton carrier, is located within the inner membrane. It is not tightly associated with any protein & it can diffuse within the hydrophobic core of the membrane bilayer. Complex II (Succinate dehydrogenase): Oxidation of succinate in the citric acid cycle is catalyzed by this complex, and the reducing equivalents are transferred via the FADH2 and a group of iron, sulfur proteins into the ubiquinone pool. This complex does not pump proton. Complex III (Cytochrome bC1 Complex): This complex oxidizes reduced ubiquinone (ubiquinol) and transfers the electron via an iron-sulfur center, two b-type cytochromes, a membrane bound cytochrome C1 to cytochrome C, four protons or electron pair are pumped by complex III. Cytochrome C is a small protein loosely attached to the outer surface of the inner membrane and serve as a mobile carrier to transfer electrons between complex III & IV Complex IV (Cytochrome C oxidase): This complex contains two copper centers (CuA & CuB) and cytochromes a & a3. Complex IV is the terminal oxidase & brings about the 4-electron reduction of O2 to two molecules of H2O. 2 protons are pumped per electron pair. [Both structurally and functionally, ubiquinone and the cytochrome bc1 complex are very similar to plastoquinone and the cytochrome b6t complex in the photosynthetic electron transport chain]  The cyanide sensitive pathway of plant mitochondrial electron transport chain. Some electron transport enzymes are unique to plant mitochondria - Two NAD(P)H dehydrogenases, both Ca2+ dependent, attached to the outer surface of the inner membrane facing the intermembrane space can oxidize cytosolic NADH and NADPH. Electrons from those external NAD(P)H dehydrogenases – NDex (NADPH) & NDex (NADPH) enter the main electron transport chain at the level of the of the ubiquinone pool. Plant mitochondria have two pathways for oxidizing matrix NADH. Sensitive pathway (inhibited by rotenone & piericidin) Resistant pathway (Plant mitochondria have rotenone- resistance dehydrogenases.) Role: When complex I is overloaded, then this pathway bypasses the reaction e.g.- under photorespiratory condition. An NADPH dehydrogenase, NDin (NADPH) is present on matrix surface. Plants have an alternative respirating pathway for the reduction of O2. It includes alternative oxidase. Unlike cytochrome, C oxidase is insensitive to inhibition by cyanide, azide etc. Alternative pathway has yet to be characterized, current evidence indicates that electrons feed off the main electron transport chain into the alternative pathway at the level of the ubiquinone pool. The cyanide resistant terminal oxidase associates with the alternative pathway catalyzes a 4-electron reduction of O2 to H2O and is specifically inhibited by several compounds, like salicylhydroxamic acid (SHAM) & n- propyigallate. Because electron branch to the alternative pathway from the ubiquinone pool two sites of energy conservation (at complex III & IV) are bypassed, and there is no evidence for an energy conservation site on the alternative pathway between ubiquinone and oxygen. The tree energy that would normally be stored as ATP is lost as heat when electrons are shunted through the alternative pathway. If cyanide (IMM) is added to activity respiring animal tissue cytochrome C oxide is inhibited & the respiration rate drops quickly to less than 1% of its initial level. Plant tissues display a level of cyanide-resistant respiration that can represent 10 to 25% and in some tissues up to 100% of the uninhibited control rate.  The alternative pathway can function as an energy overflow pathway, oxidizing respiratory substrates that accumulate in excess of those needed for growth, storage or ATP synthesis. The electron flow through the alternative pathway only when the activity of the main pathway is saturated. In vivo, saturation may occur if the respiration rate exceeds the cells demand for ATP. The alternative oxidase can be active before the cytochrome pathway is saturated. It can be activated by a variety of stress like phosphate deficiency chilling drought. Two types 1. Growth respiration (actual new growth = W2-W1) = Net photosynthesis (Pn) = Gross photosynthesis – Respiration Growth respiration  Pn = KPn (K = Constant) 2. Maintain respiration – depends upon the biomass of the organism- means mass of the living tissue. Maintain respiration  W (biomass) = CW (C = Constant) 𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐂𝐨𝟐 𝐫𝐞𝐥𝐞𝐚𝐬𝐞𝐝/𝐞𝐯𝐨𝐥𝐯𝐞𝐝 Respiratory Quotient (RQ) = 𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐎𝟐 𝐚𝐛𝐬𝐨𝐫𝐛𝐞𝐝/𝐭𝐚𝐤𝐢𝐧𝐠 It may be defined as the ratio between the volume of Co2 released or given out and O2 taken in simultaneously by a given weight of tissue in a particular period of time at standard temperature and pressure. a) In case of carbohydrates, RQ is 1 C6H1206 + 6CO2 → 6CO2 + 6H2O 6 𝐶𝑜 RQ = 6 ( 𝑂 2 ) = 1 2 b) R.Q of fats and lipids and protein, R.Q is less than 1 C18H36O2 + 26O2 → 18CO2 + 18H2O 18 𝐶𝑜2 RQ = ( )  0.7 26 𝑂2 Protein’s R.Q = (0.5 - 0.9) [found in germinating pea & gram seeds, Ideal is 0.8] c) Organic acids (more than 1) C4H6O5 + 3O2 → 4CO2 + 3H2O d) Succulents/ CAM plants 𝐶𝑜2 0 𝑂2 = 𝑥 = 0 (eg. Bryophyllum and opuntia) 2C6H12O6 + 302 → 3C4H6O5 + 3H20 [RQ = 0/3 = 0] e) Anaerobic respiration C6H12O6 → 2C2H5OH + 2CO2 [RQ = 2/0 = ∞] [In animal total ATP production = 40, net gain = 38; in glycolysis (cytosol), 1 NADH = 3 ATP, so 2NADH = 6ATP. In plant system total ATP production = 38, net gain = 36; in glycolysis (cytosol) 1 NADH = 2ATP, so 2NADH = 4ATP] Factor affecting respiration – A. Internal Factor : Protoplasmic factor: Amount of protoplasm plays an important Role. In younger cells there are more protoplasm more reaction and hence more respiration (higher rate in younger plants than older ones). Concentration of respiratory substrate: more substrates more respiration. No food for longer time results in less respiration. When there is photosynthesis, respiration is more. Age: In younger plants and germinating seedlings, respiration is more (many processes start together during germinating for which energy is required). During ripening of fruits, respiration is very high. This is climacteric rise of rate of respiration. Hydration: Less water, results in less respiration. All enzymes and hydration for activity. In its absence the enzymes remain inactive. B. External factor: Temperature: higher temperature more respiration range is (0 – 45oC). In this case Vant Hoff value of Q10 optimum is 30oC. Light: In presence of light, there is photosynthesis which forms respiratory substrate hence more respiration. O2 concentration of atmosphere: In presence of O2 anaerobic respiration in absence fermentation takes place. O2 is necessary for terminal oxidation. Min conc. Of O2 required for aerobic respiration. This is known as extinction point. Below this aerobic respiration stops. At a particular point, or conc. Both aerobic & anaerobic respiration takes place. This is called as transition point. When aerobic respiration increases, conc. Of citric acid & ATP increases which has inhibiting effect on the phosphofructo kinase enzyme of glycosis as a result glycolysis is slowed. This is “Pastern effect” – inhibition of glycolysis by higher conc. Of O2. CO2 concentration: When CO2 conc. is more (glucose is converted into starch) stomata is closed, hence there is no O 2 supply & no respiration (High CO2 conc. Is used for storage of fruits & vegetables exception potato tubers) Mineral salt: Cu, Fe, Zn, Mn, Ca, k, Mg important part of the enzymes & the enzymes are more active & respiration also increased. Linderburg & Burstrom (1933) they observed that when the mineral salt is more than the respiration also increased that is known as salt respiration. When k+ play important role is the formation of starch protein from simple sugar & when K+ is less then respiration will be more. Mechanical stimulation: - The distribution act as stimulator & respiration rate is not increased. By disturbing the plant continuous then the respiration rate is not increased. Injury: When the plant will be injured then to recover this the cell division, biosynthesis increase, the rate of respiration also increases of higher rate biosynthesis process. Organic substances: - cyanides act as inhibitor of cytochrome oxidase. Azides- acts as inhibitors of ETC. →Mitochondrial respiration during photosynthesis is to supply carbon metabolites for biosynthetic reactions for ex, by formation of 2-oxoglutarate needed for nitrogen assimilation. Leaf mitochondria typically have high capacities of non- phosphorylating pathway in the ETC. By NADH with lower ATP yield, mitochondria can maintain a higher 2- oxoglutarate production by the respiratory pathways without being restricted by the cytosolic demand for ATP.