Cellular Respiration

A. Respiration

Cellular respiration is an enzyme controlled process of biological oxidation of food materials in a living cell, using molecular O₂, producing CO₂ and H₂O, and releasing energy in small steps and storing it in biologically useful forms, generally ATP.

(1) Use of energy : Cellular activities like active transport, muscle-contraction, bioluminescenes, homothermy locomotion, nerve impulse conduction, cell division, growth, development, seed germination require energy. Main source of energy for these endergonic activities in all living organisms including plants, comes from the oxidation of organic molecules.

The energy released by oxidation of organic molecules is actually transferred to the high energy terminal bonds of ATP, a form that can be readily utilized by the cell to do work. Once ATP is formed, its energy may be utilized at various places in the cell to drive energy- requiring reactions. In these processes, one of the three phosphate groups is removed from the ATP molecule. Thus the role of ATP as an intermediate energy transforming compound between energy releasing and energy consuming reactions.

(2) Significance of respiration : Respiration plays a significant role in the life of plants. The important ones are given below :

(i) It releases energy, which is consumed in various metabolic process necessary for life of plant.

(ii) Energy produced can be regulated according to requirement of all activities.

(iii) It convert in soluble foods into soluble form.

(iv) Intermediate products of cell respiration can be used in different metabolic pathways e.g.

Acetyl- CoA (in the formation of fatty acid, cutin and isoprenoids) ; - ketoglutaric acid (in the formation of glutamic acid) ; Oxaloacetic acid (in the formation of aspartic acid, pyrimidines and alkaloids); Succinyl- CoA (synthesis of pyrrole compounds of chlorophyll).

(v) It liberates carbon dioxide, which is used in photosynthesis.

(vi) Krebs cycle is a common pathway of oxidative breakdown of carbohydrates, fatty acids and amino acids.

(vii) It activates the different meristematic tissue of the plant.

B. Respiratory substrate or Fuel.

In respiration many types of high energy compounds are oxidised. These are called respiratory substrate or respiratory fuel and may include carbohydrates, fats and protein.

(1) Carbohydrate : Carbohydrates such as glucose, fructose (hexoses), sucrose (disaccharide) or starch, insulin, hemicellulose (polysaccharide) etc; are the main substrates. Glucose are the first energy rich compounds to be oxidised during respiration. Brain cells of mammals utilized only glucose as respiratory substrate. Complex carbohydrates are hydrolysed into hexose sugars before being utilized as respiratory substrates. The energy present in one gram carbohydrate is – 4.4 Kcal or 18.4 kJ.

(2) Fats : Under certain conditions (mainly when carbohydrate reserves have been exhausted) fats are also oxidised. Fat are used as respiratory substrate after their hydrolysis to fatty acids and glycerol by lipase and their subsequent conversion to hexose sugars. The energy present in one gram of fats is 9.8 Kcal or 41kJ, which is maximum as compared to another substrate.

The respiration using carbohydrate and fat as respiratory substrate, called floating respiration (Blackmann).

(3) Protein : In the absence of carbohydrate and fats , protein also serves as respiratory substrate. The energy present in one gram of protein is : 4.8 Kcal or 20 kJ. when protein are used as respiratory substrate respiration is called protoplasmic respiration.

C. Types of respiratory organism.

Organism can be grouped into following four classes on the basis of their respiratory habit -

(1) Obligate aerobes: These organisms can respire only in the presence of oxygen. Thus oxygen is essential for their survival.

(2) Facultative anaerobes : Such organisms usually respire aerobically (i.e., in the presence of oxygen) but under certain condition may also respire anaerobically (e.g., Yeast, parasites of the alimentary canal).

(3) Obligate anaerobes : These organism normally respire anaerobically which is their major ATP- yielding process. Such organisms are in fact killed in the presence of substantial amounts of oxygen (e.g., Clostridium botulinum and C. tetani).

(4) Facultative aerobes : These are primarily anaerobic organisms but under certain condition may also respire aerobically.

D. Types of respiration.

On the basis of the availability of oxygen and the complete or incomplete oxidation of respiratory substrate, the respiration may be either of the following two types : Aerobic respiration and Anaerobic respiration

Aerobic respiration

It uses oxygen and completely oxidises the organic food mainly carbohydrate (Sugars) to carbon dioxide and water. It therefore, releases the entire energy available in glucose.

(686 Kcal)

It is divided into two phases : Glycolysis, Aerobic oxidation of pyruvic acid

E. Glycolysis / EMP pathway

(1) Discovery : It is given by Embden, Meyerhoff and Parnas in 1930. It is the first stage of breakdown of glucose in the cell.

(2) Definition : Glycolysis ( Gr. glykys= sweet, sugar; lysis= breaking) is a stepped process by which one molecule of glucose (6c) breaks into two molecules of pyruvic acid (3c).

(3) Site of occurrence : Glycolysis takes place in the cytoplasm and does not use oxygen. Thus, it is an anaerobic pathway. In fact, it occurs in both aerobic and anaerobic respiration.

(4) Inter conversions of sugars : Different forms of carbohydrate before entering in glycolysis converted into simplest form like glucose, glucose 6-phosphate or fructose 6-phosphate.

Steps of glycolysis : Glycolysis consists of 9 steps. Each step is catalysed by a specific enzyme. Most of the reaction are reversible.

(i) First phosphorylation : The third phosphate group separates from adenosine triphospate (ATP) molecule, converting the latter into adenosine diphophate (ADP) and releasing energy. With this energy, the phosphate group combines with glucose to form glucose 6-phosphate, The reaction is catalysed by the enzyme, hexokinase or glucokinase in the presence of Mg₂⁺. Thus, a molecule of ATP is consumed in this step. This glucose 6-phosphate (phosphoglucose) is called active glucose.

(ii) Isomerisation : Glucose 6-phophate is changed into its isomer fructose 6-phophate by rearrangement. The rearrangement is catalysed by an enzyme, phophoglucose-isomerase or phosphohexose isomerase.

Glucose 6-phosphate Fructose 6-phosphate

Fructose 6-phosphate may be formed directly from free fructose by its phosphorylation in the presence of an enzyme fructokinase, Mg ²⁺ and ATP

(iii) Second phosphorylation : Fructose 6-phosphate combines with another phosphate group from another ATP molecule, yielding fructose 1, 6-diphosphate and ADP , The combination is catalysed by an enzyme phosphofructokinase in the presence of Mg²⁺ and appears to be irreversible. This phosphorylation, thus, consume another molecule of ATP. Excess of ATP inhibits phosphofructokinase.

phosphorylation reaction activate the sugar and prevent its excape from the cell. They go uphill, increasing the energy content of the products.

(iv) Cleavage : Fructose 1,6-diphosphate now splits into two 3-carbon, phosphorylated sugars : dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PGAL), or glyceraldehyde 3-phosphate (GAP). The reaction is catalyzed by an enzyme aldolase. DHAP is converted into PGAL with the aid of an enzyme phosphotriose isomerase.

Fructose 1,6-diphosphate 3-phophoglyceraldehyde+Dihydroxyacetone phosphate

Dihydroxyacetone phosphate 3- phosphoglyceraldehyde

Oxidative decarboxylation of pyruvic acid : If sufficient O2 is available, each 3-carbon pyruvate molecule (CH₃COCOOH) enters the mitochondrial matrix where its oxidation is completed by aerobic means. It is called gateway step or link reaction between glycolysis and Kreb's cycle. The pyruvate molecule gives off a molecule of CO2 and releases a pair of hydrogen atoms from its carboxyl group (–COOH), leaving the 2 carbon acetyl group (CH₃CO–). The reaction is called oxidative decarboxylation, and is catalyzed by the enzyme pyruvate dehydrogenase complex (decarboxylase, TPP, lipolic acid, transacetylase, Mg²⁺) . During this reaction, the acetyl group combines with the coenzyme A (CoA) to form acetyl coenzyme A with a high energy bond (CH₃CO~CoA). Most of the free energy released by the oxidation of pyruvate is captured as chemical energy in high energy bond of acetyl coenzyme A. From a pair of hydrogen atoms released in the reaction, to electrons and one H+ pass to NAD+, forming, NADH+ H⁺ . The NADH forms 3 ATP molecules by transferring its electron over ETS described ahead.

Decarboxylation and dehydration :

**TPP=Thiamine pyrophosphate

**LAA=Lipoic acid amide

Acetyl CoA is a common intermediate of carbohydrate and fat metabolism. Latter this acetyl CoA from both the sources enters Kreb's cycle. This reaction is not a part of Kreb's cycle.

(2) Kreb's cycle / TCA cycle / Citric acid cycle

(i) Discovery : This cycle has been named after the German biochemist in England Sir Hans Krebs who discovered it in 1937. He won Noble Prize for this work in 1953. Krebs cycle is also called the citric acid cycle after one of the participating compounds.

(ii) Site of occurrence : It takes place in the mitochondrial matrix.

(iii) Steps in Kreb's cycle : Kreb's cycle consists of 8 cyclic steps, producing an equal number of organic acids. Each step is catalyzed by a specific enzyme. In Kreb's cycle, the entrant molecule is 2-carbon acetyl CoA and the receptor molecule is 4- carbon oxaloacetate.

(a) condensation : Acetyl coenzyme A reacts in the presence of water with the oxaloacetate normally present in a cell, forming 6-carbon citrate and freeing coenzyme A for reuse in pyruvate oxidation. The high-energy bond of acetyl CoA provides the energy for this reaction. The reaction is catalyzed by the citrate synthetase enzyme. The citrate has 3-carboxyl group. Hence, Krebs cycle is also called tricarboxylic acid cycle, or TCA cycle after its first product.

(b) Reorganisation (Dehydration) : Citrate undergoes reorganisation in the presence of an enzyme, aconitase , forming 6-carbon cisaconitate and releasing water.

(c) Reorganisation (Hydration) : Cisaconitate is further reorganised into 6-carbon isocitrate by the enzyme, aconitase, with the addition of water.

(d) Oxidative decarboxylation I : This is a two stage process :

Stage I : Hydrogen atoms from isocitric acid react with NAD to form NAD. 2H forming oxalosuccinic acid. The pair of hydrogen atoms give two electrons and one H+ to NAD⁺ forming NADH +H⁺. The enzyme isocitrate dehydrogenase catalyses the reaction in the presence of Mn^2+. NADH generates ATP by transferring its electron over the ETS.

Stage II : Decarboxylation of oxalosuccinic acid occurs forming -ketoglutaric acid, which is a first 5-C carbon molecule of Kreb's cycle.

(e) Oxidative decarboxylation II : This is also a 2 stage process :

Stage I : Coenzyme A reacts with -ketoglutarate, forming 4-carbon succinyl-coenzyme A and releasing CO₂ and a pair of hydrogen atoms. The reaction is catalysed by -ketoglutarate dehydrogenase complex enzyme. the pair of hydrogen atoms pass two electrons and one H+ to NAD+, forming NADH + H⁺

Stage II : Succinyl –coenzyme A splits into 4-carbon succinate and coenzyme A with the addition of water. The coenzyme A transfers its high energy to a phosphate group that joins GDP (Guanosine diphosphate), forming GTP (Guanosine triphosphate). The latter is an energy carrier like ATP. This is the only high-energy phosphate produced in the Krebs cycle. The stage 2 reaction is catalysed by succinyl-CoA synthetase enzyme. The formation of GTP is called substrate level phosphorylation.

In a plant cell, this reaction produce ATP from ADP and GTP from GDP or ITP (Inosine triphosphate) in animals.

Oxygen to oxidize a carbon atom to CO₂ is taken in steps 4 and 5 from a water molecule.

(f) Dehydrogenation : This process converts succinate into 4-carbon fumarate with the aid of an enzyme, succinate dehydrogenase, and liberates a pair of hydrogen atoms. The latter pass to FAD+ (Flavin adenine dinucleotide), forming FADH₂. Hydrogen is carried by FAD in the form of whole atoms.

Succinate+FAD⁺ Fumarate +FADH2

(g) Hydration : This process changes fumarate into 4-carbon maltate in the presence of water and an enzyme, fumarase.

Fumarate +H₂O Maltate

(h) Dehydrogenation : This process restores oxaloacetate by removing a pair of hydrogen atoms from maltate with the help of an enzyme maltate dehydrogenase. The pair of hydrogen atoms pass two electrons and one H+ to NAD+ , forming NADH+H+.

Maltate +NAD⁺ Oxaloacetate +NADH +H+

Oxaloacetate combines with acetyl coenzyme A to form citrate, and so the cycle continues.

E.Electron transport system :

The electron transmitter system is also called electron transport chain (ETC), or cytochrome system (CS), as four out of these seven carriers are cytochrome. It is the major source of cells energy, in the respiratory breakdown of simple carbohydrates intermediates like phosphoglyceraldehyde, pyruvic acid, isocitric acid, ketoglutaric acid, succinic acid and malic acid are oxidised. The oxidation in all these brought about by the removal of a pair of hydrogen atoms (2H) from each of them. This final stage of respiration is carried out in ETS, located in the inner membrane of mitochondria (in prokaryotes the ETS is located in mesosomes of plasma membrane). The system consists of series of precisely arranged seven electron carriers (coenzyme) in the inner membrane of the mitochondrion, including the folds or cristae of this membrane. These seven electron-carriers function in a specific sequence and are :

Nicotinamide adenine dinucleotide (NAD), Flavin mononucleotide (FMN), Flavin adenine dinucleotide (FAD), Co-enzyme-Q or ubiquinone, Cytochrome-b, Cytochrome-c, Cytochrome-a and Cytochrome-a3,

The first carrier in the chain is a flavoprotein which is reduced by NADH2. Coenzyme passes these electron to the cytochromes arranged in the sequence of b-c-a-a3, finally pass the electron to molecular oxygen. In this transport, the electrons tend to flow from electro-negative to electro-positive system, so there is a decrease in free energy and some energy is released so amount of energy with the electrons goes on decreasing. During electron-transfer, the electron-donor gets oxidised, while electron-acceptor gets reduced so these transfers involve redox-reaction and are catalysed by enzymes, called reductases. Oxidation and reduction are complimentary. This oxidation-reductiion reaction over the ETC is called biological oxidation.

here, electron-donor and electron –acceptor form redox pair.

During the electron transfers, the energy released at some steps is so high that ATP is formed by the phosphorylation of ADP in the presence of enzyme ATP synthetase present in the head of F1-particles present on the mitochondrial crista. This process of ATP synthesis during oxidation of coenzyme is called oxidative phosphorylation, so ETS is also called oxidative phosphorylation pathways.

From the cytochrome a₃, two electrons are received by oxygen atom which also receives two proton (H+) from the mitochondrial matrix to form water molecule. So the final acceptor electrons is oxygen. So the reaction

(called metabolic water) is made to occur in many steps through ETC, so the most of the energy can be derived into a storage and usable form.

(i) Two route systems of ETC : The pairs of hydrogen atoms from respiratory intermediates are received either by NAD+ or FAD coenzymes which becomes reduced to NADH2 and FADH₂. These reduced coenzyme pass the electrons on to ETC. Thus, regeneration of NAD+ or FAD takes place in ETC. There are two routes ETC :

(a) Route 1 :NADH₂ passes their electrons to Co-Q through FAD . In route 1 FAD is the first electron carrier. 3 ATP molecules are produced during the transfer of electron on following steps :

NAD to FAD

Cyt b to Cyt c and

Cyt a to Cyt a₃

(b) Route 2 :FADH₂ passes their electron directly to FAD. 2 ATP molecules are produced during the transfer of electron on following steps.

Cyt b to Cyt c and

Cyt a to Cyt a₃

(ii) Structure of mitochondria in relation to oxidative function : On inner side of mitochondria elementary particles or F0-F1 complex of ATPase complex or elementary particle (oxysomes) are found. Previously it was considered that elementary particles contain all the enzyme of oxidative phosphorylation and electron transport chain.

Component of electron transport chain are located in the inner membrane in the form of respiratory chain complexes. For complexes following theories are given :

(a) Four complex theory : According to Devid green electron transport chain contains 4 complexes-

Complex I : Comprises NADH dehydrogenase and its 6 Iron Sulphur centers (Fe-S).

Complex II : Consists of Succinate dehydrogenase and its 3 Iron Sulphur centers.

Complex III : Consists of cytochrome b and c, and a specific Iron-Sulphur centers.

Complex IV : Comprises cytochromes a and a3.

F.Anaerobic respiration

Anaerobic respiration first studied by Kostychev (1902), Anaerobic respiration is an enzyme-controlled, partial break down of organic compounds (food) without using oxygen and releasing only a fraction of the energy. It is also called intra-molecular respiration (Pfluger, 1875). Anaerobic respiration occurs in the roots of some water-logged plants, certain parasitic worms (Ascaris and Taenia), animal muscle and some microorganisms (bacteria, moulds). In microorganisms anaerobic respiration is often called fermentation.

Higher organism like plants can not perform anaerobic respiration for long. It is toxic because accumulation of end products, insufficient amount of available energy and causes stoppage of many active process.

(1) Process of anaerobic respiration : In this process pyruvate which is formed by glycolysis is metabolised into ethyl alcohol or lactic acid and CO₂ in the absence of oxygen. Glycolysis is occurs in cytoplasm so the site of anaerobic respiration is cytoplasm.

C₆H₁₂O₆ → 2C₂H₅OH + 2CO₂ +⁵²Kcal/218.4 kJ

(i) Formation of ethyl alcohol : When oxygen is not available, yeast and some other microbes convert pyruvic acid into ethyl alcohol. This is two step process as explained below

(a) In the first step pyruvic acid is decarboxylated to yield acetaldehyde and CO_2. In the presence of Mg⁺⁺ and TPP (Thiamine pyrophosphate) pyruvate carboxylase.

(b) In the second step acetaldehyde is reduced to ethyl alcohol by NADH₂ formed in the glycolysis.

(ii)Production of lactic acid : In this process hydrogen atoms removed from the glucose molecule during glycolysis are added to pyruvic acid molecule and thus lactic acid is formed.

Lactic acid is produced in the muscle cells of human beings and other animals. During strenuous physical activity such as running, the amount of oxygen delivered to the muscle cells may be insufficient to keep pace with that of cellular respiration. Under such circumstances lactic acid is formed which accumulates in the muscle cells and causes muscle fatigue.