Chapter 20 Respiration in Plants by Teaching Care online coaching classes

  Introduction.

All organisms require continuous input of energy to carry on life process. These energy comes from cellular activities. All the cellular activities can be grouped into two categories : anabolism (biosynthetic activities of the cell) and catabolism (breaking- up process of the cell). The anabolic activities are endergonic (utilizes energy in cellular activities), while the catabolic activities are usually exergonic (energy releasing process by oxidation of food material). The sum of total catabolic and anabolic reactions occurring at any time in a cell is called metabolism.

Respiration is a vital process, includes the intake of oxygen. Chemically it is catabolic and brings about the oxidation and decomposition of organic compounds like carbohydrate, fat, protein in the cells of plants and animals with the release of energy. Oxidation of organic compounds by respiration, resulting in the release of chemical energies water and carbon dioxide. The overall process may be states according to the following general equation:

 

C6 H12O6 + 6CO2  ¾¾enz¾ym¾es ®

6CO2

• 6H₂O+ energy

 

glucose

carbondioxide

Water

(ATP)

 

In this reaction, six molecules of oxygen taken up and six molecules each of CO₂ and H₂O are formed with energy derived from respiration of each molecule of sugar oxidation. The plant cell is able to do chemical work in synthesizing energy- rich materials such as fat and hydrocarbon, osmotic work such as uptake and accumulation of salt and mechanical work such as involved in growth.

Respiration

Definition of 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.

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

oxidation of organic molecules.

 

Reactions releasing energy

 

Inorganic

 

 

ATP

Reactions consuming energy

 

Synthesis of proteins, lipids, carbohydrates

 

Glucose Lipids Proteins

phosphate

P

 

 

 

 

ADP

Osmotic work

Growth, differentiation and development Active absorption

Cyclosis Translocation

 

Fig. ATP cycle : ATP is an intermediate energy-transfer compound between energy-releasing and energy consuming reactions

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.

• Significance of respiration : Respiration plays a significant role in the life of The important ones are given below :

 

 

 

• It releases energy, which is consumed in various metabolic process necessary for life of
• Energy produced can be regulated according to requirement of all activities.
• It convert in soluble foods into soluble
• Intermediate products of cell respiration can be used in different metabolic pathways g.

Acetyl- CoA (in the formation of fatty acid, cutin and isoprenoids) ; a – 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).

• It liberates carbon dioxide, which is used in
• Krebs cycle is a common pathway of oxidative breakdown of carbohydrates, fatty acids and amino
• It activates the different meristematic tissue of the

• Comparison between respiration and photosynthesis : Photosynthesis associated with manufacturing of food, while respiration associated with releasing of energy from this Comparison between respiration and photosynthesis is given below :

Photosynthesis	Respiration
Occurs only in chlorophyll containing cells of plants.	Occurs in all plant and animal cells.
Takes place only in the presence of light.	Takes place continually both in light and in the dark.
During photosynthesis, radiant energy is converted into potential energy.	During respiration, potential energy is converted into kinetic energy.
Sugars, water and oxygen are products.	CO2 and H2O are products.
Synthesizes foods.	Oxidizeds foods.
CO2 and H2O are raw materials.	O2 and food molecules are raw materials.
Photosynthesis is an endothermal process.	Respiration is an exothermal process.
Stores energy.	Releases energy.
It includes the process of hydrolysis, carboxylation etc.	It includes the process of the dehydrolysis, decarboxylation, etc.
Results in an increase in weight.	Results in a decrease in weight.
It is an anabolic process.	It is a catabolic process.
Require cytochrome.	Also require cytochrome.

• Exchange of gases in photosynthesis and respiration : Respiration is continually going on in all living cells and oxygen is being continually absorbed and carbon dioxide The intake of oxygen (Liberated by photosynthesis) and liberation of carbon dioxide (evolved in respiration) takes place through the stomata and lenticels. The real process of respiration consists in the oxidation of organic substances which takes place in the protoplasm of the living cells and the gaseous exchange is an outward manifestation and an accompaniment of respiration. The intensity of gaseous exchange depends upon the intensity of respiration. It is comparatively rapid in meristematic and growing tissues where the formation of new cells and cell wall material requires a large supply of energy and is comparatively slow in mature cells due to the slowness of metabolic activities.

 

Fig. Showing gas exchange due to photosynthesis and respiration

Compensation  point  : It is that value or point in light intensity and atmospheric CO₂ concentration when rate of photosynthesis is just equivalent to the rate of respiration in photosynthetic organs so that there is no net

gaseous exchange. The value is 2.5- 100 ft candles/ 26.91-1076.4 lux in shade plants and 100-400 ft candles/ 1076.4-4305.6 lux in case of  sun plants. It is called light compensation point. There is, similarly, a

CO₂ compensation point. Its value is 25-100 ppm (25-100 ml.l^-1 ) in C₃ plants and 0-5 ppm (0-5 ml.l ^-1 ) in C₄

plants. A plant cannot survive for a long at compensation point because the nonphotosynthetic parts and dark respiration will deplete organic reserve of the plant.

CO₂ intake in photosynthesis balanced with CO₂ release in respiration = Compensation point.

• Comparison between respiration and combustion : According Lavosier cell respiration resembles the combustion (e.g., burning of coal, wood, oil etc.) in the breakdown of complex organic compounds in the presence of oxygen and production of carbon dioxide and energy, but there are certain fundamental differences between the two processes:

Differences between cell respiration and combustion

 

S.No.	Characters	Cell respiration	Combustion
(i)	Nature of process	Biochemical and stepped process.	Physico-chemical and spontaneous process.
(ii)	Site of occurrence	Inside the cells.	Non-cellular.
(iii)	Control	Biological control.	Uncontrolled.
(iv)	Energy release	Energy released in steps.	Large amount of energy is released at a time.
(v)	Temperature	Remain within limits.	Rises very high.
(vi)	Light	No light is produced.	Light may be produced.
(vii)	Enzymes	Controlled by enzymes.	Not controlled by enzymes.
(viii)	Intermediates	A number of intermediates are produced.	No intermediate is produced.

 Phases of respiration.

There are three phases of respiration :

 

 

 

• External respiration : It is the exchange of respiratory gases (O₂ and CO₂) between an organism and its
• Internal or Tissue respiration : Exchange of respiratory gases between tissue and extra cellular environment .

Both the exchange of gases occur on the principle of diffusion.

• Cellular respiration : It is an enzymatically-controlled stepped chemical process in which glucose is oxidised inside the mitochondria to produce energy-rich ATP molecules with high-energy bonds.

So, respiration is a biochemical process.

 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.

• 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 Kcal or 18.4 kJ.
• Fats : Under certain conditions (mainly when carbohydrate reserves have been exhausted) fats are also 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).

• Protein : In the absence of carbohydrate and fats , protein also serves as respiratory substrate. The energy present in one gram of protein is : 8 Kcal or 20 kJ. when protein are used as respiratory substrate respiration is called protoplasmic respiration.

 Types of respiratory organism.

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

• Obligate aerobes : These organisms can respire only in the presence of oxygen. Thus oxygen is essential for their
• 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).
• Obligate anaerobes : These organism normally respire anaerobically which is their major ATP- yielding Such organisms are in fact killed in the presence of substantial amounts of oxygen (e.g., Clostridium botulinum and C. tetani).
• Facultative aerobes : These are primarily anaerobic organisms but under certain condition may also respire

 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.

C6 H12O6 + 6O2  ¾¾^enz¾^ym¾^es ® 6CO2 + 6H2O + energy (686 Kcal)

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

Glycolysis / EMP pathway

• Discovery : It is given by Embden, Meyerhoff and Parnas in It is the first stage of breakdown of glucose in the cell.
• 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).
• Site of occurrence : Glycolysis takes place in the cytoplasm and does not use oxygen. Thus, it is an anaerobic In fact, it occurs in both aerobic and anaerobic respiration.
• 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. Then these sugars are metabolized into the The flow chart that showing inter conversion of sugar are given below :

 

 

Starch      UDPG                    Sucrose

+ UDP

 

Mannose

Glucose

Fructose

Starch                   Galactose

+H₃PO₄

+ATP

 

+ATP

hexokinase

+ATP

hexokinase

+ATP

hexokinase

Phosphorylase

Glucose

hexokinase

Galactose

 

1-phosphate

6-phosphate

 

 

 

 

Mannose 6-phosphate

 

 

Glucose

6-phosphate

Isomerase

 

 

 

Fructose

6-phosphate

Isomerase

 

 

+ATP

Phosphoglucomutase

 

Glucose

• phosphate

 

Phosphohexokinase

 

Fructose 1,6-phosphate

To glycolysis

Fig : Schematic conversion of complex carbohydrates before entering into glycolysis

 

 

 

 

 

 

 

 

 

 

(5)

Glucose (6c sugar) ATP ADP   Hexokinase

Glycolysis cycle

 

 

 

(– 1 ATP)                                                             1. Phosphorylation

 

 

 

Phosphoglucoisomerase

2. Isomerisation

 

 

 

 

First phase : Phosphorylation of glucose and its conversion into glyceraldehyde

3-phosphate

 

Fructose-1-6-diphosphate (6c sugar)

Fructose-6-phosphate (6c sugar)

ATP ADP

 

 

Phosphofructokinase

 

 

(– 1 ATP)

 

 

3. Phosphorylation

 

 

 

 

 

Dihydroxyacetone phosphate (3c sugar)

Lysis

Fructose diphosphate aldolase

4. Cleavage

 

 

 

Glyceraldehyde-3-phosphate

Phosphotriose isomerase

 

 

 

 

2×Glyceraldehyde-3-phosphate (3 carbon)

2P(from H₃PO₄)

 

2NAD

2NADH+2H⁺

 

Glyceraldehyde phosphate dehydrogenase

 

5. Phosphorylation and Dehydrogenation

 

 

 

 

Second phase : Conversion of glyceraldehyde

3-phosphate into pyruvate and couple formation of ATP

2ADP

2ATP

Phosphoglycerate kinase

 

 

2×2-Phosphoglycerate

2×3-Phosphoglycerate (3 carbon)

Phosphoglycerate mutase

 

 

Enolase

(+2ATP)

6. Dephosph- orylation

 

 

7. Rearrangement

 

 

 

8. Dehydration

 

 

 

2ADP

2ATP

Pyruvate kinase

(+2 ATP)

9. Dephosphorylation Net gain = 2 ATP

 

2×Pyruvate (3 carbon)

Fig : Glycolysis: A molecule of glucose breaks into two molecules of pyruvate in nine steps. Enzymes that catalyze the reactions 1-9 are sequentially listed on the right.

 

(6)  Enzymes of glycolysis and their co-factors

 

 

 

 

S. No.	Enzyme	Coenzyme (s) and cofactor	Activator (s)	Inhibitor (s)	Kind of reaction catalyzed
(i)	Hexokinase	Mg2+	ATP4-, Pi	Glucose 6-phopshate	Phosphoryl transfer
(ii)	Phosphogluco-isomerase	Mg2-	–	2-dioxyglucose 6-phosphate	Isomerization
(iii)	Phosphofructo-kinase	Mg2+	Fructose 2, 6- diphosphate, AMP, ADP, cAMP, K+	ATP 4-, citrate	Phosphoryl transfer
(iv)	Aldolase	Zn2+ ( in microbes)	–	Chelating agents	Aldol cleavage
(v)	Phosphotriose isomerase	Mg2+	–	–	Isomerization
(vi)	Glyceraldehyde 3-phosphate dehydrogenase	NAD	–	Iodoacetate	Phosphorylation coupled to oxidation
(vii)	Phosphoglycerate kinase	Mg2+	–	–	Phosphoryl transfer
(viii)	Phosphoglycerate mutase	Mg2+       2,3-diphos phoglycerate	–	–	Phosphoryl shift
(ix)	Enolase	Mg2+ , Mn2+, Zn2+, Cd2+	–	Fluoride+ phosphate	Dehydration
(x)	Pyruvate kinase	Mg2+, K+	–	Acetyl CoA, analine, Ca2+	Phosphoryl transfer

• Steps of glycolysis : Glycolysis consists of 9 steps. Each step is catalysed by a specific enzyme. Most of the reaction are
• 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 + ATP ¾¾^Hex¾^okin¾^a¾^se ®Glucose 6 – phosphate + ADP

Mg ++

 

• 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

 

Glucose 6-phosphate

Phosphogluco isomerase

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

Fructose + ATP ¾¾^Fruc¾^tok¾^ina¾^se ®Fructose 6 – phosphate + ADP

Mg 2+

 

• 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.

Fructose 6 – phosphate + ATP ¾¾^Pho¾^sph¾^ofru¾^cto¾^– ®Fructose 1,6 – diphosphate + ADP

kinase, Mg ²⁺

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

• 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

Aldolase

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

 

 

Dihydroxyacetone phosphate

Phosphotriose isomerase

3- phosphoglyceraldehyde

 

• Phosphorylation and Oxidative dehydrogenation: In phosphorylation, 3-phosphoglyceraldehyde combines with a phosphate group derived from inorganic phosphoric acid (H₃PO₄) found in cytosol, not form ATP, forming1, 3-diposphoglycerate, or diphosphoglyceric The reaction occurs with the aid of a specific enzyme.
• In dehydrogenation, a pair of hydrogen atom separate from a molecule of 3-phosphoglyceraldehyde. Their separation releases a large amount of A part of this energy is stored in newly formed phosphate bond of 1,3-diphosphoglycerate, making it a high energy bond. Separation of hydrogen is catalysed by an enzyme, 3- phosphoglyceraldehyde dehydrogenase.
• As stated above, two hydrogen (H) atoms (2 proton and 2 electrons) separate from 3- phosphoglyceraldehyde. Of these, one complete hydrogen atom (proton and electron) and one additional electron are picked up by NAD⁺ which gets reduced to NADH. The remaining one hydrogen proton or ion (H⁺) remains free in the

2H ⁺ + 2e ^– + NAD ⁺ ® NADH + H ⁺

NADH is a high-energy substance, carrying the rest of the energy released by separation of hydrogen atoms from 3- PGAL. Energy is actually released by transfer of electrons from 3-PGAL to NAD. The NADH provides energy to convert ADP to ATP by passing its electrons over the electron transmitter system if oxygen is available.

The overall reaction is as under –

3 – PGAL + NAD⁺ + Pi 2-  ¾¾^3-P¾^hos¾^pho¾^glyc¾^e¾^r– ®1,3 – diphosphoglycerate + NADH + H ⁺

aldehyde dehydrogenase

 

• Dephosphorylation or ATP generation (First) : High-energy phosphate group on carbon 1 of 1,3 diphosphoglycerate is transferred to a molecule of ADP, converting it into an ATP 1, 3- diphosphoglycerate changes to 3-phosphoglycerate due to loss of a phosphate group. The reaction is catalysed by an enzyme diphosphoglycerokinase. Formation of ATP directly from metabolites is known as substrate level phophorylation.

Diphosphoglycero-

1, 3-diphosphoglycerate +ADP                        3-phosphoglycerate + ATP

kinase + Mg ²⁺

 

 

 

 

 

• Isomerisation/ Rearrangement  :  The phosphate group on the third carbon of 3-phosphoglycerate shifts to the second carbon, producing 2-phosphoglycerate. This change is aided by the enzyme

 

3-phosphoglycerate

Phosphoglycero- mutase

2-phosphoglycerate

 

 

• Dehydration : 2-phosphoglycerate loses a water molecule in the presence of an enzyme, enolase and Mg²⁺, and changes into phosphoenol The latter undergoes molecular rearrangement that transforms its phosphate group into a high-energy phosphate bond.

Enolase

 

2-phosphoglycerate

Mg 2+

Phosphoenol pyruvate +H₂O

 

• Dephosphorylation or ATP generation (Second) : High-energy phosphate group of phosphoenol pyruvate is transferred to a molecule of ADP with the help of an enzyme, pyruvate kinase in the presence of Mg²⁺ and K⁺. This produces simple 3-carbon pyruvate and a molecule of

 

~ phosphoenol pyruvate +ADP

   Pyruvate kinase        

Mg ²⁺, K⁺

Pyruvate +ATP

 

All enzymes, reactants, intermediates and products of glycolysis are dissolved in the cytosol. Their interaction depends on random collisions brought about by kinetic movements.

• Special features of glycolysis : The special features of glycolysis can be summarised as follows :
• Each molecule of glucose produces 2 molecules of pyruvic acid at the end of the
• The net gain of ATP in this process is two ATP molecules (four ATPs are formed in glycolysis but two of them are used up in the reaction).
• During the conversion of 1, 3-diphosphoglyceraldehyde into 1, 3-diphosphoglyceric acid one molecule of NADH₂ is formed. As each molecule of glucose yields two molecules of 1,3-diphosphoglyceric acid, hence, each molecule of glucose forms 2 molecules of NADH₂.
• During aerobic respiration (when oxygen is available) each NADH₂ forms 3 ATP and H₂O through electron transport system of mitochondria. In this process ½ O₂ molecule is utilized for the synthesis of each water

In this way during aerobic respiration there is additional gain of 6 ATP in glycolysis

 

2ATP +

(net gain)

6 ATP

(addition gain)

®   8 ATP

(total net gain)

 

• Reaction of glycolysis do not require oxygen and there is no output of CO₂.
• Overall reaction of glycolysis represented by following reaction :

C6 H12O6 ® 2C3 H4 O3 + 4 H

Pyruvate

• Total input and output materials in glycolysis :

 

 

 

1 molecule of glucose (6 C)	2 molecules of pyruvate (2×3 C)
2 ATP	4 ATP
4 ADP	2 ADP
2 × NAD +	2× NADH + 2H+
2 Pi	2×H2O

Important Tips

• Lavosier (1783) found that respiration in animals involves intake of O₂ and liberation of CO₂. Dutrochet is belived to have used the term of respiration for the first time, while book “cellular respiration” was written by
• Energesis : An old term of
• Glucose oxidation is very rapid process of complete oxidation of a glucose molecules takes only one second.
• Only 5% of total energy of glucose is released during
• Utility of phosphorylation during glycolysis : It traps glucose with in the cell as glucose 6-p is negatively
• Splitting of fructose 1,6-diP into 3-PGAL and dihydroxyacetone P is called rate determining step of
• Glucose 6-phosphate called Rohinsonester, fructose 6-phosphate called Newberg’s ester and fructose 1,6-diphosphate called Harden and Young’s ester.
• B.Cs gets energy only by glycolysis because they lacks mitochondria.
• Phosphofructokinase called regulatory enzyme of glycolysis, it is inhibited by high concentration of ATP and is stimulated by ADP and
• Preparatory phase of  glycolysis involves conversion of one molecule of glucose into two molecules of 3-PGAL and involves the use of 2 ATP molecules, while pay-off phase of glycolysis involves conversion of 2 molecules of 3-PGAL into two molecules of pyruvate and involves production of four ATP Preparatory phase causes activation of glucose, while pay-off phase involves extraction of energy from the activated glucose.
• Formation of 1,3-diphosphoglyceraldehyde called non enzymatic phosphorylation.

 

Aerobic oxidation of pyruvic acid

• Oxidative decarboxylation/ Formation of acetyl
• Kreb’s cycle/TCA cycle/Citric acid
• Electron transport system
• 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 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 :

 

 

 

 

Pyruvic dehydrogenase

CH3CO.COOH+ CoA.SH + NAD ¾¾^mul¾^tien¾^zym¾^{e co}¾^mpl¾^ex¾® CH3 .CO.S.CoA+ NAD.2H + CO2

 

(Pyruvic acid)

(CoA)

**TPP

**LAA

(acetyl – S-CoA)

 

 

**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

• 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
• Site of occurrence : It takes place in the mitochondrial

(iii)

Proteins

Fat

Kreb’s cycle

 

 

 

 

Glycine

2 Acetyl CoA

Pyruvic acid

Pyruvic dehydrogenase

1/2 O₂ H₂O

Aceto acetyl CoA

CoA

 

NAD NADH₂

 

CO₂

Alanine

Amino acids

 

 

 

 

 

 

 

Aspartic acid

 

 

 

Oxalo acetic acid

NADH₂ NAD

H₂O¬1/2 O₂

Malic acid

 

 

 

 

 

Malic dehydrogenase

Synthetase

 

 

 

 

 

 

Aconitase

 

H₂O

Citric acid

CoA

 

 

Fe++

Cis-aconitic acid

H₂O

 

 

 

 

 

 

 

 

 

 

 

 

ATP ADP

H₂O

 

 

 

Succinic acid

Fumaric acid

H₂O¬1/2 O₂ FADH₂ FAD

 

 

H₂O

GTP GDP

 

Succinyl CoA

4C

Fumarase

 

 

 

Succinic dehydrogenase

 

 

 

Succinic thiokinase

 

 

3          3

ATP

2     POOL     3 1 3

Aconitase

 

 

 

 

 

 

 

Isocitric dehydrogenase

H₂O Fe++

 

Isocitric acid

6C

 

1/2 O₂®H₂O

 

NAD NADH₂

 

Mn++

 

 

Oxalo succinic acid

6C

 

 

CO₂

 

 

NADH₂

NAD

Ketoglutaric dehydrogenase

11

H₂O¬1/2 O₂

a-Ketoglutaric acid

5C

Decarboxylase Mn++

 

CO₂

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(iv)  Enzymes of Kreb’s cycle

 

Step	Enzyme	(Location in mitochondria)	Coenzyme(s) and cofactor (s)	Inhibitor(s)	Type of reaction catalyzed
(a)	Citrate synthetase	Matrix space	CoA	Monofluoro-acetyl- CoA	Condensation
(b)	Aconitase	Inner membrane	Fe2+	Fluoroacetate	Isomerization
(c)	Isocitrate dehydrogenase	Matrix space	NAD+, NADP+, Mg2+, Mn2+	ATP	Oxidative decarboxylation
(d)	a -ketoglutarate dehydrogenase complex	Matrix space	TPP,LA,FAD,CoA, NAD+	Arsenite,Succinyl- CoA, NADH	Oxidative decarboxylation
(e)	Succinyl-CoA synthetase	Matrix space	CoA	–	Substrate level phosphorylation
(f)	Succinate dehydrogenase	Inner membrane	FAD	Melonate, Oxaloacetate	Oxidation
(g)	Fumarase	Matrix space	None	–	Hydration
(h)	Malate dehydrogenase	Matrix space	NAD+	NADH	Oxidation

• Steps in Kreb’s cycle : Kreb’s cycle consists of 8 cyclic steps, producing an equal number of organic 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.

• 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 The citrate has 3-carboxyl group. Hence, Krebs cycle is also called tricarboxylic acid cycle, or TCA cycle after its first product.

Oxaloacetate + Acetyl CoA + H 2 O ¾¾^Citr¾^a¾^te ®Citrate + CoA(Reused)

Synthetase

• Reorganisation (Dehydration) : Citrate undergoes reorganisation in the presence of an enzyme,

aconitase , forming 6-carbon cisaconitate and releasing water.

Citrate ¾¾^Aco¾^nita¾^se ®Cisaconitate + H2O

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

 

 

 

 

Cisaconitate + H2O ¾¾^Aco¾^nita¾^se ®Isocitrate

• 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 Mn²⁺. NADH generates ATP by transferring its electron over the ETS.

Isocitric acid + NAD ¾¾^Isoc¾^itric¾^deh¾^ydro¾^gen¾^a¾^se ®Oxalosuccinic acid + NAD.2H(or NADPH.2H)

Mn2+

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

2

Oxalosuccinic acid ¾¾^Car¾^boxy¾^la¾^se ®a – Ketoglutaric acid + CO

Mn2+

 

• Oxidative decarboxylation II : This is also a 2 stage process :

Stage I : Coenzyme A reacts with a -ketoglutarate, forming 4-carbon succinyl-coenzyme A and releasing

CO₂ and a pair of hydrogen atoms. The reaction is catalysed by a -ketoglutarate dehydrogenase complex enzyme. the pair of hydrogen atoms pass two electrons and one H⁺ to NAD⁺, forming NADH + H⁺

a – ketoglutarate + CoA + NAD⁺ ¾¾^a-k¾^etog¾^luta¾^ra¾^te ®Succinyl – CoA + CO2  + NADH + H ⁺

dehydrogenase

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.

Succinyl – CoA + H 2 O + GDP / ADP ¾¾^Suc¾^ciny¾^l–C¾^o¾^A ® Succinate + CoA + GTP / ATP

Synthetase

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.

• 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

 

Succinate+FAD⁺

Succinate

dehydrogenase

Fumarate +FADH₂

 

• Hydration : This process changes fumarate into 4-carbon maltate in the presence of water and an enzyme,

 

Fumarate +H₂O

Fumarase

Maltate

 

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

 

Maltate +NAD⁺

Maltate

dehydrogenase

Oxaloacetate +NADH +H⁺

 

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

 

 

 

(vi)  Summary of Kreb’s cycle

• All the enzymes, reactants, intermediates and products of TCA cycle also are found in aqueous solution in the matrix, except the enzyme a-ketoglutarate dehydrogenase and succinate dehydrogenase which are located in the inner mitochondrial Both are called mitochondrial marker enzyme.
• Oxidation of one mole of acetyl CoA uses 4 molecules of water and releases one molecule of water.
• Liberates 2 molecules of carbon
• Gives off 4 pairs of hydrogen
• Produces one GTP/ ATP molecule during the formation of
• One mole of acetyl CoA gives 12 ATP during oxidation in Krebs
• Regenerates oxaloacetate used in last cycle for

The above summary is for one molecule of acetyl coenzyme A. There are two acetyl coenzyme A molecules formed from one molecule of glucose by glycolysis and oxidative decarboxylation of pyruvate. The entire Krebs cycle may be represented by the following equation –

2Acetyl coenzyme A + 8H2O + 6 NAD⁺ + 2FAD⁺ + 2GDP / ADP + 2Pi

® 4CO2 + 2H2O + 6 NADH + 2FADH2 + 2GTP / ATP + 6H ⁺

(vii)  Difference between Glycolysis and Kreb’s cycle

 

Glycolysis	Kreb’s cycle
It takes place in the cytoplasm.	It takes place in the matrix of mitochondria.
It occurs in aerobic as well as anaerobic respiration.	It occurs in aerobic respiration only.
It consists of 9 steps.	It consists of 8 steps.
It is a linear pathway.	It is a cyclic pathway.
It oxidised glucose partly, producing pyruvate.	It oxidises acetyl coenzyme A fully.
It consumes 2 ATP molecules.	It does not consume ATP
It generates 2 ATP molecules net from 1 glucose molecules.	It generates 2 GTP/ATP molecules from 2 succinyl coenzyme A molecules.
It yields 2 NADH per glucose molecule.	It yields 6 NADH molecules and 2 FADH2 molecules from 2 acetyl coenzyme A molecules.
It does not produce CO2.	It produces CO2.
All enzyme catalysing glycolytic reactions are dissolved in cytosol.	Two enzymes of Krebs cycle reactions are located in the inner mitochondrial membrane, all others are dissolved in matrix.

• Product form during aerobic respiration by Glycolysis and Kreb’s

(a)  Total formation of ATP

 

ATP formation in Glycolysis
	Steps	Product of reactions	In terms of ATP
ATP formation by substrate phosphorylation	1, 3-diphosphoglyceric acid (2 moles) ® 3 phosphoglyceric acid (2 moles) Phosphoenolpyruvic acid (2 moles) ® Pyruvic acid (2 moles)	2 ATP 2 ATP	2 ATP 2 ATP
		Total	4 ATP

 

 

 

ATP formation by oxidative phosphorylation or ETC	1, 3 – disphosphoglyceraldehyde (2 moles) 1, 3 – diphosphoglyceric acid (2 moles)		2 NADH2		6 ATP
	Total ATP formed		4 + 6 ATP =		10 ATP
ATP consumed in Glycolysis	Glucose (1 mole) ® Glucose 6 phosphate (1 mole) Fructose 6 phosphate (1 mole) ® Fructose 1, 6-diphosphate (1 mole)		–                           1 ATP   –                           1 ATP		–  1 ATP   –  1 ATP
			Total		2 ATP
	Net gain of ATP = total ATP formed – Total ATP consumed		10 ATP – 2ATP		8 ATP
ATP formation in Kreb’s cycle
ATP formation by substrate phosphorylation	Succinyl CoA (2 mols) ® Succinic acid (2 mols)	2 GTP		2 ATP
		Total		2 ATP
ATP formation by oxidative phosphorylation or ETC	Pyruvic acid (2 mols) ® Acetyl CoA (2 mols) Isocitric acid (2 mols) ® Oxalosuccinic acid (2 mols) a-Ketoglutaric acid (2 mols) ® Succinyl CoA (2 mols) Succinic acid (2 mols) ® Fumaric acid (2 mols) Malic acid (2 mols) ® Oxaloacetic acid (2 mols)	2 NADH2   2 NADH2   2 NADH2   2 FADH2   2 NADH2		6 ATP     6 ATP   6 ATP   4 ATP   6 ATP
		Total		28 ATP
	Net gain in Kreb’s cycle (substrate phosphorylation + oxidative phosphorylation)	2ATP + 28 ATP		30 ATP
Net gain of ATP in glycolysis and Kreb’s cycle	Net gain of ATP in glycolysis + Net gain of ATP in Kreb’s cycle	8 ATP + 30 ATP		38 ATP
Over all ATP production by oxidative phosphorylation or ETC	ATP formed by oxidative phosphorylation in glycolysis + ATP formed by oxidative phosphorylation or ETC.	6 ATP + 28 ATP		34 ATP

22 ATP produced by oxidation of NADH₂ and FADH₂ in Kreb’s cycle and 6 ATP comes from oxidative decarboxylation of pyruvic acid.

(b)  Formation and use of water

 

Formation of water molecules
Formation of water molecules in glycolysis	2 phosphoglyceric acid (2 mols)  ¾¾–H¾2¾O ® 2 phosphoenol pyruvic acid (2 mols) 1, 3-diphosphoglyceraldehyde  ¾¾–H¾2¾O ®	2H2O
		2H2O

 

 

 

	1, 3 diphosphoglyceric acid
	Total water molecules formed in glycolysis	4H2O
Formation of water molecules in kreb’s cycle	One molecule of water in each of the five oxidation reactions (these reactions occur twice as there are two molecules of pyruvic acid).	10 H2O
	Other than oxidation reaction Citric acid (2 mols) ® Cis-aconitic acid (2 mols)	2H2O
	Total water molecules formed in Kreb’s cycle	12 H2O
	Total water molecules formed in aerobic respiration (Glycolysis + Kreb’s cycle)	16 H2O
Use of water molecules
Use of water in Glycolysis	3-phosphoglyceraldehyde  (2 mols)  ¾¾+H¾2¾O ®  1, 3 diphosphoglyceric acid (2 mols)	2H2O
	Total water molecule used in glycolysis	2H2O
Use of water in Kreb’s cycle	Oxaloacetic acid (2 mols)  ¾¾+H¾2¾O ®  Citric acid (2 mols)	2H2O
	Cis aconitic acid (2 mols)  ¾¾+H¾2¾O ®  Isocitric acid (2 mols)	2H2O
		2H2O
	Succinyl CoA (2 mols)  ¾¾+H¾2¾O ®  Succinic acid (2 mols)	2H2O
	Fumaric acid (2 mols)  ¾¾+H¾2¾O ®  Malic acid (2 mols)
	Total water molecules used is Kreb’s cycle	8H2O
	Total water molecules used in aerobic respiration (Glycolysis + Kreb’s cycle)	10H2O
Net gain of water molecules in aerobic respiration	Number of water molecules formed – Number of water molecules used = ( 16 H2O – 10H2O)	6H2O

 

• Evolution of carbon dioxide

 

Pyruvic acid (2 mols)  ¾¾–C¾O¾2  ®  Acetyl CoA (2 mols) Oxalosuccinic acid   ¾¾–C¾O¾2  ®  a ketoglutaric acid (2 mols) a Ketoglutaric acid (2 mols)  ¾¾–C¾O¾2  ®  Succinyl CoA (2 mols)	2CO2 2CO2 2CO2
Total CO2 molecules released in aerobic respiration	6CO2

 

(d)  Use of O₂ (Oxygen)

 

Use of oxygen in Glycolysis	+ 1 O2 1, 3-diphosphoglyceraldehyde (2mols)  ¾¾2¾ ®  1, 3-diphosphoglyceric acid (2 mols)	1O2
Use of oxygen in Kreb’s cycle	Five oxidation reactions of Kreb’s cycle (2 times)	5O2
	Total O2 molecules required for aerobic respiration	6O2

 

 

 

 

• Energy storage and energy transfer : In respiration energy released takes in the form of chemical energy, stored in a form called ATP . Energy transfer of biological oxidation hinges on the formation of labile high energy phosphate bonds of ATP. Nicotinamide adenine dinucleotide phosphate (NAD), Flavin adenine dinucleotide (FAD), Guanosine triphosphate are also the product of respiration and converted to ATP by electron transport

(a) Adenosine triphosphate : An energy intermediate :

There are several compounds like NAD, FAD, GTP and ATP are known as energy yielding compounds. The best known, and probably the most important of these are adenosine triphosphate (ATP). It serves as the energy currency of the cells.

• Structure of ATP : Adenosine triphosphate is a

 

nucleotide consisting of three main constituents;

High energy bonds

N

N

Adenine

 

• A nitrogen contain purine base
• A five carbon sugar ribose
• Three inorganic phosphate groups

The bonds attaching the last two phosphate to the

 

O

||

O —  P

| O

O              O

||              ||

~ O —  P ~ O — P

|               |

O              O

N        N

 

~ O — CH₂

O

 

H       H H

 

 

 

 

 

Ribose

 

rest of the molecule are high energy bonds (~)  contain

Inorganic phosphate

H

OH      OH

 

more than twice the energy of an average chemical bond.

Fig : Structure of ATP

 

 

 

 

• ATP  hydrolysis   :      The energy is usually released from ATP by hydrolysing the terminal phosphate Each molecule on hydrolysis yields

ADP, one inorganic phosphate group (Pi) and about 7.28 Kcal energy.

High energy bond

 

Adenosine   P      P      P H₂O Hydrolysis

 

ADP further hydrolysed to AMP and inorganic phosphate, releasing 7.3 Kcal energy per molecule (of ATP). Above process represented by following reactions.

Adenosine

+30.6 Kj mol

 

Work

 

 

Adenosine triphosphate ¾¾^hyd¾^roly¾^sis ® Adenosine diphosphate(ADP) + Pi + 7.3Kcal…..

hydrolysis

 

Adenosine diphosphate ®  Adenosine monophosphate(AMP) + Pi + 7.3Kcal.

Fig : Hydrolysis of ATP

 

• Phosphorylation : The ATP hydrolysis reactions are reversible because ATP are synthesized from ADP, Pi and energy (take up for the bond formation). The addition of phosphate group to ADP and AMP called Energy required for the bond formation is equal to the energy released in hydrolysis.

 

 

P                   P

Energy yield of

30.6 kJ or more from respiration

Adenosine

P      P

ADP

 

Adenosine               P        P ATP

Fig : phosphorylation

 

 

The significant role of ATP as an intermediate energy transfer compound

• Major functions of ATP : ATP molecules receive the energy, which released in exergonic reactions and make this energy available for various endergonic reactions. Some of the important process in which ATP is utilized are as follows :
• Synthesis of carbohydrates, proteins, fats,
• Translocation of organic
• Absorption of organic and inorganic
• Protoplasmic

 

 

Fig : Schematic representation of the role of ATP

(b) Nicotinamide  dinucleotide  phosphate/  Nicotinamide  dinucleotide  (NADP/NAD)  :    It is called universal hydrogen acceptor, produced during aerobic respiration (glycolysis+ Kreb’s cycle) and also in anaerobic respiration, work as coenzyme in ATP generation Via electron transport system. NADP have one additional phosphate.

Structure of   NAD   =   Nicotinamide-adenine-dinucleotide   (formerly   called   coenzyme   I   or CO-I

 

Diphosphopyridine dinucleotide = DPN) is shown below :

[Nicotinamide]

 

 

 

O

O      P       OCH₂

O              O

 

 

 

 

It plays a crucial role in dehydrogenation processes. Some dehydrogenases do not work with NAD, but react with NADP (Nicotinamide adenine dinucleotide phosphate). Formerly called Coenzyme II or Triphosphopyridine nucleotide = TPN Nicotinamide is a vitamin of B group.

First NAD and NADP both functions as hydrogen acceptors. Later H ions and electrons (e^_) from these are transported through a chain of carriers and after being released at the end of a chain react with O₂ and from H₂O (see Electron Transport chain). During the release of 2 electron from 2H⁺ atoms from NAD. 2H and their reaction with O₂ to form water, 3 ATP molecules are synthesized.

 Important Tips                                                                                                                                                                                                          

• Krebs cycle is the central pathway of the cell respiration where the catabolic pathways converge upon it an anabolic pathways diverge from it, so called amphibolic
• Acetyl Co~A, also called active
• Number of oxidation steps(dehydrogenation) for pyruvic acid is 5.
• In Kreb’s cycle, acetyl CoA undergoes two decarboxylation and four dehydrogenation. Krebs cycle catabolises about 80-90% of
• ATP discovered by Lohmann (1929), term was coined by Lohmann (1931) while ATP cycle was discovered by Lipmann (1941).
• Allosteric inhibition or negative feedback by accumulation of NADH
• Production of 36 ATP molecules from the oxidation of glucose is only an estimate as :
• NADH₂ may be used in some other metabolic
• NADH₂ do not always produces 3 ATP More active muscle cells produces more while less active fat cell produce less ATP molecules.
• Not all the proteins are routed through F₀ –F₁
• So realistic aerobic respiratory efficiency ranges between 22% to 38%, as realistic power limit is 21 ATP
• 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, a – 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,

Glucose

 

Diphospholyceraldehyde                                               or

 

Diphosphoglyceric acid Pyruvic acid Acetyl-CoA

 

 

 

 

(Kreb’s cycle)

Isocitric acid Oxalosuccinic acid

ADP+iP

 

ATP

 

2

ADP+iP

 

 

Oxaloacetic acid

a-Ketoglutaric acid

NAD2H         2e

FAD

CoQ

19

RED     2e

2Fe+++

2Fe⁺⁺ 2e

2Fe+++

2Fe⁺⁺

2e 2Fe⁺⁺⁺            O

 

Succinal-S CoA

2e                        2 Cyt b

2 Cyt c₁         2 Cyt c

2 Cyt a

2 Cyt a₃

 

Malic acid

Succinic acid

NAD⁺

2H⁺

FADH₂

2H⁺

CoQ_OX

2Fe⁺⁺ 2e

2Fe+++

2Fe⁺⁺

2Fe+++

2Fe⁺⁺

2e ½ O2

 

 

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.

Electron – donor ® e^– + electron – acceptor

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.

ADP + Pi ¾¾^ATP¾^Sy¾^nthe¾^ta¾^se ® ATP

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

 

H  + 1 O

2      2   2

® H 2

O (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.

• 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 Thus, regeneration of NAD⁺ or FAD takes place in ETC. There are two routes ETC :
• Route 1 : NADH₂ passes their electrons to Co-Q through FAD . In route 1 FAD is the first electron 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

• 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

• 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

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

• 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.

Outer             Inner membrane    membrane

 

NADH

Complex-I

 

Complex-III

 

 

 

Complex-IV ATP

 

Succinate

                              O₂

 

 

 

 

 

Outer chamber

Stalk Base-piece

Complex-II

Head piece

 

Fig. Four complexes of Oxysome

 

• Five complex theory : According to Hatefi, (1976), Complex I to Complex IV are related to the electron
• Complex V related to mainly with ATP synthesis, so it is called ATPase /ATP syntheses
• The head piece (F₁) of the oxysome consists of 5 hydrophobic subunits ( a, b ,g ,d ,e ), which are responsible for ATPase
• The stalk (F₀) contain F₅ (oligomycin sensitivity conferring protein) e. CSCP and F₆. F₀ are related to the proton channel and embeded fully in thickness of inner mitochondrial membrane.
• Five complex e. I, II, III, IV, V, have been isolated from mitochondrial membrane by chemical treatment.
• Complex I : NADH/NADPH : CoQ reductase Complex II : Succinate : CoQ reductase

Complex III : Reduced CoQ (CoQH₂) : cytochrome C reductase Complex IV : Cytochrome C oxidase

 

 

 

 

 

 

 

 

 

 

Complex V : ATPase

• Cytochrome C and Q are mobile components of the respiratory

C-side

 

 

Stalk

M-side

 

 

F5

d          g

 

 

 

 

Head piece

(F₁ ATPase proper)

b        a

 

ATPase inhibitor protein

 

• Oxidative phosphorylatio^Fn^ig.:^FiT^veh^ce^omp^pro^lec^xe^esss^ofo^Of^xyA^soT^mP^e synthesis during oxidation of reduced coenzymes in ETC is called oxidative Peter Mitchell (1961) proposed the chemiosmotic

Intermembrane space

Inner mitochondrial membrane

 

 

2H

Matrix

 

NADH

+H-+

 

mechanism of ATP synthesis (Noble prize in 1978) which states that ATP synthesis occurs due to H⁺– flow through a membrane. It involves two steps :

2H⁺

2H

 

2e^– FeS

FMN

 

FeS

NAD⁺

 

• Development of proton gradient. At each step of ETC, the

2H⁺

QH             2e^–

2                    O

2H⁺

 

electron- acceptor has a higher electron –affinity than the electron-donor. The energy from electron-transport is used to move the proton (H⁺) from the mitochondrial matrix to inter-membranous or outer chamber. Three pairs of protons are pushed to outer chamber during the movement of electrons along route I while two pairs of protons are moved to outer chamber during the movement of electrons along route–II. This generates a

+

 

 

 

 

 

2H⁺

2e^– Cy b

 

 

QH₂

 

2e^– Cy

 

 

2e^– FeS

 

 

2e^–

 

 

 

 

 

2H⁺

 

1/2O₂

 

pH-gradient across the inner mitochondrial membrane with protons (H )

concentration higher in the outer chamber than in the mitochondrial matrix.

Cy c

2e^– Cya-a₃

2H⁺

H₂O

 

This difference in H⁺ concentration across the inner mitochondrial

Fig : Proton flow

 

membrane is called proton-gradient( D pH). Due to proton gradient, an electrical potential (Dy) is developed across the inner mitochondrial membrane as the matrix is now electronegative with respect to the intermembranous (outer) chamber. The proton gradient and membrane electric potential collectively called proton motive force.

• Proton flow : Due to proton-gradient, the protons returns to the matrix while passing through proton channel of F₀–F₁ This proton gradient activates the enzyme ATP synthetase or F₀ – F₁ ATPase

ATP synthetase controls the formation of ATP from ADP and inorganic phosphate in the presence of energy.

2H+                  Inner

 

 

 

Important Tips

• Cytochromes were discovered by MacCunn and term cytochrome given by P.Kailin.
• Iron-Sulphur is the component of ETC (complex I, II, III) and helps in transfer of electrons from FMNH₂ to coenzyme Q. Thus, deficiency of iron direct affect ETC or oxidative phosphorylation.
• Cytochromes are Iron-containing (Iron porphyrin protein) electron transferring (electrons picked up and release by Fe) except cytochrome a₃. Cytochrome a₃ contains both Iron and Copper, in this Fe picks the electrons and through Cu it hands over electron to oxygen, so cytochrome a₃ called terminal electron
• Cytochrome P-450 : It occurs in E R and takes part in hydroxylation
• ETC inhibitors
• Dinitrophenol (2,4-DNP) : It prevents synthesis of ATP from ADP because it directs electrons from CoQ to Q
• Cyanide : It prevents flow of electrons from Cyt a₃ to oxygen.
• Carbon monoxide : It functions like cyanide.
• Antimycin A: Transfer of electron from Cyt b to Cyt c₁ is
• Rotenone : It checks flow of electrons from NADH /FADH₂ to
• Action of ATPase needed Na⁺ and K⁺.
• Amount of energy released in ETC :
• 2 Kcal during transfer of electrons from NAD to FMN.
• 2 Kcal during transfer of electrons from Cyt b to Cyt c.
• 5 Kcal during transfer of electrons from Cyt a to Cyt a_3.

 

• Role of shuttle system in energy production : Glycolysis occurs in the cytoplasm outside the mitochondrion in which 2NADH₂ molecules are produced but ETC is located along inner mitochondrial membrane, so NADH₂ of glycolysis must enter inside the mitochondrion to release energy. But the inner mitochondrial membrane is impermeable to NADH₂. In mitochondrial membrane, there are 2 shuttle-system, each formed of carrier-molecule.

These shuttle systems are :

• Malate-Aspartate shuttle : It is more efficient and results in the transfer of electron from NAD. 2H in cytosol to NAD inside the mitochondrion, via 2H dehydrogenase as follows :

Electrons are transferred from NAD. 2H in cytosol to malate which traverses the inner mitochondrial membrane and reoxidised to form NAD. 2H thus resulting in the formation of oxaloacetate . Oxaloacetate does not readily cross the inner mitochondrial membrane and so a transamination reaction is needed to form aspartate which

 

23

 

 

 

 

does traverse this barrier. As a result 3 ATP molecules are generated for each pair of electrons. Thus if this shuttle is predominant there is a gain of 38 ATP molecules by complete oxidation of one molecule of glucose.

 

 

 

Cytosol

NAD.2H          NAD

 

 

Oxaloacetate  Malate Aspartate

Outer membrane

Inner

membrane        Matrix

Malate            dehydrogenase

Malate          NAD

 

Oxaloacetate       NAD.2H Aspartate

 

Fig : Malate-Aspartate shuttle

 

 

• Glycerol-Phosphate shuttle : It is less efficient and results in the reduction of FAD inside the

If this shuttle predominates the electrons from NAD. 2H are transferred to FAD inside the mitochondrion as follows. NAD. 2H reacts with dihydroxyacetone phosphate (DHAP) in cytosol to form glycerol phosphate which diffuses through outer mitochondrial membrane to the outer surface of inner membrane. There glycerol phosphate reacts with membrane dehydrogenase to form dihydroxyacetone phosphate (DHAP) which returns to cytosol. In this process FAD is reduced to FADH₂. Electrons from FADH₂ directly pass to Q and other components of ETC and results in the synthesis of 2 ATP for each molecule of FADH₂. In this case complete oxidation of glucose will result in a gain of 36 ATP molecule.

 

 

Cytosol

NAD.2H          NAD

Outer membrane

Inner

membrane      Matrix

Glycerol phosphate dehydrogenase

 

DHAP

Glycerol phosphate

Glycerol phosphate

 

DAPH

FAD

 

 

 

FADH₂

 

 

Fig : Glycerol-Phosphate shuttle

 

Which shuttle predominates depends on the particular species and tissues envolved, for example : 38 ATP are formed in kidney, heart and liver cell while 36 ATP molecules are formed in muscle cells and nerve cells. In these cells glycerol-phosphate shuttle is predominant and 2 ATP formed from NADH_2.

 Other pathways of glucose oxidation.

(1)  Entner-Doudoroff pathway

• Discovery : Entner-Doudoroff path discovered by Entner & This pathway is also called glycolysis of bacteria.

Certain bacteria such as Pseudomonas sacchorophila, P. fluorescens, P. lindeneri and P. averoginosa lack phosphofructokinase enzyme. They can not degrade glucose by glycolytic process.

 

 

ATP ADP

Glucose (6C)

Glyceraldehyde 3-phosphate

Pyruvic acid (3C)

24

 

Glucose-6-phosphate (6C)

 

 

 

 

 

• Description : In this pathway the glucose molecule first phosphorylated to Glucose-6-phosphate by ATP. Then it oxidised to 6-phosphogluconic acid by NADP which itself reduced to NADPH₂ by the electrons released. The NADPH₂ is channeled through ETS system to produce 3-molecules of ATP per NADPH₂ molecule through ETS system to produce 3 molecules of ATP per NADH₂ molecule and 1,6-phosphogluconic acid is channeled to pyruvic The main reaction are :

• Glucose – 6 – phosphate + NADP ® 6 – Phosphogluconolactone + NADPH ₂
• 6-phosphoglyconolactone ® 2-Keto-3-deoxyphospho-6-gluconic acid
• 2-Keto-3-deoxyphosphogluconic acid ® Pyruvic acid + Glyceraldehyde-3-phosphate
• Glyceraldehyde-3-phosphate ® Pyruvic acid

The glyceraldehyde 3-phosphate by EMP pathway gets converted into pyruvic acid which can be further used up in the process.

(2)  Pentose phosphate pathway

• Discovery : It is also called as Hexose monophosphate (HMP) shunt or Warburg Dickens pathway or direct oxidation It provides as alternative pathway for breakdown of glucose which is independent of EMP pathway (glycolysis) and Krebs cycle. Its existence was suggested for the first time by Warburg et al. (1935) and Dickens (1938). Most of the reaction of this cycle were described by Horecker et al.(1951) and Racker (1954).
• Occurrence : Pentose phosphate pathway that exists in many organisms. This pathway takes place in the cytoplasm and requires oxygen for its entire

 

 

ADP

NADPH+H⁺

NADPH+H⁺

 

Fig :  Hexose mono2p5hosphate shunt

 

 

 

 

• Description  :    There are two types of evidences is support of the existence of such an alternative pathway-works on the inhibiting action of malonic acid on the Krebs cycle and studies with the radioactive (C¹⁴).

Twelve molecules of NADH₂ formed in the reaction can be oxidised back to 12 NADP with the help of the cytochrome system and oxygen of the air.

12 NADPH 2  + 6O2  ¾¾^Cyt¾^ochr¾^om¾^e ®12H 2 O + 12NADP

System

 

In this electron transfer process, 36 molecules of ATP are synthesized. The reaction can be summarised as follows :

 

• 6 Glucose + 6 ATP ¾¾^hex¾^okin¾^a¾^se ® 6 Glucose – 6 – phosphate
• 6 Glucose – 6 – phosphate + 6 NADP + 6H2O ¾¾^deh¾^ydro¾^gen¾^a¾^se ® 6 phosphogluconic acid + 6NADPH2

oxidative

• 6 Phosphogluconic acid + 6NADP ¾¾^dec¾^arbo¾^xyla¾^tio¾ⁿ ® 6 ribulose – 5 – phosphate + 6 CO 2 + 6NADPH 2

dehydrogenase

 

• 2 Ribulose – 5 – phosphate ¾¾^isom¾^era¾^se ® 2 ribose – 5 – phosphate
• 5 Ribulose – 5 – phosphate ¾¾^isom¾^era¾^se ® 4 xylulose – 5 – phosphate
• 2 Xylulose – 5 – phosphate + 2 ribose – 5 – phosphate

¾¾^tran¾^sket¾^ola¾^se ® 2 sedoheptulose – 7phosphate + 2 glyceraldehyde – 3 – phosphate

• 2 Sedoheptulose – 7 – phosphate + 2 glyceraldehyde 3 – phosphate

¾¾^tran¾^sket¾^ola¾^se ® 2 fructose – 6 – phosphate + 2 erythrose – 4 – phosphate

• 2 Erythrose – 4 – phosphate + 2 xylulose – 5 – phosphate

¾¾^tran¾^sket¾^ola¾^se ® 2 fructose – 6 – phosphate + 2 glyceraldehyde 3 – phosphate

• 2 Glyceraldehyde – 3 – phosphate + H 2 O ¾¾^aldo¾^la¾^se ® fructose – 6 – phosphate + H 3 PO4

 

Sum total of the reaction :

6 Glucose-phosphate + 12 NADP+7H₂O

(iv)  Significance of PPP

 

¾¾® 5 Fructose-6-phosphate+6 CO₂ + 12NADPH₂ +H₂ PO₄

 

• It is the only pathway of carbohydrate oxidation that gives NADPH₂, Which is needed for synthetic action like synthesis of fatty acid (in adipose tissues) and amino acids (in liver).

 

 

 

• It synthesizes 3C-glyceraldehyde-3-P, 3C-dihydroxy acetone phosphate, 4C-erythrose-4-P, 5C-ribulose phosphate, 5C-xylulose phosphate, 5C-ribose phosphate, 6 C-Fructose 6-phosphate, 7C-sedoheptulose-7-
• It is the major pathway by which necessary ribose and deoxyribose are supplied in the biosynthesis of nucleotides and nucleic
• Erythrose 4 phosphate for the synthesis of lignin, oxine, anthocyanine and aromatic amino acid (phenylalanine, tyrosine, and tryptophan).
• Young growing tissues appears to use to the Krebs cycle as the predominant pathway for glucose oxidation, while aerial parts of the plants and other tissues seem to utilise the PPP as well as the Krebs
• It gives 6 CO₂, required for
• Ribulose five phosphate is used in photosynthesis to produce RuBP which act as primary CO₂ acceptor in

C₃ cycle.

(3)  Comparison of the different pathway of glucose metabolism

 

 

Phosphoglyceraldehyde
4ATP	4H

2 Pyruvic acid EMP

Pathway (Glycolysis)

Phosphoglyceraldehyde
2ATP	2H

Pyruvic acid

Pentose phosphate pathway

+

 

 

Phosphoglyceraldehyde 2ATP 2H

E.D.

pathway

 

Fig : A comparison of the different pathways of glucose metabolism

 

• Cyanide resistant pathway : Cyanide-resistant respiration seems to be widespread in higher plant tissues. Cyanide prevents flow of electron from Cyt a₃ to oxygen, so called ETC inhibitor. In these plant tissues resistance is due to, a branch point in the ETS preceeding the highly cyanide-sensitive The tissues lacking this branch point, or alternate pathway and blockage of cytochromes by cyanide, inhibits the electron flow.

Significance

• The role of alternative pathway is that it may provide a means for the continued oxidation of NADH and operation of the tricarboxylic acid cycle, even through ATP may not be sufficiently drained

 

 

• It is significant in respiratory climateric of ripening fruits and leads to the production of hydrogen peroxide and super oxide, which in turn enhances the oxidation and breakdown of
• Necessary activities in the ripening process because peroxides are necessary for ethylene

 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.

• 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 Glycolysis is occurs in cytoplasm so the site of anaerobic respiration is cytoplasm.

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

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

 

 

 

 

 

Anaerobic

2ADP+2(Pi)

 

2×pyruvic acid CH3COCOOH In the presence of oxygen

2ATP (Net gain)

 

In absence of oxygen Respiration

 

 

 

Kreb’s cycle

 

 

 

 

NAD⁺ NADH+H⁺        

CO₂

 

 

 

 

NAD⁺ NADH+H⁺       

Alcoholic fermentation

 

Fig : Summary of anaerobic respiration pathways

 

 

28

 

 

 

 

• In the first step pyruvic acid is decarboxylated to yield acetaldehyde and CO₂. In the presence of Mg⁺⁺ and

TPP (Thiamine pyrophosphate) pyruvate carboxylase.

CH3 COCOOH ¾¾^Pyru¾^v¾^ic ® CH3 CHO+ CO2

 

(Pyruvic acid)

Carboxylase

Mg ²⁺ +TPP

(Acetaldehyde)

 

• In the second step acetaldehyde is reduced to ethyl alcohol by NADH₂ formed in the

CH3 CHO+ NADH 2  ¾¾^Alco¾^ho¾^lic ® C2 H5 OH+ NAD

 

(Acetaldehyde)

dehydrogenase

(Ethyl alcohol)

 

• 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

CH3 COCOOH ® CH3 CHOH.COOH+ NAD

(Pyruvic acid)                              (Lactic acid)

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.

• Pasteur effect : Two types of respiration –anaerobic and aerobic respiration produce carbon dioxide in the ratio of 1:3 as shown in the

 

Anaerobic Respiration :

C6 H12 O6  ® 2C2 H5 OH + 2CO2

(Two molecules of CO₂ are produced)

 

Aerobic Respiration :

C6 H12O4 + 6O2 ® 6H2O + 6CO2

(Six molecules of CO₂ are produced)

 

Pasteur noted that when oxygen is given to the running anaerobic respiration the output of CO₂ is not similar to aerobic respiration, i.e. during aerobic respiration the ratio 1:3 does not always appear to be true. In several cases the amount of carbon dioxide is much less in comparison to normal aerobic respiration as shown above. For such cases it is considered that the presence of oxygen may sometimes lower down the rate of breakdown of sugar. The phenomenon is named as ‘Pasteur’s effects’ after the name of great scientist and the process may be defined as “the inhibition of sugar breakdown due to the presence of oxygen under aerobic condition” and the reaction is called Pasteur reaction. Dixon (1937) stated that the Pasteur effect is the action of oxygen is checking the high rate of loss of carbohydrate and in suppressing or diminishing the accumulation of products of fermentation.”

Pasteur’s effect is said to occur due to many reasons. Some of them are :

• Pasteur reaction inhibits some glycolytic enzyme and stops
• Formation of excess of CO₂ from degradation of compounds other than respiratory
• Increased glycolysis with decreased oxygen
• Occurrence of partial oxidative glycolytic products and oxidative anabolism (resynthesis, a process corresponding HMP pathway).

(3)          Connection between aerobic and anaerobic respiration

 

 

 

 

 

 

Glycolysis or EMP pathway

Carbohydrate       Glucose (C6H12O6)   29 2 mols of pyruvic acid

Glycolysis or EMP pathway

 

 

 

Oxidation of pyruvic acid

 

 

 

The glycolysis is the common phase and its products pyruvic acid is the common intermediate of the aerobic and anaerobic respiration.

• Fermentation : Fermentation is a kind of anaerobic respiration carried out by microorganisms fungi and bacteria. In microorganism the term anaerobic respiration is replaced by fermentation (Cruickshank, 1897) ; which is known after the name of its major product, g., alcohol fermentation, lactic acid fermentation.

Gay Lussac was the first to provide following reaction for the fermentation of sugar.

C₆H₁₂O₆ = 2CO₂+2CH₃CH₂OH+ 52 Kcal

Louis Pasteur (1822-1895) supported Gay Lussacs reaction and concluded that fermentation occurred and concluded that fermentation occurred only when living Yeast cells were present. Buchner (1897) found that yeast extract could perform fermentation of sugary solution. The enzyme complex present in yeast which could perform fermentation was named as zymase. Because of the latter, fermentation is also called zymosis. Besides zymase yeast cells also contain enzymes like sucrose and maltose which can ferment sucrose and maltose respectively. Direct fermentation of starch by yeast is not possible as it lacks amylase enzyme.

The fermentation is of two types : Homofermentive (one product) and heterofermentive (two or more than two types of products). Alcoholic fermentation may occur in almost, any moist sugar containing medium or sugar solution, such as fruit juice, which is inoculated with yeast or which is left exposed to air. The examples of fermentation :

• Butyric acid fermentation : It occurs in butter which has turned rancid. Bacteria like Clostridium butyricus and Bacillus butyricus are responsible for fermenting sugars and lactic acid into butyric acid to the following equation :

C6 H12O6 ® C4 H8O2 + 2H2 + 2CO2

(hexose)           (butyric acid)

2C3 H6O3 ® C4 H8O2 + 2H2 + 2CO2

(lactic acid)             (butyric acid)

• Lactic acid fermentation : In this process the lactose sugar, present in the milk, is converted into lactic acid which provides a distinctive sour taste to the Two bacteria viz., Bacterium lactic acidi and B.acidi lactici take part in this process.

C12H22O11 + H2O ® C6 H12O6 + C6 H12O6

 

(lactose)

(glucose)

(galactose)

 

C6 H12O6 ® 2C3 H6O3

(hexose)             (lactic acid)

 

 

 

 

• Acetic acid fermentation : It is different from other types of fermentation as it utilises atmospheric Acetic acid fermentation is catalysed by Acetabacter aceti, and A. xylinum which oxidised ethyl alcohol into acetic acid.

C2 H5 OH+ O2 ® CH3 COOH+ H 2 O + energy

(ethyl alcohol)                          (acetic acid)

• Importance of fermentation : Anaerobic respiration is advantageous in many ways :

• It supplement, the energy provided by aerobic respiration during intense muscular
• Brewing industry produces beers and wines by fermentation of sugary solution with yeast (saccharomyces cerevisiae).
• Baking industry uses CO₂ released by Yeast cells in alcoholic fermentation in raising the dough and making bread
• Dairy industry produces yogurt, cheese and butter by fermenting milk sugar lactose to lactic acid with lactic acid bacteria (Streptococcus lactis). Lactic acid coagulates the milk protein casein and fuses droplets of milk
• Tea and tobacco leaves are cured (freed of bitterness and imparted pleasant flavours) by fermentation with certain
• Vinegar is produced by fermenting molasses with yeast to ethyl alcohol which is then oxidised to acetic acid with aerobic acetic acid bacteria (Acetobacter aceti).
• Bacterial fermentation is also used for tanning hides (removal of fat, hair and other tissues).
• Retting of hemp fibers is achieved by fermentation with Pseudomonas
• Ensilase, a nutritive fodder for cattle, is prepared by fermentation with bacteria in air-tight-chambers called

silos.

(5)  Efficiency of respiration

• Efficiency of aerobic respiration : We used the generally accepted amount of 12,000-14,000 calories

per mole of ATP approximately 456,000-532,000 calories are generated from one mole of glucose. One mole of glucose contains about 686,000 calories(686 Kcal) of energy in the form of bonds. When one molecule of glucose is oxidised to carbon dioxide and water 673,000 calories of energy released.

However the actual amount of energy available from each ATP (rest of energy is lost as heat, and so on) is approximately 7,3000 calories (7.28 Kcal) or–34 kJ.

Therefore, actual energy yield from one mole of glucose is :

= 38×7.3=277.4 Kcal

 

 

So the percent of aerobic respiration =

277.4 ´ 100

686

 

= 40.43%

Thus, efficiency of aerobic respiration = 40% approx.

Out of 686 Kcal. of one mole glucose, only 277.4 Kcal. is trapped in the form of ATP.

• Efficiency of anaerobic respiration : In anaerobic respiration of carbohydrate by glycolysis apparently 2ATP molecules are formed per glucose Therefore, efficiency of anaerobic respiration will be :

C6 H12O6 ® 2C2H5OH + 2CO2 + 52 Kcal.

 

Percent of anaerobic respiration =

2 ´ 7.3 ´ 100 = 28.07%

52

 

 

 

 

• Efficiency of alcoholic fermentation : By Yeast only two molecules of ATP are generated per glucose molecule and efficiency will be, therefore,

C6 H12O6 ® 2C2H5OH + 2CO2  + 56Kcal.

 

 

Percent of fermentation =

2 ´ 7.3 ×100 =26.07%

56

 

(6)  Difference between respiration and fermentation

 

Respiration	Fermentation
It may occur both in the presence and absence of oxygen.	It does not require oxygen.
Occurs only in living cells.	It does not occur with in the living cells. It requires only enzymes and substrate.
Sugar is oxidised and CO2 and H2O are formed as end products.	Different substances oxidise to form alcohol or organic acids.
Complete oxidation of substrate occurs, hence produces large amount of energy.	Incomplete oxidation of substrate occurs and hence less energy is produced.
It can occur in any living cell.	It occurs mainly with the help of yeast or bacterial cells.

Many microbiologists have distinguished Aerobic respiration, Anaerobic respiration and Fermentation from one another principally by the means through which hydrogen is removed from various substrates (the hydrogen donor) and by nature of ultimate substrate accepting this hydrogen (the hydrogen acceptor).

Aerobic Respiration	Anaerobic Respiration	Fermentation
Molecular oxygen is the ultimate electron acceptor for biological oxidation. The ETS serves to transfer electrons from oxidisable donor to molecular oxygen. The early enzymatic steps involve dehydrogenation whereas the final steps are mediated by a group of enzyme called cytochromes. Ultimately the electrons are transferred to oxygen which is reduced to water. During aerobic respiration ATP is generated by coupled reaction	The ultimate electron acceptor is an inorganic compound other than oxygen. The compounds accepting the hydrogen (electrons) are nitrates, sulphates, carbonates or CO2. Anaerobic respiration produces ATP through phosphorylation reaction involving electron transfer systems. (mechanism not known)	The final electron acceptors are organic compounds. Both electron donors (oxidizable substrate) and electron acceptors (oxidizing agent) are organic compounds and usually both substrates arise from same organic molecules during metabolism. Thus part of the nutrient molecule is oxidised and part reduced and the metabolism results in intramolecular electron rearrangement. ATP is generated by substrate level phosphorylation. This reaction differs from oxidative phosphorylation because oxygen itself is not required for ATP generation.

 

 Fat, Protein and Salt respiration.                                                                                                                      

• Fat respiration : Fats are stored as triglycerides in cells, (in animal-adipose tissues and in plants-seeds). They break up into fatty acids and glycerol in the cytoplasm before use in respiration. Glycerol converted into Dihydroxy acetonephosphate and enters into glycolysis. The conversion of fatty acid into carbohydrate is called

b oxidation. It convert in acetate units of acetyl CoA to glyoxylate and malate (malic acid) takes place in microbodies, termed glyoxysomes. The glyoxysomes are contain all the necessary enzymes for b oxidation of fatty acid to acetyl CoA and subsequent conversion of the acetate units to malic acid (malate) and succinic acid

(succinate), the cycle is known as Glyoxylate cycle.

 

 

 

• Energy output : A molecule of 18-carbon stearic acid on complete oxidation produces 147 high-energy A 6-carbon glucose molecule yields 36 or 38 ATP. With this rate, an 18-carbon molecule is expected to give 3 times more energy (36 or 38×3=108 or 114 ATP) but it provides about 4 times more energy (36 or 38

×4=144 or 152 ATP) than 6-carbon glucose produces.

(ii)  Glyoxylate cycle

• Discovery : Kornberg and Krebs discovered first this cycle in the bacterium Later, the reaction of b -oxidation of fatty acids and its conversion of acetyl CoA to glyoxylate and malate occurs in glyoxysomes given by Beevers.
• Occurrence : It occurs in seed rich in fats convert stored fats to carbohydrates during The cycle does not appears to be present in those seeds that store starch rather than fat. Glyoxylate activity in germination seeds ceases as soon as the fat reserves have been used up. The fact that plants convert fatty acid to carbohydrates is due to operation of two unique glyoxysome enzyme not known to be present in animals : isocitrate lyase and malate synthetase.

 

 

Phosphoenol pyruvate
Hexose phosphates

Fig : Conversion of storage fat to carbohydrates in germinating

 

 

 

 

 

 

 

• Description : The cycle starts with fatty acids are derived from lipase-mediated enzymolysis of triglycerides occurring in lipid bodies called oleosomes. The fatty acids undergo b oxidation in the glyoxysome with the formation of acetyl

The acetyl CoA reacts with oxaloacetate to form citrate and then isocitrate. The isocitrate is cleaved to succinate and glycoxylate. This reaction is catalyzed by isocitrate lyase. The glyoxylate combines with acetyl CoA to form malate, the reaction being catalyzed by malate synthetase. The malate in the glyoxysome is oxidized to oxaloacetate, which initiates the cycle again by combining with acetyl CoA derived from b oxidation of fatty acids. The succinate produced moves out of the glyoxysome and into the mitochondrion, where it is converted through

the conventional Krebs cycle reactions to oxaloacetate.

The increase of oxaloacetate (OAA) provides ample substrate for amino acid production and carbohydrate formation by reverse glycolysis. Conversion of OAA to phosphoenolpyruvic acid and other glycolytic intermediates takes place in the cytoplasm.

• Protein respiration : The proteins split into amino acids in the cytoplasm for use in The amino acids enter respiratory routes in two ways : Deamination and Transamination.
• Deamination : In deamination, an amino acid loses its amino group (-NH₂) and changes into a keto The latter may further change into a pyruvic acid or acetyl coenzyme A. Pyruvic acid is oxidised to acetyl coenzyme

1. The latter enters the Krebs cycle.

• Transamination : In transamination, an amino group of an amino acid is transferred to an appropriate keto acid, forming a new amino acid and a new keto acid. The keto acids so formed are normal participants of glycolysis or Krebs cycle. Of the all amino acids of plant cell only glutamic acid is believed to be oxidised directly by the enzyme, glutamic acid dehydrogenase into a-ketoglutaric acid and ammonia in the presence of a- ketoglutaric acid enters the Kreb’s cycle to undergo cyclic degration and oxidation.

 

 

Glucose phosphate

 

 

Fructose

Fructose phosphate

 

 

Lactic acid

Fructose diphosphate

 

 

Glycerol

Phosphoglyceraldehyde

 

 

Phosphoglyceric acid

 

 

 

 

 

CO₂

Keto acids

 

Pyruvic acid

34

Deamination

NH₃

 

Fatty acids

Oxidation

Acetyl coenzyme A

 

 

• Salt respiration : This respiration is discovered by Lundegarth and Burstram monovalent chlorides of Na, K and divalant chlorides of Li, Ca and Mg are responsible for salt According to Lundegarth amount of anion absorbed by plant cells rather than to the absorption of cations of salts, so it is also called anion respiration.

When a fresh water plant transferred to the salty water the rate of respiration increase due to salt respiration.

The cause of increase in the rate of respiration during absorption of minerals by roots is also salt respiration.

 Respiratory quotient / Respiration ratio.

R.Q. is the ratio of the volume of CO₂ released to the volume of oxygen taken in respiration and is written as

R.Q.= Volume of CO2 evolved = CO2

 

Volume of O2 absorbed        O2

Value of R.Q. varies with substrate. Thus the measurement of R.Q. gives some idea of the nature of substrate being respired in a particular tissue.

When carbohydrates are completely oxidised the value of R.Q. is unity (=one). If fats and proteins are the substrate the value of R.Q. is less than unity (0.5 to 0.9) and when organic acids are substrate the value is more than unity (1.3 to 4.0).

In succulent like Opuntia, Bryophyllum where there is incomplete oxidation of carbohydrates no CO₂ is produced, hence the value of R.Q. is zero.

R.Q. is usually measured by Ganong’s respirometer.

• When carbohydrates are the respiratory substrate (=germinating wheat, oat, barley, paddy grains or green leaves kept in dark or tubers, rhizomes, )

 

C6 H12 O6

+ 6O2

® 6CO2

• H 2O

; CO2

O

= 6 = 1 (Unity)

6

 

Glucose                                                                  2

• When fats are the respiratory substrate (=germinating castor, mustard, linseed, til seeds)-for fatty substances Q. is generally less than one .

 

• C18

H36 O2

+ 26O2

® 18CO2

+ 18H

₂O ;

CO2 O

= 18 = 0.7 (Less than unity)

26

 

Stearic acid                                                                        2

 

2C51

H98 O6

+ 145O2

® 102CO₂

+ 98H

₂O ;

CO2 O

= 102 = 0.7 (Less than unity)

145

 

Tripalmitin                                                                                 2

• When protein are the respiratory substrate (=germinating gram, pea, bean, mung seeds)- value of Q. is less than unity (0.5-0.9).

(4)  When organic acids are the respiratory substrate

 

• C4

H6 O5

+ 3O2

® 4CO2

• 3H

₂O ;

CO2 O

= 4 = 1.33 (More than unity)

3

 

Malic acid

 

 

2(COOH)₂

Oxalic acid

• O2

® 4CO2

• 2H

₂O ;

2

 

CO2 O2

= 4 = 4 (More than unity)

1

 

 

 

Organic acid	R.Q.
Succinic acid	1.14
Taurtric acid	1.6
Acetic acid	1

• When there is incomplete oxidation of carbohydrates (In the respiration of succulents e.

Bryophyllum, Opuntia)

 

2C6

H12 O6

+ 3O2

® 3C4

H6 O5

• 3H

₂O ;

CO2 O2

= 0 = 0 (Zero)

3

 

• Respiration in the absence of O₂ (in anaerobic respiration)

 

C6 H12 O6

¾¾Zym¾a¾se ® 2C2

H₅OH + 2CO₂ ;

CO2 O2

= 2 = ¥ (Infinite)

0

 

 Factors affecting rate of respiration

Many external and internal factors affecting the rate of respiration are as follows :

 

(1)  External factors

• Temperature : Temperature is the most important factor for respiration. Most of the plants respire normally between 0^oC to 30^oC. With every 10⁰C rise of temperature from 0^oC to 30^oC the rate of respiration increases 2 to

10

2.5 times (i.e., temperature coefficient (Q ^o) is = 2 to 2.5), following Vant Hoff’s Law. Maximum rate of respiration takes place at 30^oC, there is an initial rise, soon followed by a decline. Higher the temperature above this limit, more is the initial rise but more is the decline and earlier is the decline in the rate of respiration. Probably this is due to denaturation of enzymes at high temperature.

45°

40°

35°

 

30°

25°

 

 

 

20°

 

10°

 

0°

0     1       2       3       4      5

Time in hours

 

Fig : Interrelationship between respiratory rates of germinating pea seeds, temperature and time

 

 

 

Below 0^oC the rate of respiration is greatly reduced although in some plants respiration takes place even at- 20^oC. Dormant seeds kept at –50^oC survive.

• Supply of oxidisable food : Increase in soluble food content readily available for utilization as respiratory substrate, generally leads to an increase in the rate of respiration upto a certain point when some other factor becomes
• Oxygen concentration of the atmosphere : Respiration is aerobic or anaerobic depending upon the presence or absence of

oxygen. Air has 20.8% oxygen which is more than enough keeping in

 

view the requirements of plants. Due to this if the amount of oxygen in the environment of plants is increased or reduced upto quite low values the rate of respiration is not effected. On decreasing the amount of oxygen to 1.9% in the environment aerobic respiration become negligible (extinction point of aerobic respiration) but anaerobic respiration takes place.

5            10          15           20          25

Oxygen %

Fig : Effect of oxygen concentration on the rate of respiration. On the right of arrow – Rate of respiration increases with increases in oxygen concentration. On the left of arrow -Rate of anaerobic respiration increases again with decrease in oxygen concentration and ethyl alcohol produced and CO₂ is released

 

Oxygen poisoning : The significant fall in respiration rate was observed in many tissues in pure O₂, even at

N.T.P. This inhibiting effect was also observed in green peas when they are exposed to pure oxygen exerting a pressure of 5 atm- the respiration rate fall rapidly. The oxygen poisoning effect was reversible, if the exposure to high oxygen pressure was not too prolonged.

 

• Water : With increase in the amount of water the rate of respiration In dry seeds, which have 8-12% of water the rate of respiration is very low but as the seeds imbibe water the respiration increases. The life of seeds decreases with increase of water. This increase is slow at first but very rapid later. This is very clearly seen in the tissues of many xerophytes. As the water contents of such plants is increased, often there is no great immediate effect upon the rate of respiration. Minor variation in water content of well- hydrated plant cell do not appear to have very great influence upon the rate of respiration.

Figure shows that in wheat grains rate of respiration increases with increase of water to 16-17%. The rate of respiration of seeds increase with increase of water because water causes hydrolysis, and activity of respiratory enzymes is increased. Also oxygen enters the seed through the medium of water.

12

 

10

 

8

 

6

 

4

 

2

 

 

12     13     14     15      16      17

Percentage of moisture

Fig : Effect of moisture on the rate of respiration of germinating wheat grains

 

• Light : Respiration takes place in night also which shows that light is not essential for But light

effects the rate of respiration indirectly by increasing the rate of    ₆₀

photosynthesis due to which concentration of respiratory substrates is increased. More the respiratory substrate more is the rate of

respiration.                                                                            40

In case of blue green algae (Anabaena) respiration rate was

found to depend upon light and the effect was also influenced by   20

O₂-concentration.

 

37

0         20                40                60                80

CO₂ Concentration %

Fig : Effect of CO₂ concentration on the rate of respiration of germinating mustard seeds

 

 

 

• Carbon dioxide (CO₂) : If the amount of CO₂ in the air is more than the usual rate of respiration is Germination of seeds is reduced and rate of growth falls down. Heath, (1950) has shown that the stomata are closed at higher cone. of CO₂, due to which oxygen does not penetrate the leaf and rate of respiration is lowered.

This fact is made use of in storage of fruits. Air containing 10% CO₂ (in atmosphere it is only 0.03%) retards respiratory break down and therefore reduces sugar consumption and thus prolongs the life of the fruit.

• Inorganic salts : The chlorides of alkali cations of Na and K, as also the divalent cations of Li, and Ca and Mg, generally increase the rate of respiration as measured by the amount of CO₂ Monovalent chlorides of K and Na increases the rate of respiration, while divalent chlorides of Li, Ca and Mg causes less increase in respiration.
• Injury and effects of mechanical stimulation : Wounding or injury almost invariably results in an increase in the rate of respiration . Whenever a plant tissue is wounded the sugar content of the wounded portion is suddenly This increase in the sugar content is responsible for the observed temporary increase in the respiration rate.

A purely mechanical stimulation of respiration has been demonstrated in leaves of a number of species by Audus (1939,40,46). A gentle rubbing, touching, handling and bending of the leaf blade was sufficient to induce a marked rise in the respiration rate (20 to 183%) which persisted for several days. If the treatment was repeated at intervals, the stimulus gradually lost its effect in increasing the rate of respiration.

• Effect of various chemical substances : Certain enzymatic inhibitors like cyanides, azides, carbon monoxide, iodoacetate, malonate etc. reduce the rate of respiration even if they are present in very low

However, various chemical substances such as chloroform, ether, acetone, morphine, etc., brings about an increase in respiratory activity. Ripening fruits produce ethylene and this is accompanied by an increase in respiration rate. Other volatile products responsible for the flavour (aroma) e.g., methyl, ethyl, amyl, esters of formic, acetic, caproic and caprylic acid also associated with increased respiration rate, reach a maximum during ripening of fruits.

• Pollutants : High concentration of gaseous air pollutants like SO₂ , NO_X and O₃ inhibit respiration by damaging cell membrane. These gaseous pollutant causes increase in pH which in turn affects the electron transport system thus inhibiting

Heavy metal pollutant like lead (Pb) and cadmium(Cd) inhibit respiration by inactivating respiratory enzymes.

(2)  Internal Factors

• Protoplasm : The rate of respiration depends on quality and quantity of protoplasm. The meristematic cells (dividing cells of root and shoot apex) have more protoplasm than mature cells. Hence, the meristematic cells have higher rate of respiration than the mature cells. Respiration rate high at growing regions like floral and vegetative buds, germinating seedlings, young leaves, stem and root
• Respiratory substrate : With the increase of in the amount of respiratory substrate, the rate of respiration

 Experiments of respiration

• Demonstration of aerobic respiration in plant tissues : Experimentally, aerobic respiration is proved by respiroscope. As a result of this experiment water column arises (also mercury) due to absorption of evolved CO₂ in respiration by KOH.

Fig : Respiroscope for demonstration of aerobic respiration in plants

• Demonstration of anaerobic respiration : In this experiment anaerobic condition created by filling the test tube with After completion of experiment the level of mercury goes down, because CO₂ evolved by the respiration push the mercury level down. When KOH is introduced mercury level will again rise up to top.

CO₂ gas

 

 

Fig : Demonstration of the evolution of CO₂ in anaerobic respiration

 

 

(3)  To prove that CO₂ is evolved in

aerobic     respiration     :     In   following apparatus, when suction pump is started it

 

To aspirator

 

 

Lime

 

 

Block cloth

 

 

 

Lime

 

Air

 

pulls air through absorbed by KOH of U- tube. CO₂ of air free of CO₂ enters bottles C. If the calcium hydroxide water does not turns milky it is an indication that all CO₂ has been absorbed by KOH. The results

water

cover

Potted plant

water                  ‘U’ Tube

 

Caustic potash

 

Glass plate

 

shows that lime water in bottle A turns milky proved that liberation of CO₂ takes place in aerobic respiration

(A)                      (B)                       (C)                   (D)

Fig : Apparatus to demonstrate that CO₂ is evolved in aerobic respiration

 

 

 

 

• Demonstration of liberation of heat energy during respiration : In this experiment bottle A filled with boiled seed and bottle B filled with germinating seed. After 24 hours temperature of both thermometer noted. Observation shows rise in temperature in bottle B because these seeds are

 

 

 

• (B)

Fig : Demonstration of evolution of heat in respiration (A) Dry seeds

• Germinating seeds

• Measurement of rate of respiration by Ganong’s respirometer : The apparatus consists of three parts :

 

• A bulb for the respiring material, with
• A graduated manometer
• A levelling or reservoir tube connected with manometer tube by a stout rubber

2 ml of respiring material are put into the bigger bulb of the respirometer and 10% KOH in placed in the manometer tube. Experiment start with the turning the glass stopper at the top. Respiration now takes place in a closed space and the absorption of

CO₂ liberated shown by rise in KOH solution in the graduated tube.

Stopper

 

Lateral hole Bulb/Reservior

Seeds

 

 

 

 

Graduated manometer tube (KOH solution)

 

[For the measurement of R.Q. saturated solution of NaCl is

first place in manometer tube. (Pure water not used, as it absorbed CO₂)].

Fig : Ganong’s respirometer

 

Different respiratory substrates (carbohydrate, fat seed) are taken. In the graduated tube will remain more or less and unaltered showing that the volumes of CO₂ evolved and oxygen absorbed are the same and R.Q. =1. If solid KOH pellets than added to salt solution in the tube, the accumulated CO₂ is absorbed and can be measured from the reading in the tube.

• Demonstration of fermentation : For this experiment glucose, baker’s yeast and water are taken in Kuhne’s tube. As a result the level of solution falls in the upright arm and the solution gives alcoholic smell, proves that alcoholic fermentation of glucose takes

Carbon dioxide

 

 

 

40

Glucose and yeast

 

 

 

Important Tips

• Glyoxylate cycle is called adaptation of Kreb’s
• Effect of cyanide poisoning can be minimised by immediate supply of ATP.
• Cut fruit and vegetable : They often turn brownish due to oxidation of tannins or orthodihydroxyphenols to orthoquinones/polyphenols by means of phenolases and Laccase(e.g., Apple, Potato, Cauliflower, Cabbage, Banana, Peach). This can be prevented by sprinkling ascorbic acid (vit.C), vacuum packing, sugar syrup, steam boiling water or potassium
• In prokaryotes aerobic cell respiration of glucose always produces 38 ATP molecules, as NADH₂ molecules formed during glycolysis are not enter the
• The main place of metabolism is cytoplasm, maximum reaction like glycolysis, fat oxidation into acetyl CoA, protein oxidation into a – ketoglutaric acid, ED pathway and pentose phosphate pathway occurs in Only Krebs cycle and ETC occurs in mitochondria.
• Pentose phosphate pathway called connective link between photosynthesis and fat
• The low temperature and high CO₂ concentration used in cold storage of fruits and tubers increases the rate of
• The potato growing in hilly areas are bigger in size because in hilly areas temperature is low. Respiration decreases on low temperature therefore in potato complete oxidation of carbohydrate not takes place and carbohydrate/ starch in potato tuber accumulates and increases the
• Animals cells respire anaerobically during straneous condition forms lactic acid, and fungi respire anaerobically so the requirement for respiration of fungi considered similar to the animal cells.

 

• The Q. at compensation point = CO2

O2

= Zero ( CO₂

and O₂

equal at compensation point).

 

• Temperature affects germinating seeds because hydration makes enzyme more sensitive to
• Glucose before converting glycogen in muscles and liver converted into glucose 6-phosphate needed Glycogen also before utilization converted into glucose –6-phosphate process called glycogenolysis.
• Thiamine pyrophosphate is the active form of vitamin B₁ (Thiamine) work as coenzyme of pyruvate carboxylase
• Climateric fruits : Those fruits which show a high rate of respiration during their ripening g., Apple, Banana. In these fruits rise of respiration called climatric rise.
• NADH produces during the glycolysis are utilized in formation of ethyl alcohol from acetaldehyde during anaerobic The net gain of ATP is 2 ATP in anaerobic respiration which comes from the process of glycolysis.
• Aldolase and triose phosphate isomerase enzyme are common for EMP and C₃
• In white muscles fibers lactic acid accumulated more faster than red muscle fiber because red muscle fiber have more myoglobin (O₂ storing pigment) as compared to white muscles fiber so white muscle get fatigued