In this tutorial, we have discussed ‘Aerobic and Anaerobic Respiration in Plants‘. The common pathway of aerobic respiration consists of three steps – glycolysis, Krebs cycle, and terminal oxidation.

— All the cellular activities are grouped into two categories: anabolism (biosynthetic activities of the cell) and catabolism (breaking up processes of the cell).

— The anabolic activities are endergonic (energy-dependent), while catabolic activities are usually exergonic (energy-producing).

—The sum total of catabolic and anabolic reactions occurring at any time in a cell is called metabolism.

What is Respiration in Plants

— Definition: Respiration is an exergonic and enzymatically controlled catabolic physicochemical process that involves the step-wise biological oxidation of organic substances in all types of living cells. 

— By respiration, high energy compounds, i.e., carbohydrates (glycogen, starch, sucrose, glucose, etc.), proteins, fats, and other organic substances are oxidized broken down in a step-wise manner into simple substances, and energy is released. The released energy is stored in the form of ATP, which is utilized further (by ATP break down) in various biosynthetic reactions in the cell, so ATP is commonly called energy currency.

Thus, both energy-yielding and energy-consuming reactions occur within the living cell. 

— The dry weight of a plant decreases due to respiration.

— All living organisms (except viruses) respire.

(01) External Respiration

That is actually a gaseous exchange process or breathing process in most animals whereby O₂ is usually absorbed from the atmosphere and CO₂ is usually evolved.

(02) Cellular Respiration / Internal Respiration

Cellular respiration, i.e., oxidative breakdown of food materials within the cell, releases energy and biochemical intermediates. This energy is used in the synthesis of ATP (adenosine triphosphate). The biochemical intermediates are used for the synthesis of organic compounds that take part in growth, repair, and metabolism.

Every living cell oxidises its food to release energy. About 50% of the energy liberated during cellular respiration is used for the synthesis of biomolecules and other life activities like absorption of minerals, maintenance of cell permeability, uptake of materials from other cells, osmotic work, growth, development, cell division, etc. Energy is not released in a single step in respiration nor it is directly used for various cellular activities. Instead, energy is liberated in a controlled fashion in several steps and is mostly stored in ATP molecules. Thus 114.5 kcal of energy is liberated for each molecule of oxygen used in respiration. It is employed in the synthesis of 6 molecules of ATP.

Definition: Respiratory substrates are those organic substances which are oxidised during respiration to liberate energy inside the living cells.

➢ The common respiratory substrates are carbohydrates, proteins, fats, and organic acids. The most common respiratory substrate is glucose. It is a hexose monosaccharide. Another related compound is fructose. Glucose is formed from storage carbohydrates like starch in most plants and glycogen in animals and fungi.

➢ Fats are used as respiratory substrates by a number of organisms because they contain more energy as compared to carbohydrates. However, fats are not directly used in respiration. Instead, they are first broken down to intermediates common to glucose oxidation, viz., acetyl CoA, glyceraldehyde phosphate.

➢ Proteins are used in respiration only rarely, as during germination of protein rich seeds and spores. Proteins are hydrolysed to form amino acids from which organic acids are produced through deamination. In animals, excess amino acids are regularly deaminated to produce organic acids. Organic acids enter Krebs Cycle, e.g., aspartic acid, glutamic acid. At other times, proteins are employed as respiratory substrates under starvation conditions only when carbohydrates and fats become unavailable. Respiration involving proteins as the respiratory substrate is called protoplasmic respiration as compared to floating respiration which uses carbohydrates and fats. Protoplasmic respiration cannot be continued for long as it depletes the protoplasm of structural and functional proteins as well as liberates toxic ammonia.

Respiration is of two main types, aerobic and anaerobic (fermentative).

Aerobic Respiration

The word aerobic means that oxygen is needed for this chemical reaction.

Aerobic respiration is that type of respiration in which organic food, such as glucose, is completely oxidized with the help of oxygen (as a terminal oxidant) into carbon dioxide and water.

In living things, oxidation occurs by the removal of hydrogen atoms (e^– + H⁺). In aerobic respiration, Every carbon atom of the respiratory substrate is oxidised to form carbon dioxide (CO₂). Hydrogen atoms eventually reduce oxygen, forming water (H₂O). Thus, the end result of aerobic respiration is carbon dioxide (CO₂) and water (H₂O).

686 kcal or 2870 kJ of energy is liberated per mole of glucose. The value was previously calculated to be 673 kcal. One kcal is equal to 1000 calories. It is that amount of energy (as heal) that can raise the temperature of one litre of water by 1°C.

Anaerobic Respiration

Anaerobic respiration is a type of respiration where oxygen is not used as an oxidant and the organic food is broken down incompletely to liberate energy without oxygen being used as an oxidant. Energy is liberated during the breaking of bonds between various types of atoms. The common products of anaerobic respiration are CO₂, ethyl alcohol, and lactic acid.

Anaerobic respiration is the only mode of respiration in some micro-organisms. In higher organisms, it occurs as a temporary measure. Anaerobic respiration cannot continue for long in higher organisms because

(i) It yields a small amount of energy.

(ii) More substrate is decomposed so that little is left for growth and repair.

(iii) Some of the end products and intermediates of anaerobic respiration are toxic in higher concentrations.

(iv) Several physiological processes of higher organisms are linked to aerobic respiration, e.g., active absorption of minerals, protoplasmic streaming, etc.

Aerobic respiration is an enzymatically controlled release of energy in a stepwise catabolic process of complete oxidation of organic food into carbon dioxide and water with oxygen acting as a terminal oxidant. 

The chemical reactions of the aerobic respiration of glucose are grouped into four stages: glycolysis, formation of acetyl coenzyme A, Krebs cycle, and terminal oxidation. In eukaryotes, the first stage (glycolysis) takes place in the cytosol, and the remaining stages take place inside mitochondria. Most bacteria and archaea also carry out these processes, but because prokaryotic cells lack mitochondria, the reactions of aerobic respiration occur in the cytosol and in association with the plasma membrane. 

➢ Stage 1. Glycolysis: During glycolysis, one six-carbon molecule of glucose is broken into two molecules of the three-carbon compound pyruvate. During this process, ATP is produced from ADP and Pi, and nicotinamide adenine dinucleotide (NAD⁺) is reduced to form NADH (reduced nicotinamide adenine dinucleotide). NADH is a reduced molecule that transfers energy by transferring electrons.

➢ Stage 2. Oxidation of Pyruvate to acetyl coenzyme A: Each pyruvate enters a mitochondrion and is oxidized to a 2-carbon group (an acetyl group) that combines with coenzyme A, forming acetyl coenzyme A. NADH is produced, and carbon dioxide is released as a waste product. 

➢ Stage 3. Krebs cycle / Citric acid cycle / Tricarboxylic acid cycle (TCA cycle):  The acetyl group of acetyl coenzyme A  combines with a 4-carbon molecule (oxaloacetate) to form a 6-carbon molecule (citrate). In the course of the cycle, citrate is recycled to oxaloacetate, and carbon dioxide is released as a waste product. During this sequence of reactions, more ATP and NADH are produced, and flavin adenine dinucleotide (FAD) is reduced to form FADH₂ (reduced flavin adenine dinucleotide) 

Krebs cycle occurs in the mitochondrial matrix.

➢ Stage 4. Oxidative Phosphorylation (Electron Transport Chain and Oxidative phosphorylation): Electrons from NADH and FADH₂ move through a series of proteins that together are called an electron transport chain (ETC). The energy released in this chain of redox reactions is used to create a proton gradient across a membrane; the ensuing flow of protons back across the membrane is used to make ATP. Because this mode of ATP production links the phosphorylation of ADP with the oxidation of NADH and FADH₂, it is called oxidative phosphorylation.

In eukaryotes, the electron transport chain is embedded in the mitochondrial inner membrane, while in prokaryotes it is found in infoldings of the plasma membrane.

(01) GLYCOLYSIS PATHWAY (Gk. glycos – sugar, lysis – splitting)

— It is also called EMP pathway because it was discovered by three German scientists  — Gustav Embden, Otto Meyerhof, and J. Parnas in 1930.

— Glycolysis is the process of partial oxidation of glucose or similar hexose sugar into two molecules of pyruvic acid through a series of ten enzyme mediated reactions releasing some energy (as ATP) and reducing power (as NADH₂). The term glycolysis means “splitting of sugar“.

— It occurs in cytosol or cytoplasm. Glycolysis is common to both aerobic and anaerobic modes of respiration. It is the first stage of the breakdown of glucose in aerobic respiration and the only step in glucose breakdown in anaerobic respiration.

— Glycolysis has two phases, Preparatory phase and Payoff phase. In the preparatory phase, glucose is broken down to glycerealdehyde 3-phosphate. In the payoff phase, the latter is changed into pyruvate producing NADH and ATP.

► PREPARATORY PHASE (ENERGY SPENDING PHASE)

● Step 1: Phosphorylation of Glucose by ATP

Respiratory substrate (glucose or fructose) is formed by hydrolysis of starch or sucrose. Hydrolysis of starch occurs with the help of enzymes amylase and maltase. It yields glucose. Sucrose is hydrolyzed by enzyme invertase to form glucose and fructose.

Glucose is phosphorylated to glucose-6-phosphate by ATP in the presence of enzyme hexokinase (Meyerhof, 1927) or glucokinase (e.g., liver), and Mg²⁺.

● Step 2: Synthesis of Fructose-6-phosphate.

Glucose-6-phosphate is changed to its isomer fructose-6-phosphate with the help of the enzyme phosphohexose isomerase in a reversible rearrangement.

Fructose-6-phosphate can also be produced directly by phosphorylation of fructose with the help of the enzyme fructokinase.

● Step 3: Formation of Fructose-1, 6-biphosphate.

Fructose-6-phosphate is further phosphorylated by means of ATP in presence of enzyme phosphofructo-kinase and Mg²⁺. The product is fructose-1, 6-biphosphate.

In plants, a pyrophosphate (ppi) dependent phosphofructokinase has been discovered (Kruger, 1997) which carries out the conversion of fructose-6-phosphate into fructose-1, 6-biphosphate.

● Step 4: Splitting of Fructose-1, 6-Biphosphate.

Fructose-1, 6-biphosphate splits up enzymatically to form one molecule each of 3-carbon compounds, glyceraldehyde-3- phosphate (=GAP) or 3-phosphoglyceraldehyde (=PGAL) and dihydroxy acetone-3-phosphate (=DHAP) by an enzyme aldolase.

● Step 5: Isomerisation of DHAP.

Dehydroxyacetone-3-phosphate is isomerized (rapidly inter-converts) to 3-phosphoglyceraldehyde with the help of the enzyme triose phosphate isomerase.

► PAY OFF PHASE (ENERGY CONSERVING PHASE)

● Step 6: Oxidation and Phosphorylation.

In the presence of the enzyme glyceraldehyde-3-phosphate dehydrogenase, the 3-phosphoglyceraldehyde (or, glyceraldehyde-3-phosphate) is oxidized through the removal of hydrogen and the addition of phosphate from an inorganic source to form 1, 3-biphosphoglycerate. NAD⁺ is a hydrogen acceptor. It produces NADH + H⁺.

● Step 7: Substrate Level Phosphorylation (Formation of ATP).

One of the two phosphates of 1,3-biphosphoglycerate is linked by high energy bond. It can synthesize ATP and form 3-phosphoglycerate with the help of the enzyme is phosphoglycerate kinase. The direct synthesis of ATP from metabolites is called substrate level phosphorylation.

● Step 8: Isomerization.

3-phosphoglycerate is changed to its isomer 2-phosphoglycerate by enzyme phosphoglyceromutase.

● Step 9: Dehydration.

Through the agency of enzyme enolase, 2-phosphoglycerate is converted to phosphoenol pyruvate (PEP). A molecule of water is removed in the process. Mg²⁺ is required.

● Step 10: Formation of Pyruvate.

Phosphoenolpyruvate (PEP), possesses a high-energy phosphate (P) bond similar to the bonds in ATP. At the last step of glycolysis, with the help of enzyme pyruvate kinase, PEP’s phosphate group is transferred enzymatically to ADP, the energy in the bond is conserved, and ATP is created. This is an example of substrate level phosphorylation.

(02) OXIDATION OF PYRUVATE TO ACETYL-CoA

— Mitochondria have two membranes, called the outer membrane and inner membrane. Portions of the inner membrane fill the interior of the organelle with sac-like structures called cristae. Short tubes connect the cristae to the rest of the inner membrane. The regions between the outer and inner membranes and within the cristae make up the intermembrane space. The region enclosed within the inner membrane is the mitochondrial matrix. 

— The pyruvic acid formed at the end of glycolysis does not enter the next step of citric acid cycle directly. Rather, it moves across the mitochondrial outer membrane through small pores and is transported into the matrix through a carrier protein in the inner membrane. Once it is inside the matrix, a sequence of reactions occurs inside an enormous and intricate enzyme complex called pyruvate dehydrogenase [made up of decarboxylase, lipoic acid, thiamine pyrophosphate (TPP), transacetylase, and Mg²⁺]. In eukaryotes, this complex is located in the mitochondrial matrix. In bacteria and archaea, pyruvate dehydrogenase is located in the cytosol.

— In the presence of oxygen, pyruvate undergoes a process known as oxidative decarboxylation. As pyruvate is being processed, one of its carbons is oxidized to CO₂ and NAD⁺ is reduced to NADH. The remaining two-carbon acetyl unit (–COCH₃) reacts with a coenzyme A (CoA), yielding acetyl coenzyme A (acetyl-CoA), which enters the citric acid cycle.  Coenzyme A is sometimes abbreviated as CoA-SH to call attention to its key sulfhydryl functional group.

Coenzyme A is manufactured in the cell from one of the B vitamins, pantothenic acid.

— This step is called link reaction or transition reaction or gateway step as it links glycolysis with Krebs cycle. Acetyl CoA is the final product of the pyruvate-processing step in glucose oxidation. Pyruvate, NAD⁺, and CoA go in; CO₂, NADH, and acetyl CoA come out. Keep in mind that these NADH molecules will be used later (during electron transport) to form additional ATP molecules. 

— Like glycolysis, pyruvate processing is regulated by feedback inhibition. When the products of glycolysis and pyruvate
processing are in abundant supply, the process shuts down. Pyruvate processing stops when the pyruvate dehydrogenase complex becomes phosphorylated and changes shape. The rate of phosphorylation increases when one or more of the products are at a high concentration. 

In contrast, a high concentration of reaction substrates— which indicates low ATP supplies—results in more dephosphorylated and active forms of the pyruvate dehydrogenase complex.

Pyruvate processing is thus under both positive and negative regulation. Large supplies of products inhibit the enzyme complex; large supplies of reactants and low supplies of products stimulate it.

— In the absence of oxygen, the fermentation path uses the reduction of all or part of pyruvate to oxidize, NADH back to NAD⁺. This regenerates NAD⁺, allowing glycolysis to continue. Direct reduction of pyruvate, as in muscle cells, produces lactate. In yeast, carbon dioxide is first removed from pyruvate, producing acetaldehyde, which is then reduced to ethanol. 

(03) KREBS CYCLE or TRICARBOXYLIC ACID CYCLE (TCA) or CITRIC ACID CYCLE (CAC)

— Krebs cycle was discovered by Hans Adolf Krebs (1937, 1940, Nobel Prize 1954). It occurs inside the matrix of mitochondria.

— The cycle is also named as citric acid cycle (CAC) because the first stable product of this cycle is citric acid.

— The cycle is also called as tricarboxylic acid (TCA) cycle because the first product citric acid has 3 carboxylic
groups (Tricarboxylic acid).

— Krebs cycle is stepwise oxidative and cyclic degradation of activated acetate ( (Acetyl CoA) derived from pyruvate.

— Acetyl CoA acts as substrate entrant in Krebs cycle, and the acceptor molecule is 4-carbon compound oxaloacetate.

— All enzymes of Kreb’s cycle are soluble in the mitochondrial matrix, but succinate dehydrogenase (SDH) is found attached to the inner mitochondrial membrane.

— The cycle consists of 10 enzymatic steps which involves two decarboxylations and four dehydrogenations. About 65-70% of the ATP is synthesized by Krebs cycle. This cycle of reactions are each regulated by a specific enzyme. 

The various components of ten stepped Krebs cycle are as follows:

1. Formation of Citrate (Condensation): Acetyl CoA (2-carbon compound) combines with oxaloacetate (OAA; 4-carbon compound) in the presence of condensing enzyme citrate synthase to form a tricarboxylic 6-carbon compound called citric acid. It is the first product of Krebs cycle. CoA is liberated.

2. Formation of Cis-aconitate (Dehydration): Citrate undergoes a reorganization in the presence of iron-containing enzyme aconitase first forming cis-aconitate and releasing water.

3. Formation of Isocitrate (Hydration I): cis-aconitate is then converted into isocitrate with the addition of water while attached to enzyme aconitase. This results in the interchange of hydrogen and hydroxyl groups in the molecule.

4. Formation of Oxalosuccinate (Dehydrogenation I): Isocitrate is dehydrogenated in the presence of enzyme isocitrate dehydrogenase and Mn²⁺. As a result, a transient oxalosuccinate is formed intermediate and NADH (NADPH according to some workers) is produced.

5. Formation of α-Ketoglutarate (Decarboxylation I): Oxalosuccinate is unstable and undergoes decarboxylation to form 5-carbon α-ketoglutarate through enzyme decarboxylase and Mn²⁺. This step releases CO₂.

6. Formation of Succinyl-CoA (Dehydrogenation II and Decarboxylation II): α – Ketoglutarate is both dehydrogenated (with the help of NAD) and decarboxylated by an enzyme complex α-ketoglutarate dehydrogenase. The enzyme complex contains TPP,  lipoic acid, and Mg²⁺. The product combines with CoA to form succinyl-CoA.

7. Conversion of Succinyl CoA to Succinate: To continue the cycle, succinyl-CoA is converted to succinate (a 4C compound) by the enzyme succinyl CoA synthase or succinyl thiokinase. The reaction releases sufficient energy to form ATP (in plants) or GTP (in animals). This is the only step in Kreb’s cycle, the high energy phosphate bond is generated at the substrate level. 

GTP can form ATP through a coupled reaction by the action of nucleoside diphosphokinase.

8. Formation of Fumarate (Dehydrogenation III): Succinate undergoes dehydrogenation to form fumarate with the help of a membrane-based enzyme succinate dehydrogenase. Coenzyme FAD (Flavin adenine dinucleotide) is reduced to FADH₂ (reduced flavin adenine dinucleotide).

9. Formation of Malate (Hydration II): A molecule of water gets added to fumarate to form malate by the enzyme fumarase.

10. Formation of Oxaloacetate (OAA) (Dehydrogenation IV): Malate is dehydrogenated or oxidized through the enzyme of malate dehydrogenase to produce oxaloacetate (OAA). Hydrogen is accepted NADP+ / NAD+ and produces one NADPH / NADH.

 

Oxaloacetate picks up another molecule of activated acetate to repeat the cycle. Net gain of Kreb’s cycle is: 

NADH₂ and FADH₂ are reduced co-enzymes that are further oxidized in the electron transport chain (ETC) in the presence of O₂.

Overall Chemical reactions of Krebs cycle or TCA cycle are as follows: 

● Significance of Krebs Cycle

— It is a common pathway for the oxidation of carbohydrates, fats, and amino acids.

— Krebs cycle is the major pathway of the controlled release of energy.

The inputs to the citric acid cycle are acetate (in the form of acetyl CoA), water, and the oxidized electron carriers NAD+, FAD, and GDP.

The outputs are carbon dioxide, reduced electron carriers (NADH and FADH

₂), and two ATP per glucose results from substrate level ATP synthesis.

Overall, the citric acid cycle releases two carbons as CO₂ and produces four reduced electron carrier molecules.

— Krebs cycle produces reduced coenzymes in four separate steps. Another step for the formation of reduced coenzymes occurs during the formation of acetyl CoA from pyruvate.

— Reduced coenzymes are formed near the matrix side of the inner mitochondrial membrane which carries the respiratory chain for the transport of electrons and protons. The latter helps in the oxidative formation of ATP. Krebs cycle is, therefore, connected with the synthesis of most ATP molecules.

— Kreb’s cycle is an amphibolic pathway i.e., having both catabolic (breakdown) and anabolic (build-up) reactions. Since the organic substances are broken down to release energy, it is considered a catabolic pathway. At the same time, the intermediates formed in the cycle are used in several biosynthetic pathways, representing an anabolic pathway. 

— Many of the intermediates in Kreb’s cycle are involved in the synthesis (anabolism) of other important organic molecules. For Example,

➟ Acetyl CoA involves in the synthesis of fatty acids, cutin, and aromatic compounds.

➟ α-ketoglutarate involves in the synthesis of amino acids called glutamic acid (or glutamate), glutamine, proline, arginine. 

➟ Succinyl CoA takes part in the synthesis of pyrrole compounds of chlorophyll, cytochrome, and phytochrome.

➟ Oxalo Acetate (OAA) forms amino acids called aspartic acid (or aspartate), asparagine, methionine, and threonine.

Thus being catabolic, Kreb’s cycle provides a number of intermediates which are used in different anabolic pathways to form important biomolecules like glutamic acid, aspartic acid, etc. So it is called amphibolic pathway.

● Inhibitors of Krebs cycle

The important enzymes of Krebs cycle inhibited by the respective inhibitors are listed below:

(04) TERMINAL OXIDATION

— Definition: Terminal oxidation is the oxidation found in aerobic respiration that occurs towards the end of the process and involves the passage of both electrons and protons from reduced co-enzymes to oxygen. It produces water. 

— Terminal oxidation consists of 2 processes: Electron transport chain and Oxidative phosphorylation.

(A) ELECTRON TRANSPORT CHAIN (ETC)

► Definition: A series of coenzymes and cytochromes that take part in the passage of electrons from a chemical to its ultimate acceptor is called electron transport chain (ETC) or electron transport system (ETS) or mitochondrial respiratory chain.

— Electron transport takes place on cristae of mitochondria [oxysomes (F₀-F_i particles) found on the inner surface of the membrane of mitochondria].

— The passage of electrons from one electron carrier to next, till its terminal acceptor oxygen is a downhill journey, with the loss of energy at each step.

— It begins with oxidation of NADH (reduced nicotinamide adenine dinucleotide) & FADH₂ (reduced flavin adenine dinucleotide). 

When a substrate is oxidized, NAD⁺ accepts two electrons plus a hydrogen ion (H⁺), and NADH results:

FAD accepts two electrons and two hydrogen ions (H⁺) to become FADH₂:

The electrons received by NAD⁺ and FAD are high-energy electrons that are usually carried to an electron transport chain. The energy captured as electrons move down the chain will be used for ATP production inside mitochondria.

Later, this energy will be used for the production of ATP by chemiosmosis. Oxygen (O₂) finally shows up here as the last acceptor of electrons from the chain. After oxygen receives electrons, it combines with hydrogen ions (H⁺) and becomes water (H₂O).

► Components of Electron Transport Chain

The electron transport chain is comprised of five enzyme complexes (i.e., COMPLEX-I, II, III, IV, V) and two electron carriers Ubiquinone (in the internal membrane), Cytochrome c (in the external membrane). All the electron carriers and enzyme complexes of the respiratory chain, except cytochrome c, are embedded in the inner mitochondrial membrane. 

The first four protein complexes (I, II, III, and IV) mainly constitute the electron transport chain because they contain electron carriers and associated enzymes and are involved in electron transport. While the fifth complex (Complex V) is associated with oxidative phosphorylation (ATP synthesis).

Enzyme Complexes:

(01) Complex I (NADH-Q-reductase complex or NADH dehydrogenase complex) is the largest complex, with a structure consisting of 15 subunits. It contains flavin mononucleotide (FMN) and six iron-sulfur (Fe-S) centers. This complex spans the inner mitochondrial membrane and is able to translocate protons across it from the matrix side to the cytosol side.

(02) Complex II (Succinate-Q-reductase complex or succinate dehydrogenase complex) contains Flavin adenine dinucleotide (FAD) and three iron-sulfur (Fe-S) centers. In contrast to complex I, it is unable to translocate protons across the membrane. 

(03) Complex III (QH₂– cytochrome-c reductase complex or Cytochrome bc1 complex) consists of cytochrome b, cytochrome c1, and iron-sulfur (Fe-S) centers. Cytochrome c acts as a mobile carrier between complexes III and IV.

(04) Complex IV (cytochrome c oxidase complex) consists of cytochrome a and cytochrome a₃. Cytochrome a₃  also possesses two copper (Cu²⁺) centers. It is the terminal oxidase and brings about the four electron reduction of O₂ to two molecules of H₂O.

(05) Complex V (ATP synthase complex) comprising of head piece, stalk and a base piece. It is also called as F₀-F₁ or elementary particles. The F₁ and F₀ units are connected by a shaft, as well as by a stator, which holds the two units in place. The F₀ unit spins as protons pass through. The shaft transmits the rotation to the F₁ unit, causing it to make ATP from ADP and P_i. 

F₁ – It is a peripheral membrane protein complex, composed of five different subunits and contains a catalytic site for converting ADP and P_i to ATP. It is necessary for binding F₁ to the inner mitochondrial membrane.

F₀ – It is an integral membrane protein complex consisting of 3 different polypeptides that form the channel for the flow of electrons to F₁.

Electron Carriers:

(01) Ubiquinone (abbreviated Q) is a small, nonpolar, lipid molecule that moves freely within the hydrophobic interior of the phospholipid bilayer of the inner mitochondrial membrane. 

(02) Cytochrome c is a small peripheral protein that lies in the intermembrane space. It is loosely attached to the outer surface of the inner mitochondrial membrane.

5 types of Cytochromes are involved- a, a3, b, c, c1. 

Cyt.a, a3, b, and c1 are integral proteins and Cyt.c is peripheral protein.

►Mechanism of Electron Transport Chain

— Depending upon the substrates from Krebs cycle, such as NADH, electrons follow the pathway of complexes (I, III and IV); whereas for FADH₂, electrons follow the pathway of complexes (II, III and IV).

All the electron carriers and enzymes of the respiratory chain, except cytochrome c, are embedded in the inner mitochondrial membrane. During electron transport, protons are also actively transported across the membrane from the mitochondrial matrix to the intermembrane space. The transmembrane protein complexes (I, III, and IV) act as proton pumps, and as a result, the intermembrane space is more acidic than the matrix.

— Each NADH molecule transfers both protons (H⁺) and electrons (e^–) to flavin mononucleotide (FMN) of complex I (NADH-Q-reductase). Protons are pumped by complex I from the mitochondrial matrix to the intermembrane space. But the electrons are carried in the following sequence.

— Complex II (succinate dehydrogenase) passes electrons directly to Q (Ubiquinone) from FADH2, which was generated in reaction of the citric acid cycle. FADH₂, in turn, bypasses FMN. In contrast to complex I, it is unable to translocate protons across the membrane from the mitochondrial matrix to the intermembrane space. These electrons enter the chain later than those from NADH and will ultimately produce less ATP.
— Complex III (cytochrome c reductase) receives electrons from Q and passes them to cytochrome c.

— Complex IV  (cytochrome c oxidase) has two cytochromes a and a₃ containing copper (Cu²⁺). The a-a3 complex brings together two electrons (2e^–) , two protons ( 2H⁺) and one atom of oxygen ¹/₂ O₂ to produce a molecule of water. In this process, oxygen becomes the terminal acceptor, and the process is terminal oxidation. This is the place where cells actually use oxygen. Finally, the reduction of oxygen to H₂O occurs:

¹⁄ ₂ O₂ + 2 H⁺ + 2 e^– → H₂O

Notice that two protons (H⁺) are also consumed in this reaction. This contributes to the proton gradient across the inner mitochondrial membrane.

— With the help of proton pumps, the positive charge proton (H⁺) pumps from the matrix to the outer chamber of mitochondria. This pumping creates not only a concentration gradient but also a difference in electric charge across the inner mitochondrial membrane, making the mitochondrial matrix more negative than the intermembrane space. Together, the proton concentration gradient and the electrical charge difference constitute a source of potential energy called the proton-motive force (PMF). 

(B) OXIDATIVE PHOSPHORYLATION

— Oxidative phosphorylation is the synthesis of energy rich ATP molecules with the help of energy liberated during oxidation of reduced co-enzymes (NADH, FADH₂) produced in respiration.

— There is uphill proton transport from the mitochondrial matrix to intermembrane space accompanied by downhill electron transport. As a result of coupling of downhill electron transport and uphill proton transport, a proton gradient is established across the membrane i.e., there is high concentration of protons in the intermembrane space as compared to the matrix. Since the hydrophobic lipid bilayer is essentially impermeable to protons, so the potential energy of the proton-motive force cannot be discharged by simple diffusion of protons across the membrane. However, protons can diffuse across the membrane by passing through a specific proton channel, called ATP synthase [It is considered to be fifth complex (Complex V) of electron transport chain], which couples proton movement from intermembrane space back to the matrix, as a result, ATP is synthesized from ADP and P_i.

This coupling of proton-motive force and ATP synthesis is called the chemiosmotic mechanism (or chemiosmosis) and is found in all respiring cells. 

ATP synthesis is a reversible reaction, and ATP synthase can also act as an ATPase, hydrolyzing ATP to ADP and P_i:
If the reaction goes to the right, free energy is released and is used to pump H⁺ out of the mitochondrial matrix—not the usual mode of operation. If the reaction goes to the left, it uses the free energy from H⁺ diffusion into the matrix to make ATP. What makes it prefer ATP synthesis? There are two answers to this question:

➢ ATP leaves the mitochondrial matrix for use elsewhere in the cell as soon as it is made, keeping the ATP concentration
in the matrix low, and driving the reaction toward the left.

➢ The H+ gradient is maintained by electron transport and proton pumping. (Ref: Life: The Science of Biology; 9th Edition; Sadava, Hillis, Heller, Berenbaum; 2011).

— Oxidation of one molecule of NADH₂ produces 3 ATP molecules while similar oxidation of FADH₂ forms 2 ATP molecules.

Glycolysis, the citric acid cycle, and the respiratory chain are subject to allosteric regulation of the enzymes involved. When ATP levels are high, cells do not need to be generating large amounts of ATP by cellular respiration. As this process is quite complex, there are many steps that could be regulated. High levels of ATP and intermediates in the citric acid cycle feedback to inhibit glycolysis, the citric acid cycle, and the breakdown of fatty acids, slowing ATP production. Conversely, when ATP levels in the cell are low, ADP levels are high; and ADP activates enzymes in the pathways of carbohydrate catabolism to stimulate the production of more ATP. 

(01) REGULATION OF GLYCOLYSIS

The rate at which the glycolytic pathway operates is controlled primarily by allosteric regulations of three enzymes: phosphofructokinase (PFK), hexokinase, and pyruvate kinase.

(i) Phosphofructokinase (PFK): The main control point in glycolysis is the enzyme phosphofructokinase. This enzyme is allosterically inhibited by ATP or citrate, and activated by ADP or AMP. Under anaerobic conditions, fermentation yields a relatively small amount of ATP, and phosphofructokinase operates at a high rate. However, when aerobic conditions, respiration makes 16 times more ATP than fermentation does, and the abundant ATP allosterically inhibits phosphofructokinase. Consequently, the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate declines, and so does the rate of glucose utilization. 

Citrate is also an inhibitor of this enzyme, allowing the accumulation of citric acid cycle intermediates to feedback and inhibit glycolysis. In contrast, ADP is an activator of this phosphofructokinase, which makes the enzyme sensitive to levels of both ADP and ATP.

(ii) Hexokinase (HK): It is inhibited by glucose-6-phosphate (Glu-6-P), the product is formed from glucose in the first step of glycolysis with the help of enzyme hexokinase. The control of hexokinase is necessary to prevent the accumulation of Glu-6-P in the cell when flux through the glycolytic pathway is low.

(iii) Pyruvate Kinase (PK): This enzyme is activated by Fructose-1, 6-diphosphate, driving the rate of glycolysis when more substrate is present, The inhibition of Pyruvate Kinase is by ATP. The binding of the ATP to the inhibitor site reduces its affinity for 2-phosphophenol pyruvate (PEP).

(02) REGULATION OF PYRUVATE OXIDATION

Another control point involves the oxidation of pyruvate (production of acetyl CoA). If NADH is not being oxidized fast enough, the levels of NADH will rise and inhibit pyruvate dehydrogenase, preventing the conversion of pyruvate to acetyl-CoA.

(03) REGULATION OF KREBS’ CYCLE

(i) Isocitrate dehydrogenase: The main control point in the citric acid cycle is the enzyme isocitrate dehydrogenase, which converts isocitrate to α-ketoglutarate. NADH and ATP are feedback inhibitors of this reaction, while ADP and NAD+ are activators. If too much ATP or NADH accumulates, the conversion of isocitrate is slowed, and the citric acid cycle shuts down. A shutdown of the citric acid cycle would cause large amounts of isocitrate and citrate to accumulate if the production of citrate were not also slowed. 

But, as mentioned above, an excess of citrate acts as a feedback inhibitor of phosphofructokinase. Thus, if the citric acid cycle has been slowed or shut down because of abundant ATP (and not because of a lack of oxygen), glycolysis is slowed as well. Both processes resume when the ATP level falls and they are needed again. Allosteric regulation keeps these processes in balance.

(ii) α-ketoglutarate dehydrogenase: Enzyme α-ketoglutarate dehydrogenase catalyzes α-ketoglutarate to succinyl-CoA in Krebs cycle. This enzyme is inhibited by succinyl CoA and NADH, both products are derived by catalyzation. In addition, α-ketoglutarate dehydrogenase is inhibited by a high energy charge.

(iii) Citrate synthase:  Another control point in the Krebs cycle is the enzyme citrate synthetase, which catalyzes the first reaction, the conversion of oxaloacetate and acetyl-CoA into citrate. High levels of ATP inhibit citrate synthetase (as well as pyruvate decarboxylase and two other Krebs cycle enzymes), shutting down the catabolic pathway.

— Definition: Pentose phosphate pathway (PPP) is the direct oxidative pathway for catabolism of glucose through the formation of phosphorylated heptose, hexose, pentose, tetrose, and triose sugars.

— Although the aerobic breakdown of glucose mainly occurs through glycolysis and TCA cycle, there exists an alternative pathway called pentose phosphate pathway. It is so named because unlike the glycolytic pathway, it proceeds through formation of phosphorylated pentose sugars.

— This pathway is called hexose monophosphate shunt (HMS) or hexose monophosphate pathway (HMP), because it involves hexose monophosphates only and not hexose diphosphate.

— This pathway is known as pentose cycle, because in this cyclic pathway pentose sugars are formed.

— This pathway is also known as phosphogluconate pathway (because phosphogluconate is the first product of this pathway); direct oxidative pathway (because through this single pathway glucose molecules are completely oxidized instead of entering glycolysis plus TCA cycle); Warburg-Dickens-Lipmann pathway (after the names of the scientists who described it).

— Site of PPP: This pathway for oxidation of glucose occurs in certain tissues like liver, adipose tissue, lactating mammary gland, adrenal cortex, testis, ovary, etc., that are concerned with the synthesis of fatty acids or steroids. It also occurs in thyroid gland and erythrocytes. All the enzymes required for this pathway are present in the cytosol (= cytoplasm). Thus, both glycolysis and PPP operate in the cytosol. This is because some enzymes are common to these two pathways.

— Steps of PPP:

01. The first reaction of the pentose phosphate pathway is the oxidation of glucose 6-phosphate by glucose 6-phosphate dehydrogenase (G6PD) to form 6-phosphogluconolactone, an intramolecular ester. NADP⁺ is the electron acceptor, and the overall equilibrium lies far in the direction of NADPH formation. The lactone is hydrolyzed to the free acid, 6-phosphogluconate by a specific enzyme lactonase. Then, 6-phosphogluconate undergoes oxidation and decarboxylation by 6-phosphogluconate dehydrogenase to form ribulose 5 phosphate; the reaction generates a second molecule of NADPH.

02. Phosphopentose isomerase converts ribulose-5-phosphate to its aldose isomer, ribose 5-phosphate. In some tissues, the pentose phosphate pathway ends at this point, and its overall equation is :

Glucose-6-phosphate + 2 NADP⁺ + H₂O → Ribose 5-phosphate + CO₂ + 2 NADPH + 2H⁺

The net result is the production of NADPH, a reductant for biosynthetic reactions, and ribose 5-phosphate, a precursor for nucleotide synthesis. This allows the continued oxidation of glucose-6-phosphate with production of NADPH.

03. Continued recycling leads ultimately to the conversion of glucose 6-phosphate to six CO₂. Two enzymes unique to the pentose phosphate pathway act in these interconversions of sugars: transketolase and transaldolase.

Transketolase catalyzes the transfer of a two-carbon fragment from a ketose donor to an aldose acceptor. In its first appearance in the pentose phosphate pathway, transketolase transfers C-1 and C-2 of xylulose 5-phosphate to ribose 5-phosphate, forming the seven-carbon product sedoheptulose 7-phosphate. The remaining three-carbon fragment from xylulose is glyceraldehyde 3-phosphate.

04. Next, transaldolase catalyzes a reaction similar to the aldolase reaction of glycolysis; a three-carbon fragment is removed from sedoheptulose 7-phosphate and condensed with glyceraldehyde 3-phosphate, forming fructose 6-phosphate and erythrose 4-phosphate.

05. Now transketolase acts again, forming fructose 6-phosphate and glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate. Two molecules of glyceraldehyde-3-phosphate formed by two interactions of these reactions can be converted to a molecule of fructose-1, 6-bisphosphate as in gluconeogenesis, and finally phosphohexose isomerase converts fructose-1, 6-bisphosphate to glucose 6-phosphate. Transketolase requires the co-factor thiamine pyrophosphate (TPP), which stabilizes a two carbon carbanion in this reaction, just as it does in the pyruvate decarboxylase reaction.

06. For the complete oxidation of glucose-6-phosphate to 6CO₂, 6 molecules of glucose-6-phosphate are needed. Out of these, 5 molecules are regenerated with the production of 12 NADPH.

The overall reaction may be represented as :

6 Glucose-6-phosphate + 12 NADP⁺ + 6H₂O → 6CO₂ + 12 NADPH + 12 H⁺ + 5 Glucose-6-phosphate

— Significance of Pentose Phosphate Pathway

This pathway occurs both in plants and animals. The significant features are as follows:

➢ This is an alternative to TCA cycle. NADPH can undergo oxidative phosphorylation to generate 3 ATP.

➢ The pathway generates reducing power in the form of NADPH, which serves as electron and proton donor for N₂-fixation, fatty acid biosynthesis, nitrate reduction, and other processes.

➢ Pentose phosphates, such as ribose-5-phosphate produced in this pathway, serve as precursors for the synthesis of nucleotides (needed for the synthesis of ADP, ATP, DNA, RNA, etc.)

➢ In plants, erythrose-4-phosphate, an intermediate of PPP is a precursor for the biosynthesis of lignin, flavonoids, and aromatic amino acids.

Compensation Point is that value or point in light intensity and atmospheric CO₂ concentration when the rate of photosynthesis is just equivalent to the rate of respiration in the photosynthetic organs so that there is no net gaseous exchange. The value of light compensation point is 2.5-100 ft candles for shade plants and 100-400 ft candles for sun plants. The value of CO₂ compensation point is 25-100 ppm (25–100 μl.l^-1) in C₃ plants and less than 5 ppm (5 μl.l^-1) in C₄ plants. A plant cannot survive for long at the compensation point because there is a net loss of organic matter due to the respiration of nongreen organs and dark respiration.

— Definition: Respiratory quotient (R.Q.) is the ratio of volume of carbon dioxide liberated in respiration to the volume of oxygen consumed in it over a specific period of time by a unit tissue, organ or organism. The ratio is expressed as:

— Respiratory quotient can be unity, more than one, less than one, zero and infinity. 

● RQ Equal to Unity (R.Q.= 1) : Carbohydrates as a respiratory substrate

Respiratory quotient is unity if carbohydrate is a respiratory substrate and the respiration is aerobic.

Some examples—germinating wheat, oat, barley, paddy grains, tubers, rhizomes or green leaves kept in the dark, etc.

● RQ Less than Unity (R.Q. <1) : Proteins and Fats as a respiratory substrate 

RQ is less than one when respiration is aerobic but the respiratory substrate is either fat or protein. RQ is about 0.7 for most of the common fats. It occurs during the germination of seeds of castor, mustard, linseed, cotton (oily seeds).

RQ is about 0.9 in case of proteins, peptones, etc, e.g., germinating seeds of gram, pea, mung, bean.

● R.Q. Zero (R.Q. =0) : When Carbohydrates are incompletely oxidised to organic acids

It is found in cases where oxygen is consumed but CO₂ is not evolved. E.g.: Succulent plants (e.g., Opuntia). [All the CO₂ produced during respiration is fixed internally for use in photosynthetic activity during the day].

● RQ More than Unity (R.Q. >1) : Organic acids as respiratory substrate

RQ slightly more than unity is found when organic acids are broken down as respiratory substrates under aerobic conditions, e.g.,

● RQ is infinity (R.Q. = ∞) : Anaerobic respiration of Carbohydrate

In anaerobic respiration, there is no consumption of oxygen. Carbon dioxide is produced in most cases. Therefore, the respiratory quotient is infinity. Carbohydrate is the usual substrate.

An intermediate value is obtained where an organ is undergoing both aerobic and anaerobic modes of respiration.

— Importance of R.Q.

(i) Knowledge of respiratory quotient helps in determining respiratory substrate,  i.e., carbohydrate, protein or fat.

(ii) It helps in knowing the type of respiration being performed,  i.e., aerobic or anaerobic.

(iii) It provides some information about the major transformation of food materials like the synthesis of fats or conversion of fats into carbohydrates etc.

(iv) Normal nutrition or starvation condition can be known. 

(v) R.Q. values indicate that living organisms seldom use a single respiratory substrate. It is usually more than one.

— Many organisms, including humans, depend on nutrients other than glucose as a source of energy. Nutrients include carbohydrates (such as sucrose and other disaccharide sugars and starch, a polysaccharide), fats, and proteins. In fact, we usually obtain more of our energy by oxidizing fatty acids than by oxidizing glucose. Amino acids derived from protein digestion are also used as fuel molecules.

Such food nutrients like carbohydrates, proteins, and fats are first converted into substrates that can enter the respiratory pathway (i.e., they are fed into glycolysis or the citric acid cycle).

—Carbohydrates are usually first converted into glucose before they are used for respiration. 

— By the process of deamination, the proteins are degraded by proteases to individual amino acids in which the amino group (— NH₂ ) is first removed. In mammals and some other animals, the amino group is converted to urea and excreted, but the carbon chain is metabolized and eventually is used as a reactant in one of the steps of aerobic respiration. For example, amino acid alanine undergoes deamination to become pyruvate; similarly, amino acid glutamate is converted to α-ketoglutarate, and the amino acid aspartate yields oxaloacetate.

Pyruvate enters aerobic respiration as the end product of glycolysis, and α-ketoglutarate and oxaloacetate both enter aerobic respiration as intermediates in the citric acid cycle. Ultimately, the carbon chains of all the amino acids are metabolized in this way.

— Lipids are rich in energy because they are highly reduced; that is, they have many hydrogen atoms and few oxygen atoms. When completely oxidized in aerobic respiration, a molecule of a 6-carbon fatty acid generates up to 44 ATPs (compared with 36 to 38 ATPs for a molecule of glucose, which also has 6 carbons). 

Fats are broken down into glycerol and fatty acid first.

Fatty acids are oxidized and split enzymatically into 2-carbon acetyl groups that are bound to coenzyme A; that is, fatty acids are converted to acetyl CoA and enter the citric acid cycle. This process, which occurs in the mitochondrial matrix, is called β-oxidation (beta-oxidation).

Glycerol enters glycolysis after being converting it to G3P (Glyceraldehyde-3-phosphate or 3-phosphoglyceraldehyde).

► Definition: Anaerobic respiration is an enzyme mediated energy liberating, catabolic process of step-wise but incomplete breakdown of the organic substrate without using oxygen as an oxidant. Energy is liberated during breaking of bonds between various types of atoms. The common products of anaerobic respiration are CO₂, ethyl alcohol, and lactic acid.

— In microorganisms, the term anaerobic respiration is replaced by fermentation. The latter is defined as anaerobic breakdown of carbohydrates and other organic compounds to form alcohol and organic acids with the help of microbes or their enzymes.

► Mechanism of Anaerobic Respiration

— Glycolysis, the enzymatic breakdown of glucose into two molecules of pyruvate is common to both anaerobic and aerobic mechanisms of respiration.

— Here onwards, pyruvate formed is anaerobically broken down into various products depending upon the organism and the site i.e., type of tissue where it is occurring.

►Types of Fermentation

Fermentation is incomplete anaerobic breakdown of respiratory substrates (glucose) that forms lactic acid or ethyl alcohol (ethanol), which are still relatively energy-rich molecules. Because the breakdown of glucose is incomplete, much less energy is released by fermentation than by cellular respiration.

(01) Ethanol Fermentation

— It is commonly observed in fungi e.g., yeast, Rhizopus, and bacteria.

— The process involves decarboxylation of pyruvic acid in the presence of an enzyme pyruvate decarboxylase, co-enzyme thiamine pyrophosphate (TPP) and Mg²⁺ as cofactor. It produces acetaldehyde with the release of CO₂.

— Acetaldehyde is reduced to ethyl alcohol or ethanol by alcohol dehydrogenase. Hydrogen is obtained from NADH, produced during oxidation of glyceraldehyde 3-phosphate to 1, 3-biphosphoglycerate in glycolysis.
The reaction is represented as:
— The ethyl alcohol does not accumulate in cells rather is excreted, as accumulation beyond a certain limit can kill yeast cells.
— The process is utilised in brewing industry for production of alcoholic beverages like beer, rum, whisky, etc.

(02) Lactic Acid Fermentation

— It is found in lactic acid bacteria e.g., Lactobacillus, some fungi and muscles of vertebrates.

— In this process, pyruvic acid is directly reduced by NADH in the presence of enzyme lactic dehydrogenase, coenzyme flavin mono-nucleotide (FMN), and Zn²⁺ as cofactor.
— CO2 is not released in the process and there is an absence of formation of intermediate compounds.

► Why Anaerobic Respiration is not a preferred mode by living cells?

Anaerobic respiration produces very little energy (about 5%) as compared to aerobic respiration, so it is not efficient to sustain the activities of cells. The various reasons contributing to it are :

(i) There is incomplete breakdown of respiratory substrate.

(ii) At least one of the products of anaerobic respiration is organic. It can be further oxidised to release energy.

(iii) NADH produced during glycolysis is often reutilised.

(iv) ATP formation does not occur during regeneration of NAD⁺.

(v) Electron transport chain is absent.

(vi) Oxygen is not used for receiving electrons and protons.

► Significance of Anaerobic Respiration

● Advantages

(i) Anaerobic respiration is important mode of respiration during periods of oxygen deficiency.

(ii) Important role in brewing industry for the production of various types of beers, whisky and other wines (Saccharomyces cerevisiae), baking, vinegar and dairy industries (Lactococcus lactis, Streptococcus lactis).

(iii) Carbon dioxide of alcoholic fermentation is used in baking industry for making the bread spongy.

(iv) Vinegar is obtained by the fermentation activity of acetic acid bacteria.

(v) Dairy industry depends upon the action of lactic acid bacteria which convert milk sugar to lactic acid. Lactic acid coagulates the milk protein casein and the droplets of milk fat fuse.

(vi) Production of industrial alcohols and organic acids by fermentation of certain fungi e.g., Aspergillus, Penicillium and Rhizopus. 

(vii) Tea and tobacco leaves are cured (or removed of their bitterness) and provided with a fine flavour.

(viii) Retting or separation of stem fibres is carried out with the help of bacterial fermentation of softer tissues.

(ix) Ensilage or preserved fodder is prepared by keeping green chopped fodder in silo where bacterial action causes softening and release of preserving acids.

(x) Cleaning of raw hides by bacterial fermentation to make leather.

(xi) Decomposition of organic remains is carried out by fermentation. However, the fermenting organisms also spoil out food and may cause food poisoning by releasing toxins or ptomains.

● Disadvantages

(i) Low energy producing process (only 59 Kcal of energy) which is insufficient to maintain life of multicellular aerobes, though it can maintain life of unicellular eukaryotic cells as yeast. 

(ii) Accumulation of lactic acid during strenuous exercise causes muscle fatigue Accumulation of lactic acid during strenuous exercise causes muscle fatigue.

(iii) Fermentation in food stuffs may lead to production of toxic substances that may cause food poisoning. 

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TABLE: Differences between Anaerobic Respiration and Fermentation

Sl. No.	Anaerobic Respiration	Fermentation
01.	It is a type of respiration.	It is enzyme controlled breakdown and transformation of organic nutrients.
02.	It is an intracellular process.	It is both intracellular and extracellular process.
03.	It is not economically exploited.	It is economically exploited.

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TABLE: Differences between Glycolysis and Fermentation

Sl. No.	Glycolysis	Fermentation
01.	It is the first step of respiration which occurs without requirement of oxygen and is common to both aerobic and anaerobic modes of respiration.	It is anaerobic respiration or respiration which does not require oxygen.
02.	Glycolysis produces pyruvic acid.	Fermentation produces different products. The common ones are ethanol (and CO2) and lactic acid.
03.	It produces two molecules of NADH per glucose molecule.	It, generally, utilises NADH produced during glycolysis.
04.	Glycolysis forms 2 ATP molecules per glucose molecule.	It does not produce ATP.

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