Stages Involved in the Aerobic Respiration Process: 4 Stages | Plants:

Let us make an in-depth study of the four stages involved in the aerobic respiration process.

The four stages involved in the aerobic respiration process are: (1) Glycolysis (or EMP-Pathway of Glycolytic Breakdown) (2) Pyruvate Oxidation or Conversion of Pyruvic Acid to Acetyle Coenzyme A (3) TCA Cycle or Krebs’ Cycle and (4) Terminal Oxidation.

Stage I of Respiration: Glycolysis (or EMP-Pathway of Glycolytic Breakdown):

Although glycolysis occurs in all organisms except primitive bacteria, it has been studied in greatest detail in yeast and animal muscle cells. The same sequence of reactions is found in both types of cells. The chemical reactions of glycolysis were discovered mainly by the efforts of G.G. Embden (1874-1933), Otto Meyerhof (1884 -1951) and J.K. Parnas (1884 -1949), and hence this pathway is also generally referred to as EMP-pathway. Within a period of about 10 years following 1914 the essential features of the EMP pathway were established. It is common to the majority of living organisms, both plants and animals.

It occurs chiefly in the soluble part of the cytoplasm (i.e., cytosol), rather than being associated with mitochondria or other organelles. This is first stage of respiration, which includes a sequence of chemical reactions through which glucose is incompletely oxidized to yield the 3-carbon compound pyruvic acid (Glycolysis = lysis or splitting of glucose).

The essential feature of glycolysis is the breakdown of 6-carbon sugar glucose into two 3-carbon fragments (Pyruvic acid molecules). Glycolysis takes place in ten consecutive steps, each catalyzed by a different enzyme complex (Fig. 7.2). Biochemical steps 1, 3 & 10 are irreversible in glycolysis.

(i) First Half of Glycolysis (Preparatory Phase):

Step 1 (1st Phosphorylation):

In order to obtain energy in the form of ATP (Adenosine triphosphate) from the breakdown of glucose, it is first necessary to “spark off” the reaction sequence by putting ATP into it. During step 1 the primary alcohol group (- CH2OH) at position C-6 of glucose reacts with the terminal phosphate group of ATP, forming glucose-6-phosphate and ADP (Adenosine diphosphate). The reaction is catalysed by enzyme hexokinase and activator Mg++ ions.

Step 2 (Isomerization):

During second step of reactions the glucose-6-phosphate is rearranged (isomerized) to form fructose-6-phosphate. In this reaction, the aldehyde group (- CHO) at C-1 is reduced to a primary alcohol group (- CH2OH) as a result of simultaneous oxidation of the secondary alcohol group (> CHOH) at C-2 to a keto group (> CO). This isomeric change is catalysed by the enzyme phosphohexoisomerase.

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

(i) First Half of Glycolysis (Preparatory Phase):

Step 1 (1st Phosphorylation):

In order to obtain energy in the form of ATP (Adenosine triphosphate) from the breakdown of glucose, it is first necessary to “spark off” the reaction sequence by putting ATP into it. During step 1 the primary alcohol group (- CH2OH) at position C-6 of glucose reacts with the terminal phosphate group of ATP, forming glucose-6-phosphate and ADP (Adenosine diphosphate). The reaction is catalysed by enzyme hexokinase and activator Mg++ ions.

Step 2 (Isomerization):

During second step of reactions the glucose-6-phosphate is rearranged (isomerized) to form fructose-6-phosphate. In this reaction, the aldehyde group (- CHO) at C-1 is reduced to a primary alcohol group (- CH2OH) as a result of simultaneous oxidation of the secondary alcohol group (> CHOH) at C-2 to a keto group (> CO). This isomeric change is catalysed by the enzyme phosphohexoisomerase.

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

We have seen that the first three steps of glycolysis have thus converted, at the expense of two molecules of ATP, a molecule of glucose into one of fructose- 1-6-diphosphate, with a phosphate group at each end. Fructose-1-6-di-phosphate is now split into two smaller fragments.

In this step the 6-carbon sugar, fructose-1-6-di-phosphate, is split between C-3 and C-4 to form two 3-carbon fragments, one an aldehyde (glyceraldelyde-3-phosphate) and the other a ketone (dihydroxyacetone-phosphate). This reaction is carried out under the influence of an enzyme aldolase.

The EMP pathway is common to a great many microorganisms as well as higher forms. The enzyme fructose diphosphate aldolase (also called as fructose biphosphate aldolase) is one of the most critical steps in the pathway. In the absence of this enzyme, glucose or other hoxose sugars must be metabolized via one of several alternative pathways. (Not discussed here).

Step 5:

These two fragments, which may collectively be called triose phosphates, are readily inter-convertible by an isomerization under the effect of an enzyme complex Phospho-triose- isomerase. Mostly dihydroxyacetone- phosphate is converted to glyceraldehyde-3-phosphate.

(ii) Second Half of Glycolysis (Pay off Phase):

In the second half of glycolysis, comprising steps 6 to 10, there is a net gain of ATP, achieved by the oxidation of glyceraldehyde-3-phosphate, one of the triose phosphates formed in step 4. Because the two triose phosphates are inter-convertible, both follow the same pathway and steps 6 to 10 are therefore repeated twice to complete the breakdown of one molecule of glucose.

Step 6 (Dehydrogenation and Phosphorylation):

From the point of view of energy production step 6 is very important one. During this step glyceraldehyde-3-phosphate is simultaneously oxidized and phosphorylated, and the energy liberated by the oxidation of the aldehyde group is conserved to form the high-energy compound 1, 3-diphosphoglycerate, i.e., it has a high free energy of hydrolysis.

The electrons removed from the aldehyde group during its oxidation are accepted by the coenzyme NAD+ (Nicotinamide adenine dinucleotide), which is thereby reduced to give NADH2 (NADH + H+). The NAD+ is bound to the enzyme glyceraldehyde-3-phosphate dehydrogenase which catalyzes the overall reaction.

In this reaction one inorganic H3PO4 group combines at C-1 of 3-phosphogiyceraldehyde (GAP) by non-enzymatic condensation to from 1-3-diphosphogly- ceraldehyde, and then two hydrogen atoms are removed by NAD+ coenzyme 1 under the effect of enzyme glyceraldehyde-3-phosphate dehydrogenase to yield NADH and 1, 3-diphospholgyceric acid (1, 3-PGA). In this dehydrogenation energy is carried along with H atoms into NADH2.

Net Products of Glycolysis:

In glycolysis two molecules of ATP are consumed during double phosphorylation of glucose to form fructose 1: 6 diphosphate. In return four molecules of ATP are produced by substrate level phosphorylation (conversion of 1: 3 diphosphoglycerate to 3-phosphoglycerate and phosphoenol pyruvate to pyruvate). Two molecules of NADH2 are formed at the time of oxidation of glyceraldehyde 3-phosphate to 1: 3 diphosphoglycerate.

The net reaction is as follows:

Summary and Special Features of Glycolysis:

The special features of glycolysis can be summarized as follows (Fig. 7.2):

1. Each molecule of glucose produces 2 molecules of pyruvic acid at the end of the aerobic glycolysis.

2. 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).

3. During the conversion of 1, 3-diphospho- glyceraldehyde 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 ofNADH2.

4. During aerobic respiration (when oxygen is available) each NADH2forms 3ATP and H2O through electron transport system of mitochondria. In this process ½ O2 molecule is utilized for the synthesis of each water molecule.

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

5. Reactions of glycolysis do not require oxygen and there is no output of CO2.

Stage II of Respiration: Pyruvate Oxidation or Conversion of Pyruvic Acid to Acetyle Coenzyme A:

Although not itself part of the TCA cycle, the oxidative decarboxylation of pyruvic acid to acetyl CoA is the essential step by which the partially oxidized glucose molecule enters the cycle. The reaction is very complicated and involves the participation of several enzymes and coenzymes, collectively called as pyruvate dehydrogenase complex.

The most important coenzyme in this complex is coenzyme A. From the point of view of its involvement in the oxidative decarboxylation of pyruvate, the most significant feature of the coenzyme A molecule is that it is terminated by a – SH group because it is here that an acetyl group becomes attached. The molecule can thus conveniently be written in equations as CoA-SH. The oxidation which accompanies the removal of CO2 from pyruvic acid requires the presence of NAD as the oxidizing agent.

The overall reaction may be written as follow:

Acetyl CoA, which is the end product of this reaction, is another high energy compound. The energy locked in the bond between the acetyl group and the coenzyme A is ultimately responsible for driving the TCA cycle.Acetyl CoA being high energy compound acts as a link between EMP Pathway and TCA Cycle.

Stage III of Respiration: TCA Cycle or Krebs’ Cycle:

In most eukaryotes, including all the higher plants and higher animals, respiratory breakdown of pyruvate (i.e., pyruvic acid) occurs chiefly within the mitochondria by a cyclic pathway, variously known as the citric acid cycle, the tri-carboxylic acid cycle, the TCA cycle, or the Krebs’ cycle, after Sir Hans Adolf Krebs, a German (later British) biochemist who proposed its basic features in 1937.

Krebs was awarded Nobel Prize in 1953 for this work. The reaction sequence is called a ‘cyclic’ because, unlike the reactions of the glycolytic pathway, the end product (here, the 4-carbon oxaloacetic acid) combines with more of the starting material (here acetyl CoA) and reenters the reaction sequence as soon as it is formed.

The sequence is called the TCA cycle because the first intermediate in the cycle is a tri-carboxylic acid. Many prokaryotes (bacteria and blue-green algae) also have the TCA cycle, but others do not have all the necessary enzymes.

The TCA cycle, in combination with the closely linked cytochrome system, causes the complete oxidation of pyruvate to form water and carbon dioxide. Free oxygen is required, and energy is liberated. Much of the energy is transferred into ATR but some of it is lost as heat 15 molecules of ATP can be produced from ADP and phosphate ion through the respiration of one molecule of pyruvic acid (i.e., 30 molecules of ATP from 1 mole of glucose) by the TCA cycle and the associated cytochrome system.

The successive reactions of the TCA cycle (Figs.7.3) may be outlined as follows:

The TCA cycle can be divided into nine main steps, although some of them involve more than one chemical reaction.

Step 1 (Condensation):

In the initial step, acetyl-CoA reacts with oxaloacetic acid (a 4-carbon compound) in the presence of a condensing enzyme to produce citric acid (a 6-carbon compound) and to regenerate coenzyme A, which is reused to form acetyle-CoA.

Step 4 (Decarboxylation ):

Being highly unstable, the oxalosuccinic acid is converted to α- ketoglutaric acid, a 5-carbon compound, and a molecule of CO2 is released in the process. The responsible enzyme is oxalosuccinate decarboxylase, acting in the presence of manganese ions.

Step 5 (Decarboxylation and Dehydrogenation):

α-ketoglutaric acid, which resembles pyruvic acid in having a keto group (> CO) next to its acidic group (- COOH), undergoes an oxidative decarboxylation reaction with CoA, similar to that undergone by pyruvic acid, the product being a 4-carbon succinyl ~S-CoA. This is a high energy compound analogous to acetyl ~S-CoA formed from pyruvic acid. A molecule of CO2 is produced in the formation of succinyl ~S-CoA, a molecule of water is taken on, and another pair of reactive hydrogen atoms are transferred to NAD, forming NAD.2H.

Step 6 (Formation of ATP/GTP) :

Succinyl-CoA releases succinate (succinic acid) another 4- carbon compound, and CoA. The CoA is immediately available again for either of its two reactions in the TCA cycle or for other uses. A molecule of ATP is formed from ADP and phosphate ion in this step, through the intervention of another phosphate carrier called guanosine di-phosphate (GDP).

The breakdown of succinyl-CoA to succinate and CoA releases energy which is used to attach a phosphate ion to GDP forming GTP The phosphate group is then transferred from GTP to ADR forming ATP The enzyme governing this step is called succinyl-CoA synthetase (or Succmyl Thiokinase).

The remaining three steps of the TCA cycle serve to regenerate oxaloacetic acid, the initial 4-carbon acceptor of acetyl-CoA, from succinate. This involves the oxidation of the hydrocarbon group (CH2) in the α position (i.e., next to the – COOH group) of succinate to a carbonyl group (> CO).

Step 7 (Dehydrogenation) :

Succinate forms fumarate (fumaric acid) another 4-carbon compound, and releases another pair of reactive hydrogen atoms, which are fed directly into the cytochrome system (discussed in subsequent paragraphs) instead of being transferred to NAD.

The requisite enzyme is called succinate dehydrogenase. The two reactive hydrogen atoms are accepted by a coenzyme called FAD (The only functional difference between NAD and FAD in the present context is that the subsequent oxidation of each molecule of reduced FAD is coupled to the formation of two molecules of ATP, whereas the oxidation of reduced NAD results in the formation of three molecules of ATP).

Step 8 (Hydration):

Fumarate (fumeric acid) forms malate (malic acid), another 4- carbon compound, taking on a molecule of water in the process. The governing enzyme is called fumarase. No significant energy change occurs in this step. This reaction is just molecular rearrangement by the addition of the elements of water across the double bond to form malate.

Step 9 (Dehydrogenation):

In the final step malic acid gives rise to oxaloacetate (oxaloacetic acid) and releases two more reactive hydrogen atoms, which are accepted by NAD+ to form NADH2+ The oxaloacetic acid, in turn, is ready to react with another molecule of acetyl-CoA (fed in from the breakdown of pyruvic acid) completing the TCA cycle. The requisite enzyme is called malate dehydrogenase.

Oxaloacetic acid picks up another molecule of activated acetate to repeat the cycle.

A molecule of glucose yields two molecules of NADH2,2ATP and two pyruvate while undergoing glycolysis. The two molecules of pyruvate are completely degraded in Krebs’ cycle to form two molecules of ATP, 8NADH2 and 2 FADH2

In the above description TCA cycle has been described with reference to glucose, but it must be remembered that the significance of this cycle is not confined to the respiration of glucose only. All food materials, after they have been broken down into their constituent units (e.g., storage polysaccharides into monosaccharide’s; proteins into amino acids, etc.) are incompletely oxidized to form, apart from CO2 and H2O, one of only three possible substances, all of which are intermediates in the TCA cycle.

Summary of Krebs’ Cycle:

To sum up, the Krebs cycle

1. Uses 4 molecules of water and releases one molecule of water.

2. Liberates 2 molecules of carbon dioxide.

3. Gives off 4 pairs of hydrogen atoms.

4. Produces one GTP molecule during the formation of succinate,

5. Regenerates oxaloacetate used in first reaction for reuse.

6. One turn of TCA cycle releases 3 NADH2,1 FADH2, 2CO2 & one GTP molecules. In one turn it synthesizes 12 ATP molecules.

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 —

Like glycolysis. Krebs cycle also produces only two high-energy phosphate molecules. It, however, forms 6 NADH and 2 FADH2, as compared to only 2, NADH formed in glycolysis. The 6 NADH and 2 FADH2 provide energy (22 ATP) to the cell in electron transmitter chain (described ahead).

Summary of the Products of Glycolysis, Pyruvate Oxidation and Krebs’ Cycle:

Let us sum up the total products of the aerobic oxidation of glucose to CO2 from glycolysis through Krebs cycle. For each glucose molecule that starts oxidation, the Krebs’ cycle turns twice. Glycolysis produces 2NADH and 2ATP. Pyruvate oxidation gives 2 CO2 and 2 NADH.

Adding to this the yield of two turns of Krebs cycle, we get:

Glucose + 10 NAD+ + 2 FAD+ + 2ADP + 2GDP + 4Pi + 8H2O → 10NADH + 2FADH2 + 2ATP + 2GTP + 6CO2 + 6H2O + 10H+

The 6 H2O molecules shown in the right side of the equation include 2H2O released in Krebs’ cycle and 4H2O formed as the products during ATP and GTP synthesis –

2ADP + 2GDP + 4Pi → 2ATP + 2GTP + 4H2O

It may be noted that only a small amount (4 molecules) of ATP has been produced up to this stage of cellular respiration. However, the electrons carried by 10 NADH and 2 FADH2 molecules hold most of the energy released by oxidation of glucose to 6 CO2. These electrons on their transfer over the ETS of mitochondria generate 32 ATP molecules as explained ahead.

Stage IV of Respiration: Terminal Oxidation:

It is the name of oxidation found in aerobic respiration that occurs towards the end of catabolic process and involves the passage of both electrons and protons of reduced coenzymes to oxygen.

Terminal oxidation consists of two processes:

(i) Electron transport and

(ii) Oxidative phosphorylation.

(i) Electron Transport Chain (ETC) or Electron Transport System (ETS):

You have seen that the glucose molecule is completely oxidized by the end of the citric acid cycle. But the energy is not released unless NADH and FADH2 are oxidized through the electron transport system (ETS) or Electron Transport Chain (ETC) or Mitochondrial Respiratory Chain.

Here, oxidation of a compound means removal of electrons from it. This is usually accompanied by removal of hydrogen. Reduction means addition of electrons to a compound, usually accompanied by addition of hydrogen.

The metabolic pathway through which the electron passes from one carrier to another is called electron transport system (ETS) or Electron transport chain (ETC) (Fig. 7.4), and it is operative in the inner mitochondrial membrane. Four enzyme complexes (Complex I, II, III & IV) (Fig. 7.5) are involved in electron transport.

Electrons from NADH produced in the mitochondrial matrix during citric acid cycle are oxidized by an NADH dehydrogenase (Complex I), and electrons are then transferred to ubiquinone located within the inner membrane. Ubiquinone also receives reducing equivalents via FADH2 that is produced during oxidation of succinate (i.e., succinic acid) through the activity of the enzyme, succinate dehydrogenase (Complex II), in the citric acid cycle. The reduced ubiquinone (ubiquinol) is then oxidized with the transfer of electrons to cytochrome-C via cytochrome bc1 complex (Complex III).

Cytochrome-C is a small protein attached to outer surface of the inner membrane and acts as a mobile carrier for transfer of electrons between complex III and IV Complex IV refers to cytochrome c oxidase complex containing cytochromes a and and two copper (cu) centres.

When the electrons pass from one carrier to another via complex I to IV in the electron transport chain, they are coupled to ATP synthase (Complex V) for the production of ATP from ADP and inorganic phosphate (pi).

The number of ATP molecules synthesized depends on nature of the electron donor. Oxidation of one molecule of NADH gives rise to 3 molecules of ATP, while that of one molecule of FADH2 produces 2 molecules of ATR During electron transfer, the hydrogen atoms split into protons and electrons.

The electrons are carried by the cytochromes. They recombine with their protons before the final stage, when hydrogen atom is accepted by oxygen to form metabolic water. (2H + O → 2H2O). Although the aerobic process of respiration takes place only in the presence of oxygen, the role of oxygen is limited to the terminal stage of the process.

Yet, the presence of oxygen is essential, since it drives the whole process by removing hydrogen from the system. Oxygen acts as the final hydrogen acceptor. The whole process by which oxygen effectively allows the production of ATP by phosphorylation of ADP, is called oxidative phosphorylation.

Details of oxidative phosphorylation follow:

(ii) Oxidative Phosphorylation or Chemiosmotic Synthesis of ATP: Mitchell’s Hypothesis:

Oxidative phosphorylation is the synthesis of energy rich ATP’ molecules with the help of energy liberated during oxidation of reduced co-enzymes (NADH2, FADH2) produced in respiration. The enzyme required for the synthesis is called ATP synthetize.

It is considered to be the fifth complex (complex V) of electron transport chain. Synthesis of ATP molecules takes place through the process of chemiosmosis. Peter Mitchell in 1961 put forward chemiosmotic hypothesis in mitochondrial ETC. He got the Nobel Prize in 1978 for this hypothesis.

According to this hypothesis, ATP synthesis involves two steps:

(i) Creation of H+ Gradient:

The energy released during electron transfer passes H+ ions or protons from the matrix through the inner mitochondrial membrane into the inter-membrane space between the outer and inner membranes. This raises the concentration of protons outside the inner membrane above that in the matrix.

Higher concentration of positively charged protons in the inter-membrane space creates an electric potential across the inner mitochondrial membrane as the matrix now becomes negative with respect to the inter-membrane space. The proton concentration gradient and the membrane electric potential are together referred to as the proton motive force.

(ii) Proton Flow (Fig. 7.7):

Proton motive force causes the flow of protons from the inter-membrane space across the inner mitochondrial membrane down their electrochemical gradient into the matrix. Protons pass through the channel in F0 — F1 ATPase particle, where F1 catalyzes the synthesis of ATP from ADP and Pi with the electrical kinetic energy released by the downhill flow of protons.

The transfer of energy from electron flow to electrochemical proton gradient and then to the phosphate bonds of ATP is called oxidative phosphorylation. The ETS is, therefore, also known as oxidative phosphorylation pathway. The method of forming ATP by using chemical (H+ ) flow through a membrane is termed chemiosmotic ATP synthesis (Fig. 7.6).

Route 1 of ETS:

The electrochemical gradient created as an electron pair passes from NADH2 to oxygen is sufficient to drive the synthesis of 3 ATP molecules.

Route 2 of ETS:

The electron pair from FADH2 passes over only a part of the electron transport chain. Therefore, fewer H+ ions are added to the gradient. The smaller gradient generates only 2 ATP molecules for each pair of electrons.

Shuttle System:

The NADH2 formed during glycolysis is in the cytoplasm. It must reach the inner membrane of mitochondria where ETS is located so that ATP may be produced. The inner mitochondrial membrane is impermeable to NADH2. A special electron carrier system, called the shuttle system, located in the mitochondrial membrane, picks up the electrons from the hydrogen’s of NADH present in the cytoplasm, transfers them across the mitochondrial membranes, and delivers them to the electron carriers inside the mitochondrion.

There are two distinct shuttle systems: the less efficient glycerol-phosphate shuttle present in the skeletal muscle, brain cells and most of the eukaryotic cells, and the more efficient maltate-aspartate shuttle present in the heart, liver and kidney cells (Fig. 7.8). The less efficient shuttle transfers electrons from NADH to FAD, reducing the latter to FADH2. The FADH2 produces only 2 ATP molecules as stated above.

The more efficient shuttle transfers electrons from NADH of cytoplasm to NAD of mitochondrion, reducing it to NADH. The latter introduces its electron pairs into route 1 of ETS. In this route,  3 ATP molecules are formed per pair of electrons. The shuttle continuously transfers electrons from cytoplasm to the mitochondria and does not allow the accumulation of hydrogen molecules in the cytoplasm.