Cellular Respiration

Respiration is a series of metabolic reactions that take place within the cells of a living organism and it involves the breakdown of organic molecules such as glucose to release energy. These organic molecules are known as respiratory substrates. The energy released during this process is then used to synthesize molecules of ATP which will act as an immediate source of energy.

Respiration can occur in aerobic or anaerobic conditions and in either a respiratory substrate is oxidized.

Aerobic respiration occurs in the presence of oxygen and the substrate molecule is oxidized completely, releasing a lot of energy.

Anaerobic respiration takes place in the absence of oxygen and the substrate is only partially oxidized. Only a small portion of the energy contained by the substrate molecule is released.

12.1 (a). The need for energy in living organisms:

Active transport: Energy is required for the active transport of substances into and out of the cells, across a partially permeable membrane (e.g. cell surface membrane and tonoplast), against the concentration gradient. Active transport is achieved by carrier proteins each of which is specific for a particular type of molecule or ion. Since the movement of substances is against the concentration gradient, active transport requires energy provided by an ATP molecule. The energy is used to make the carrier protein change its shape, transferring the specific ions or molecules binding to it across the membrane in the process. An example of active transport is the activity of the Sodium-Potassium Pump.

Anabolic reaction: the synthesis of complex substances from simpler ones, requires energy. For example, the synthesis of polysaccharides from monosaccharides through glycosidic bonds such as synthesis of cellulose from beta glucose. Other examples include the synthesis of triglycerides from fatty acids and glycerol through the formation of ester bonds, the synthesis of polypeptides from amino acids through peptide bonds, the synthesis of nucleic acids from nucleotides through the formation of phosphodiester bonds (this also includes DNA replication, in which nucleotides are joined by condensation reactions to form polynucleotides), the synthesis of steroids from cholesterol. )

For mechanical work such as muscle contraction (heartbeat, breathing movements, walking) or cellular movements like the beating of cilia and flagella. The movement of vesicles along microtubules within the cell as well as amoeboid movement also requires energy. (flagellum is an organelle of locomotion in the cells of many living organisms including bacteria)

Electrical transmission of nerve impulses requires energy.
The heat energy released during metabolic reactions is used to maintain a constant body temperature in endotherms, for example, birds and mammals (or thermoregulation which is a mechanism of homeostasis). This is crucial because metabolic reactions are controlled by enzymes, so organisms need to maintain a suitable temperature that allows enzyme action to proceed at a rate that will sustain life.

Electric discharge as in electric eel.

Energy is also required for bioluminescence, i.e. the production of light by living organisms such as fireflies, glow worms, and some deep-sea animals requires energy

Light energy is needed for photosynthesis.

Energy is required for the maintenance, repair, and division of cells and the organelles within them require energy.

Structure of ATP:

The structure of ATP is similar to the nucleotides that make up RNA, however, it has 3 phosphate groups attached to it instead of one.

ATP is a nucleotide and consists of organic base adenine― a nitrogen-containing organic base belonging to the group called purines and pentose sugar ribose. Together they make nucleoside, adenosine. This adenosine, is combined with three phosphate groups (more correctly known as phosphoryl groups). The purine base adenine is attached to the first carbon of the pentose while on the fifth carbon of the pentose the phosphate groups are attached. The phosphate groups are linked to each other by two high-energy bonds called phosphoanhydride bonds. ATP is, therefore, an activated phosphorylated nucleotide.

A nucleotide is the basic building block of nucleic acids. DNA and RNA are polymers made up of long chains of nucleotides. A nucleotide consists of a sugar molecule (ribose in RNA and deoxyribose in DNA) attached to a phosphate group and a nitrogen-containing base.

Roles of ATP:
Binding to a protein molecule, changing its shape and causing it to fold differently, to produce movements. For example: In muscle contraction, ATP provides the energy for the filaments of striated muscle to slide past one another, therefore, shortening the overall length of the muscle fiber.

Binding to an enzyme molecule, thus, allowing an energy-requiring reaction to be catalyzed.

Transferring a phosphate group to an enzyme, making the enzyme active.

Transferring AMP to an unreactive substrate molecule, producing a reactive intermediate compound. For example, amino acids before binding to tRNA during protein synthesis.

Transferring a phosphate group to an unreactive substrate molecule so that it can react in a specific way. For example during Glycolysis and Calvin Cycle (ATP makes other molecules more reactive by transferring one of its phosphate groups to them (phosphorylation). During glycolysis glucose is phosphorylated so that it becomes unstable and can be broken down to release energy at the start of a catabolic reaction. Another example is activated nucleotides in DNA and RNA synthesis.)

Binding to a transmembrane protein so that active transport can take place across the membrane.(Transmembrane protein is a protein existing or occurring across a cell membrane)

ATP is used to regenerate RuBP during the Calvin Cycle.
ATP is needed for the conversion of GP to TP.
ATP is also needed to form vesicles necessary for the secretion of cell products.

12.1 (b). Features of ATP that make it suitable as universal energy currency:

ATP is a short-term store of energy derived from cellular respiration. It acts as an immediate donor of energy to the cell’s energy-requiring reactions in all living organisms. Hence, forming a link between the energy-yielding and the energy-requiring reactions.
The hydrolysis of a single ATP molecule releases small packets of energy at a time. This manageable amount of energy is often just the right size to fuel a particular step in a process. Thus, it will not be wasted and will not damage the cell.

ATP molecules are small, diffusible, and water-soluble. It can easily be transported around the cell from where it is made to where it is needed.

When the terminal phosphate group is removed, about 30.5kJ/mol of energy is released. This reaction is feasible and energy is released very quickly. Only one chemical reaction, hydrolysis, is required and enzyme ATPase catalyzes this reaction.

Hydrolysis of ATP can be described by the following reaction:
ATP + HշO ⇌ ADP + HვPOч

This reaction is reversible and ADP can be recycled back to ATP. Therefore, a constant supply of ATP is possible as it can be recycled from ADP, which is easily phosphorylated. There is no requirement for new raw material.

Removing the second phosphate group, giving AMP, also releases about 30.5kJ/mol of energy, but removing the last phosphate group releases only 14.2kJ/mol of energy.

Each cell makes its own ATP and each cell has only a tiny quantity of ATP in it at any one time. The cell does not import ATP, ADP, and AMP. As the reaction is reversible, interconversion of ATP and ADP occurs. The rate of turnover is enormous and ATP can be synthesized, hydrolysed and re-synthesized in a very short time.

Synthesis of ATP:

12.1 (c)(e). ATP is synthesized in substrate-linked reactions during glycolysis and in the Krebs cycle and the synthesis of ATP is associated with the electron transport chain on the membranes of mitochondria and chloroplasts

Since the synthesis of ATP from ADP involves the addition of a phosphate molecule, it is a phosphorylation reaction. This phosphorylation is catalyzed by the enzyme ATP synthase (sometimes called ATP synthetase) and occurs in three ways:

Photophosphorylation, that takes place in the grana of the chloroplasts during photosynthesis.
Oxidative phosphorylation, that takes place on the inner mitochondrial membranes of the plant and animal cells, and the cell surface membranes of bacteria.

Substrate-level-phosphorylation, that takes place in plant and animal cells when phosphate groups are transferred from donor molecules to ADP to make ATP. For example: In the formation of pyruvate, at the end of glycolysis. Substrate-level-phosphorylation is called so because ATP is made when one substrate is converted into another.

In the first two processes, ATP is synthesized using the energy released during the transfer of electrons along a chain of electron-carrier molecules in either the chloroplasts or the mitochondria. There is a difference in hydrogen ion concentration on either side of certain phospholipid membranes in the chloroplasts and mitochondria and it is essentially the flow of these hydrogen ions across these membranes that generates ATP.

12.2 (a) list the four stages in aerobic respiration and state where each occurs in eukaryotic cells

The respiration of glucose can be broken down into four stages:
Glycolysis: This takes place in the cytosol of all cells. It is an ancient biochemical pathway. It doesn’t need oxygen and can take place in both aerobic or anaerobic conditions. During glycolysis, glucose is broken down into two molecules of pyruvate which is a three-carbon compound.

Link Reaction: This takes place in the matrix of the mitochondria. Pyruvate is dehydrogenated (Hydrogen is removed) and then decarboxylated (a carboxyl group is removed which is eventually released as carbon dioxide) and converted to acetate.

Krebs Cycle: This also takes place in the matrix of the mitochondria. Acetate is decarboxylated and dehydrogenated.

Oxidative Phosphorylation: This takes place on the folded inner mitochondrial membranes (known as cristae). It involves the oxidation of reduced NAD and reduced FAD as a part of an electron transport chain and ATP synthesis by the process of chemiosmosis. Oxygen is required as a final electron acceptor and metabolic water is produced.

ATP is synthesized in substrate-linked reactions in Glycolysis and in Krebs Cycle and by Chemiosmosis in Oxidative Phosphorylation.

12.2(b) outline glycolysis as phosphorylation of glucose and the subsequent splitting of fructose 1,6-bisphosphate into two molecules of triose phosphate, which are then further oxidized to pyruvate with a small yield of ATP and reduced NAD

It is a multi-step process in which a glucose molecule, with 6 carbon atoms, is eventually split into 2 molecules of pyruvate, each with 3 carbon atoms. Glycolysis takes place in the cytosol of all living cells. In organisms that perform cellular respiration, glycolysis is the first stage of the process. However, glycolysis doesn’t require oxygen, occurring in both aerobic and anaerobic conditions.

There are two phases of glycolysis i.e. The Preparatory phase during which ATP is utilized and hexose molecule is cleaved into two molecules of triose phosphate and the Oxidative phase during which ATP is synthesized. Glucose is a very stable molecule and it needs to be activated before it can undergo lysis. One ATP molecule is hydrolyzed and the phosphate group released is attached to the glucose molecule at carbon number 6 to form Glucose-6-Phosphate. This phosphorylation of glucose is carried out by the enzyme Hexokinase.
Glucose-6-Phosphate is changed into its isomer Fructose-6-Phosphate.

Another ATP molecule is hydrolyzed and the phosphate group released is attached to Fructose-6-Phosphate at carbon number 1. This activated hexose sugar is now called fructose 1,6-bisphosphate. This step is catalyzed by the enzyme Phosphofructokinase, which can be regulated to either speed up or slow down the glycolysis pathway. This phase also prevents glucose from being transported out of the cell.

Fructose 1,6-bisphosphate splits to form two three-carbon sugars, DHAP (dihydroxyacetone phosphate) and Glyceraldehyde-3-Phosphate (triose phosphate). They are isomers of each other, but only one─Glyceraldehyde-3-phosphateㅡcan directly continue through the next steps of glycolysis pathway. DHAP is easily converted into Glyceraldehyde-3-Phosphate/Triose Phosphate.

The Preparatory phase is followed by the Oxidative phase. Two Hydrogen atoms are removed from each molecule of Glyceraldehyde-3-Phosphate/ Triose Phosphate. (The removal of hydrogen atoms is an oxidation reaction.) This process involves dehydrogenase enzymes. These are aided by the coenzyme NAD (nicotinamide adenine dinucleotide). NAD is a hydrogen acceptor which combines with the Hydrogen atoms, becoming reduced NAD. Thus, resulting in the formation of two molecules of reduced NAD, one for each molecule of Triose Phosphate. The overall reaction is exergonic and the energy that is released is then used to phosphorylate the resulting two molecules, forming 2 molecules of 1,3-bisphosphoglycerate.

Each molecule of 1,3-bisphosphoglycerate donates one of its phosphate groups to ADP making a molecule of ATP. This is known as substrate-linked or substrate-level phosphorylation. Each molecule of 1,3-bisphosphoglycerate turns into 3-phosphoglycerate which is then converted to its isomer, 2-phosphoglycerate.

Each molecule of 2-phosphoglycerate then loses a molecule of water, becoming phosphoenolpyruvate (PEP). PEP is an unstable molecule poised to lose its phosphate group in the last step of glycolysis. Each molecule of PEP readily donates its phosphate group to ADP making a second molecule of ATP. In this process, a molecule of ADP is phosphorylated (an inorganic phosphate group Pi is added to a molecule of ADP by substrate-level phosphorylation).

As it loses its phosphate, PEP is converted to pyruvate which is the final product of glycolysis.

Products of Glycolysis:

From each molecule of glucose at the beginning of Glycolysis, at the end of Glycolysis, there are:

Net two molecules of ATP. Four had been made but 2 were used during the preparatory phase of glycolysis.

Two molecules of reduced NAD. These will then carry hydrogen atoms to the inner mitochondrial membrane and be used to generate more ATP during Oxidative Phosphorylation.

Two molecules of pyruvate. In the presence of Oxygen, it will be actively transported into the mitochondrial matrix for the next stage of aerobic respiration. In the absence of Oxygen, it will be changed in the cytoplasm to lactate or ethanol.

Overall equation for Glycolysis::

Glucose + 2NAD ⟶ 2Pyruvate + 2ATP + 2 reduced NAD

THE LINK REACTION (pyruvate oxidation):

12.2(c). explain that, when oxygen is available, pyruvate made during glycolysis is converted into acetyl coenzyme A in the link reaction

When Oxygen is available, pyruvate passes by active transport through the outer and inner mitochondrial membrane into the mitochondrial matrix.

Pyruvate first undergoes decarboxylation. A carboxyl group is removed from the pyruvate molecule, which eventually leaves as carbon dioxide, leaving behind a 2-carbon molecule. This carbon dioxide produced diffuses out of the mitochondrion and out of the cell. The enzyme involved in this step is known as pyruvate decarboxylase.

The resulting 2-carbon molecule undergoes dehydrogenation by dehydrogenases and is oxidized. The hydrogen atoms that are removed are transferred to NAD to form reduced NAD.

The oxidized two-carbon molecule─an acetyl group becomes attached to coenzyme A (CoA) to form acetyl CoA.

The overall reaction for this process is:

pyruvate + NAD + CoA ⟶ acetyl CoA + reduced NAD + COշ

The function of coenzyme A is to carry acetate/acetyl groups to Krebs Cycle.

Products of Link Reaction:

If we consider the two pyruvates that enter from the glycolysis pathway (for one glucose molecule), we can summarize the link reaction as follows:

Two molecules of pyruvate that enter from glycolysis are converted into two molecules of acetyl CoA.
Two carbons are released as carbon dioxideㅡout of six originally present in glucose.

2 reduced NAD are formed from NAD

No ATP is produced. However, each reduced NAD will take hydrogen atoms to the inner mitochondrial membrane and they will be used to make ATP during oxidative phosphorylation

KREBS CYCLE (Citric acid cycle or Tricarboxylic acid cycle):

12.2 (d)(e). outline the Krebs cycle, explaining that oxaloacetate accepts the acetyl group from acetyl coenzyme A to form citrate, which is reconverted to oxaloacetate in a series of small steps explain that reactions in the Krebs cycle involve decarboxylation and dehydrogenation and the production of reduced NAD and FAD

In all eukaryotic cells, both the Krebs Cycle and the link reaction take place exclusively inside the mitochondria and these will only occur if oxygen is available i.e. in aerobic conditions.

The Krebs Cycle takes place within the matrix of the mitochondrion. It is a cycle of enzyme-catalyzed reactions.

In the first set of Krebs Cycle, the acetyl group (which is the end product of the link reaction) combines with a four-carbon molecule, Oxaloacetate, releasing the CoA and forming a six-carbon molecule known as citrate.

Citrate is converted into its isomer, isocitrate. Isocitrate is dehydrogenated and decarboxylated, leaving behind a five-carbon molecule called α-ketoglutarate. During the dehydrogenation step, NAD accepts two hydrogen atoms and gets reduced to reduced NAD. The enzyme catalyzing this step, isocitrate dehydrogenase, is vital in regulating the speed of the Krebs Cycle. During the decarboxylation step, a carboxyl group is removed from isocitrate. This is eventually released as carbon dioxide.

α-ketoglutarate is then dehydrogenated (dehydrogenation is a type of oxidation), reducing NAD to reduced NAD and Decarboxylated, releasing a molecule of carbon dioxide in the process. The remaining four-carbon molecule combines with Coenzyme A, forming an unstable compound succinyl-CoA. The enzyme catalyzing this step, α-ketoglutarate dehydrogenase, is important in the regulation of the Krebs Cycle.

In the next step, the CoA of succinyl-CoA is replaced by a phosphate group which is then used to phosphorylate a molecule of ADP from ATP, by substrate-level phosphorylation. The four-carbon molecule produced in this step is known as succinate.

In the next step Succinate is dehydrogenated, forming another four-carbon molecule called fumarate.Two hydrogen atoms are transferred to FAD, producing reduced FAD.

In the next step, water is added to the four-carbon molecule fumarate, converting it into another four-carbon molecule–malate.

In the last step of the citric acid cycle, oxaloacetate, the starting four-carbon compound is regenerated by the dehydrogenation of malate. Another molecule of NAD is reduced to reduced NAD. Regenerated oxaloacetate is ready to combine with another acetyl group made during the link reaction..

Krebs Cycle does not produce much ATP directly. However, it makes a lot of ATP indirectly by the way of reduced NAD and reduced FAD that it generates. These hydrogen carriers (electron carriers) will deposit their electrons to the electron transport chain and drive the synthesis of ATP molecules through the process of Oxidative Phosphorylation.

In a single turn of the Cycle:

Two carbons enter from the acetyl CoA, and two molecules of COշ are released.
Three molecules of reduced NAD are generated and one molecule of reduced FAD is generated.
one molecule of ATP is generated.
These figures are for one turn of the cycle, corresponding to one molecule of acetyl-CoA
Each glucose produces two acetyl-CoA molecules, so we will multiply these numbers by two for the yield per molecule of glucose.

The significance of Krebs cycle:

It produces hydrogen atoms that are carried by NAD and FAD to the electron transport chain for oxidative phosphorylation and production of ATP by chemiosmosis, which provides energy for the cell.
It regenerates oxaloacetate, which would otherwise be completely used up
It is a source of intermediate compounds used by the cells in the manufacture of other vital substances such as fatty acids, amino acids, and chlorophyll.


12.2(f) outline the process of oxidative phosphorylation and also outline the role of oxygen as the final electron acceptor

12.2(g) explain that during oxidative phosphorylation:

• high energy electrons release energy as they pass through the electron transport system
• the energy released is used to transfer protons across the inner mitochondrial membrane
• protons return to the mitochondrial matrix by facilitated diffusion through ATP synthase enzyme thus providing energy for ATP synthesis (details of ATP synthase are not required)

The last stage of aerobic respiration is Oxidative Phosphorylation and it involves the electron carriers embedded in the inner mitochondrial membrane. These membranes are folded into cristae which increases the surface area for electron carriers and ATP synthase enzymes. Oxidative Phosphorylation is made up of two closely linked components i.e. the electron transport chain and chemiosmosis. In the electron transport chain, electrons are passed from one electron carrier/acceptor to the next and oxygen acts as the final electron acceptor. It gets reduced to metabolic water. Energy is released in these electron transfers and is used to form an electro-chemical gradient. In chemiosmosis, the energy stored in this gradient is used to generate ATP.

The Electron transport chain, ETC, is a series of electron carriers/acceptors that are embedded in the inner mitochondrial membrane. ETC has two specific qualities:

Every next compound in ETC has a higher affinity for electrons. So, electrons are forced to move down the chain towards molecular oxygen which acts as the final electron acceptor, with the highest affinity for electrons. So, provides a suction force for ETC to operate
The arrangement of compounds in ETC is such that electrons move down the energy level towards Oxygen which accepts electrons at the lowest energy level. So, the flow of electrons is exergonic.

Reduced NAD and reduced FAD from other stages of respiration are passed on to ETC. Here the hydrogen atoms are removed from the two hydrogen carriers and each hydrogen atom is split into its constituent proton (H+) and electron (e-). The protons go to the solution in the matrix while the high-energy electrons are transferred from one electron carrier to another.

As the electrons travel through the chain of electron carriers, they go from a higher energy level to a lower energy level, moving from a less electronegative to a more electronegative component of the chain. This is an exergonic process and quanta of energy are released in these electron transfers. These electrons are finally accepted by molecular oxygen which acts as the final electron acceptor. At the end of the chain, the electrons reunite with the protons from which they were originally split. 4H+ and 4e- combine with molecular oxygen and reduce it to water. (This is why oxygen is needed in aerobic respiration.) The energy released from the transfer of electrons is used by some carrier proteins (complexes I III and IV) to actively pump protons from the mitochondrial matrix into the intermembrane space. (Complexes I, III, and IV of the electron transport chain are proton pumps.) As electrons move energetically downhill, the complexes capture the released energy and use it to pump protons from the matrix into the intermembrane space. This pumping generates a concentration gradient of protons between the intermembrane space and the matrix. This also means that there is an electrochemical gradient across the inner mitochondrial membrane. The gradient is sometimes referred to as the proton-motive force.

Protons can’t pass directly through the phospholipid bilayer of the membrane because its core is too hydrophobic. H+ ions can only move down their concentration gradient with the help of channel proteins that form hydrophilic tunnels across the membrane.
The protons pass back into the mitochondrial matrix through these protein channels in the inner membrane, down their concentration gradient via facilitated diffusion. Associated with each channel is the enzyme ATP synthase. As the protons pass through the channel, this enzyme uses the energy of the flowing protons to catalyze the reaction ADP + Pi ⟶ ATP. This process, in which energy stored in the proton gradient is used to make ATP, is called chemiosmosis. Once in the matrix, the protons recombine with the electrons at the end of the electron transport chain on the inner mitochondrial membrane to form hydrogen atoms, which in turn combine with oxygen to form metabolic water.

All of the electrons that enter the transport chain come from reduced NAD and reduced FAD molecules produced during earlier stages of cellular respiration: glycolysis, pyruvate oxidation, and the Krebs cycle.

ETC has 2 important functions:

Regenerates electron carriers, reduced NAD and reduced FAD pass their electrons to the electron transport chain, turning back into NAD and FAD. This is necessary because the oxidized forms of these electron carriers are used in glycolysis and the Krebs cycle and must be available to keep these processes running.

generates a proton gradient. The Electron transport chain builds a proton gradient across the inner mitochondrial membrane, with a higher concentration of H+ ions in the intermembrane space and a lower concentration in the matrix by providing energy to pump hydrogen ions into the intermembrane space. This gradient represents a stored form of energy.

Theoretically less energy is released than it should be because:
• Some protons leak across the mitochondrial membrane, this reduces the number of protons to generate the proton motive force.
•Some ATP produced is used to actively transport pyruvate into the mitochondrial matrix.
•Some ATP is used for the shuttle to bring hydrogen from reduced NAD made during glycolysis, in the cytoplasm, into the mitochondria.

12.1 (d) outline the roles of coenzyme NAD, FAD, and Coenzyme A in respiration:
Role of NAD/FAD in respiration:

Hydrogen atoms are removed from substrate molecules in oxidation reactions during glycolysis, the link reaction, and the Krebs cycle. These reactions are catalyzed by dehydrogenase enzymes. NAD/FAD are coenzymes for these dehydrogenases and act as hydrogen/electron carriers. A coenzyme is a molecule that is required for an enzyme to be able to catalyze a reaction. There are two important electron/hydrogen carriers in cellular respiration, nicotinamide adenine dinucleotide (abbreviated as NAD in its oxidized form) and flavin adenine dinucleotide (abbreviated as FAD in its oxidized form). The hydrogen atoms from substrate molecules are combined with coenzymes such as NAD. These carry the hydrogen atoms, which can later be split into hydrogen ions and electrons, to the inner mitochondrial membrane. Here, they will be involved in the process of oxidative phosphorylation which produces huge quantities of ATP. When reduced NAD or reduced FAD give their high energy electrons to the electron transport chain during oxidative phosphorylation, reduced NAD and reduced FAD are re-oxidized and regenerated. (The term ‘reduce’ means to add hydrogen, so reduced NAD/FAD has had hydrogen added to it.) These low-energy molecules cycle back to glycolysis and/or the Krebs cycle, where they pick up more high-energy electrons and the process continues. NAD/FAD is present in small quantities within the cells and is, therefore, continually recycled. The oxidation of reduced NAD to NAD allows it to be recycled and used again. Glycolysis and the Krebs cycle cannot proceed if there is no NAD present to pick up electrons as the reactions continue. The electrons pass from one electron acceptor in the ETC to another thereby releasing energy. This energy is used to pump H+ ions (protons) from the matrix to the intermembrane space. From here, they diffuse down their electrochemical gradient through ATP synthase which generates ATP.

Structure of NAD:

NAD is an organic, non-protein molecule that acts as a coenzyme for dehydrogenase enzymes helping them to carry out oxidation reactions. NAD is made of two linked nucleotides. It’s made in the body from nicotinamide (Vitamin B3), the 5-carbon sugar ribose, adenine, and two phosphates. One nucleotide contains the nitrogenous base adenine. The other contains a nicotinamide ring that can accept hydrogen atoms each of which can later be split into a hydrogen ion and an electron.
When a NAD has accepted two hydrogen atoms, it is in its reduced state. When it loses them, it is in its oxidized state. NAD is required during glycolysis, the link reaction and Krebs cycle, and during anaerobic respiration.

Role of coenzyme A in respiration:

This coenzyme is made from pantothenic acid (also known as vitamin Bཏ), adenosine (ribose and adenine), three phosphates groups, and cysteine (an amino acid). It’s function is to carry ethanoate (acetate)/ acetyl groups, made during the link reaction, onto the Krebs cycle. During the Krebs Cycle, each acetyl group combines with a molecule of oxaloacetate (A 4 carbon compound) to form a six-carbon molecule called citrate. It also carries acetate groups that have been made from fatty acids or from some amino acids onto the Krebs cycle. The reverse is also true, namely that the excess carbohydrates can be made into fats vis acetyl CoA, making it a pivotal molecule in the interconversion of major substances in eukaryotes.

Anaerobic Respiration

12.2 (j) distinguish between aerobic respiration and anaerobic respiration in mammalian tissue and in yeast cells, comparing the relative energy released by each

12.2(k) explain the generation of a small yield of ATP from respiration in anaerobic conditions in yeast and in mammalian muscle tissue, including the concept of oxygen debt

Anaerobic respiration involves the breakdown of organic molecules without oxygen. This releases much less energy as compared with aerobic respiration. Anaerobic respiration is useful in tissues that have a high demand for energy such as in muscles, in which there is not enough oxygen to produce all the energy needed by using aerobic respiration alone.

Oxygen acts as the final electron acceptor in the process of oxidative phosphorylation. If oxygen is absent, the electron transport chain (ETC) cannot function, so the Krebs cycle and the link reaction also stop. This leaves the anaerobic process of glycolysis as the sole source of ATP. The reduced NAD, generated during the oxidation of glucose, has to be reoxidized so that glycolysis can keep operating. The hydrogen atoms from the reduced NAD must be accepted to regenerate NAD. Without this, the already little supply of NAD in cells will entirely be converted to reduced NAD leaving no NAD to accept hydrogen atoms from substrates involved in the process of glycolysis. This increases the chances of survival of an organism under temporary adverse conditions.

For eukaryote cells there are two pathways to re-oxidized the reduced NAD:
• Fungi, such as yeast, use ethanol (alcohol) fermentation (plant cells, such as root cells
under waterlogged conditions, can also use this pathway).
• Animals use lactate fermentation.

The anaerobic pathway utilizes pyruvate, the final product of glycolysis.
Without the functioning ETC, there is an excess of reduced NAD and pyruvate produced during glycolysis. Pyruvate is subsequently reduced to lactate (lactic acid)/ethanol by reduced NAD, leaving NAD after the reduction. This reaction is catalyzed by the enzyme dehydrogenase. This essentially leads to the recycling of NAD.

By recycling NAD the process of glycolysis can continue as the NAD ‘stock’ has been replenished. The glycolysis pathway produces 2 net ATP molecules which can be used for energy to drive muscular contraction etc. The 2 ATP molecules are much less than would be produced by aerobic respiration, it is necessary as without anaerobic respiration there would be no other method of ATP production

Alcohol Fermentation:
In alcohol fermentation, the reduced NAD donates its electrons/hydrogen atoms to a derivative of pyruvate, producing ethanol. Going from pyruvate (3C) to ethanol (2C) is a two-step process. In the first step, a carboxyl group is removed from pyruvate and released as carbon dioxide, producing a two-carbon molecule called acetaldehyde/ethanal. This reaction is catalyzed by an enzyme called pyruvate decarboxylase (not present in mammals), which has a coenzyme (thiamine diphosphate) bound to it.

CHဒCOCOOH (pyruvate) ⟶ CHဒCHO (ethanal) + COշ (carbon dioxide)

In the second step the reduced NAD passes its electrons/ hydrogen atoms to acetaldehyde/ethanal, deoxidizing and regenerating NAD and reducing ethanal/acetaldehyde to ethanol. This step is catalyzed by the enzyme ethanol dehydrogenase. This process is irreversible.

CHဒCHO(ethanal) ⟶ CշHཏOH(ethanol)
Glucose ⟶ 2 CշHཏOH(ethanol) + 2 COշ(carbon dioxide)

Lactic Acid Fermentation:

Lactate fermentation occurs in mammalian tissue during vigorous activity, for example, running to escape a predator, when demand for ATP for muscle contraction is high and there is an oxygen deficit. Under these conditions pyruvate doesn’t enter the mitochondria and is removed by converting it to lactate.

Pyruvic acid/pyruvate + reduced NAD ↔ lactic acid/lactate + NAD

The enzyme used in this reaction is known as lactate dehydrogenase (LDH).
It is a single-step process in which pyruvate acts as the hydrogen acceptor. It accepts the hydrogen atoms from the reduced NAD which is re-oxidized and NAD is regenerated so glycolysis can continue. Lactate dehydrogenase catalyzes the oxidation of reduced NAD together with the reduction of pyruvate to lactic acid. The lactate produced is carried in the blood away from muscles, to the liver. When more oxygen is available lactate can be converted back to pyruvate and eventually glucose in a process called the core cycle. The need for oxygen during this process is the cause of oxygen debt after exercise.

(It’s not the buildup of lactate that causes muscle fatigue. Muscles can still function in the presence of lactate if the pH is kept constant by buffers. It is specifically the reduction in pH that will reduce the enzyme activity in the muscles.)

Lactate fermentation cannot happen indefinitely because:
The reduced quantity of ATP generated would not be enough to sustain vital processes over time.
Accumulation of lactic acid causes a fall in pH leading to denaturation of proteins such as enzymes..

Lactic acid fermentation is important as:
Glycolysis can continue,so at least a little amount of ATP is produced.
Another advantage of anaerobic respiration is its speed. It produces ATP very quickly. For example, it lets the muscles get the energy they need for short bursts of intense activity. Aerobic respiration, on the other hand, produces ATP more slowly.

In anaerobic respiration, only glycolysis occurs. Due to the absence of the Oxygen, Krebs Cycle, Link reaction, and Oxidative Phosphorylation come to halt. Since glucose is not fully broken down, it still contains a lot of energy. Pyruvate does not enter mitochondria to undergo further oxidation as Oxygen is not available as the final electron acceptor. ETC stops and oxidative Phosphorylation can’t occur, which produces the most ATP.

Oxygen debt:
Oxygen debt is the difference in the supply of Oxygen and demand of Oxygen during vigorous activity. This reduces the rate of aerobic respiration.
The demand for the remaining energy is fulfilled by anaerobic respiration so, the more the anaerobic respiration more will be the oxygen debt.

Even after the physical exercise, the rate of oxygen consumption in the body will remain high because oxygen is required for oxidation of lactic acid, oxygenation of myoglobin, and for regeneration of creatine phosphate.

Respiratory substrates:

A respiratory substrate is any organic molecule from which energy can be liberated to produce ATP in any living cell.
The majority of ATP made during respiration is produced during oxidative phosphorylation; when protons flow through channels associated with which is the enzyme ATP synthase, on the inner mitochondrial membranes. The protons and electrons then combine with oxygen to produce metabolic water.

The more protons there are, the more the ATP produced. It follows then that the more hydrogen atoms there are in a molecule of respiratory substrate, the more ATP can be generated when that substrate is respired. It also follows that if there are more hydrogen atoms per mole of a respiratory substrate, then more oxygen is required to respire that substance.

Energy values of respiratory substrates:

12.1(f). explain the relative energy values of carbohydrate, lipid, and protein as respiratory substrates and explain why lipids contain more energy

Most of the energy released in respiration comes from the oxidation of hydrogen to water when reduced NAD and reduced FAD transfer their electrons to the electron transport chain (ETC). The more hydrogen atoms there are (in comparison with carbon or oxygen atoms) in the structure of a substrate molecule, the greater the energy value of that substrate molecule. It is hydrogen atoms that are used to generate ATP via the ETC. The more hydrogen atoms the more reduced NAD and reduced FAD that can be made. This will generate ATP in oxidative phosphorylation.

Fatty acids have more C-H bonds and thus more hydrogens per unit mass/per molecule than carbohydrates, so lipids have a greater energy value per unit mass/per molecule (lipid provides more than twice as much energy per gram as carbohydrate or protein).

For proteins, the number of hydrogen atoms per mole accepted by NAD and then used in oxidative phosphorylation is slightly more than the number of hydrogen atoms per mole of glucose, so proteins release slightly more energy than equivalent masses of carbohydrate.

The net energy released by biological macromolecules are arranged in this order:
Lipids > Alcohols > Proteins > Carbohydrates.

The energy values of these respiratory substrates are found using a calorimeter. A known mass of substrate is completely burnt in the presence of oxygen. The energy given off as heat is then used to raise the temperature of a known volume of water.

12.1(f) define respiratory quotient (RQ) and find RQs from equations for respiration
Respiratory Quotient (RQ): can be used to determine which respiratory substrate being used in respiration. It also shows whether or not anaerobic respiration is taking place.

It is the ratio of moles/volume of Carbon dioxide released per unit time to the moles/volume of oxygen absorbed during the same time interval.

For example, when glucose (a carbohydrate) is oxidized:

C₆H₁₂O₆ + 6O₂ ⟶ 6CO₂ + 6H₂O

RQ is therefore: 6/6 = 1.0

Lipids, however, have more hydrogen atoms in their structure relative to carbon. Hence, a greater volume of oxygen is required to oxidize a lipid completely and their RQs are therefore lower than those of carbohydrates.

Proteins have a very varied structure depending on the number and types of each amino acid in the protein molecule. Their RQs are ,therefore, equally varied but most have values around 0.9

There is no Respiratory Quotient for lactic acid fermentation as no carbon dioxide is liberated.

If only alcoholic fermentation is occurring, RQ will be infinity. High values of RQ (or more than one) indicate that both aerobic and anaerobic respiration are taking place..

RQ value of less than 0.7 indicates that Carbon dioxide is being used in some metabolic process.

RQ of carbohydrates is 1.0
RQ of lipids is 0.7
RQ of proteins is 0.9
(RQ is more accurately known as respiratory exchange ratio RER)

The usefulness of RQs in determining the substrate that is being respired is limited because:

Substances are rarely oxidized completely and partial oxidation gives a different value.
Organisms rarely, if ever, respire a single food substance, and the RQ, therefore, reflects the proportions of the different substrates that are being respire.

Most resting animals have an RQ of between 0.8 and 0.9. Although these values suggest that proteins were being respired, we know that protein is used only in extreme situations such as starvation or prolonged fasting. We must assume, therefore, that these values are due to a combination of carbohydrates(1.0) and lipids (0.7) being respired.

Importance of oxygen in aerobic respiration:

In the Electron transport chain, electrons are transferred to a series of electron acceptors/carriers. Oxygen is the final acceptor of electrons in this series. It also acts as the final acceptor for hydrogen. Molecular oxygen, after accepting electrons, reacts with protons to form metabolic water. Oxygen has a very high electronegativity, therefore, it has a strong affinity for electrons and will effectively draw electrons away from other atoms. When Oxygen is not available, the electron transport chain will cease to operate because oxygen provides the suction force needed for electrons to move. Ultimately, the electron carriers/acceptors of ETC will remain in their reduced states as ETC ceases to function. Due to no exergonic flow of electrons, hydrogen ions (protons) cannot be pumped into intermembrane space. No proton motive force will be generated and Oxidative Phosphorylation will not take place.

Since the electron acceptors remain in their reduced state hydrogen atoms from reduced NAD and reduced FAD can’t be accepted so they can’t be re-oxidized to pick up more hydrogen atoms produced during aerobic processes of (cellular) respiration. Dehydrogenases in the mitochondria won’t work as their coenzymes are not present and the link reaction and Krebs cycle will stop. Pyruvate will not enter the mitochondria.

However, glycolysis can still continue, so long as pyruvate produced at the end of glycolysis can be removed and reduced NAD can be converted back to NAD.

12.2(i) describe the relationship between the structure and function of the mitochondrion

All eukaryotic cells contain mitochondria. Their function is performing aerobic respiration and generating ATP. The number of mitochondria in a cell depends on the energy demand of that cell. Higher the energy demand the more the number of mitochondria.
Mitochondria are surrounded by 2 membranes forming an envelope. The space between the membranes is called the intermembrane space.

It has proteins, some of which form channels or carriers that allow the passage of molecules such as pyruvate while other proteins are enzymes. The outer membrane contains transport proteins known as porins which form wide aqueous channels allowing easy access of small water-soluble molecules from the surrounding cytoplasm into the intermembrane space.

The outer membrane is smooth but the inner membrane is folded inwards to form finger-like cristae. Mitochondria from active cells have longer and more densely packed cristae than mitochondria from less active cells, so to house more ETCs. The inner membrane is selectively permeable and allows only certain ions and molecules to enter the matrix. Because it is impermeable to small ions, protons accumulate in the intermembrane space, building up a proton gradient which represents a stored form of energy.

The inner membrane is folded to form finger-like cristae which project into the interior solution called matrix, bounded by the inner membrane. The presence of cristae increases the efficiency of respiration because it increases the surface area available for the reactions to take place. This allows greater space for processes that happen across this membrane. There can be more ATP synthase and ETCs hence more ATP via oxidative phosphorylation.

The inner membrane is the site of ETC and has electron carriers embedded in it. Its presence on the inner membrane makes the specific arrangement of electron carriers close together possible so there is a more efficient flow of electrons and ATP formation can happen.
The inner membrane contains ATP Synthase (sometimes referred as stalked particles) which generates ATP in the matrix. The ATP synthase enzymes protrude from the inner membrane into the mitochondrial matrix, forming a channel that allows protons to pass through them. Protons flow down a proton gradient, through the ATP synthase enzymes, from the intermembrane space into the matrix of mitochondria. This flow is called chemiosmosis. The force of this flow (the proton motive force) drives rotation of part of the enzyme and allows ADP and Pi (inorganic phosphate) to be combined to form ATP.

The matrix is the site of the link reaction and Krebs cycle. It contains:

• the enzymes that catalyze the reactions in these processes.
• molecules of coenzyme NAD
• oxaloacetate (4C): the compound that accepts acetate from the link reaction
• several identical copies of looped mitochondrial DNA, some of which code for mitochondrial enzymes and other proteins.
•70S ribosomes which will perform protein synthesis for mitochondrial purposes.
•Phosphate granules are present which can provide inorganic phosphate groups for ATP formation if needed.

The space between 2 membranes of the envelope usually has a lower pH than the matrix of the mitochondrion as a result of protons that are released into the intermembrane space by the activity of the electron transport chain, from the matrix through the inner membrane. This establishes a proton gradient between the intermembrane space and the mitochondrial matrix. The facilitated diffusion of protons from the intermembrane space into the matrix through ATP synthase results in the synthesis of ATP.

As mitochondria are double membranous organelles, a compartment in the form of intermembrane space is available where hydrogen ions can be pumped to generate a proton motive force for chemiosmosis to take place.

Mitochondria can be moved around within cells by the cytoskeleton/microtubules. In some types of cells, the mitochondria are permanently positioned near a site of high ATP demand, for example at the synaptic knobs of neurons. However, they have been moved to that position by the microtubules.

12.2(l) explain how rice is adapted to grow with its roots submerged in water

Rice plants are grown partly submerged in paddy fields. Oxygen levels in the paddy fields are low as oxygen is used up by the respiring bacteria in the mud and also as oxygen is not very soluble in water.
There are two advantages of growing rice plants in paddy fields. Flooding brings about chemical changes in the soil that increases the supply of soil nutrients required by the rice plants. It also reduces competition from weeds. Rice plant does not grow well when it has to compete with weeds for the resources that it needs.

The main adaptive feature of the rice plant to grow with its roots submerged in water is the presence of loosely packed cells forming a tissue called aerenchyma. Rice stems contain a large number of interconnected air spaces (hollow aerenchyma) running down the length of the stem and into the roots. This allows oxygen (some of which was formed in the plant from photosynthesis) to penetrate through to the roots which are submerged in water, supplying oxygen for aerobic respiration. This decreases the chances that anaerobic respiration will occur.

This is also supplemented by the air that is trapped in between the ridges of the underwater leaves. These leaves have a hydrophobic corrugated surface that holds a thin layer of air in contact with the surface of the leaf.

Many rice roots are very shallow; this allows access to oxygen that diffuses into the surface layer of the waterlogged soil. Thus, allowing access to higher levels of oxygen in surface water.

The part of the stem of the rice plant which is submerged in water secretes the hormone ethene which will stimulate the release of another hormone gibberellin, which causes the elongation of stems, elevating the leaves above water. Hence, the top parts of the leaves and flower spikes are always held above the water. This allows oxygen and carbon dioxide to be exchanged through the stomata present on the leaves so they can carry out more photosynthesis and oxygen will be available to roots for more respiration.

The cells of the submerged roots have to rely mostly on alcoholic fermentation. This can result in ethanol accumulation in the plant tissues. Ethanol is toxic to the plant cells. Cells in the roots of rice plants show a very high tolerance to ethanol. When oxygen levels fall too low these plants can respire anaerobically for longer periods of time.
They produce much more alcohol dehydrogenase which breaks down ethanol. This allows the plants to grow actively even when oxygen is scarce by using ATP produced by alcoholic fermentation.