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During the process, a proton gradient is created when the protons are pumped from the mitochondrial matrix into the intermembrane space of the cell, which also helps in driving ATP production. Often, the use of a proton gradient is referred to as the chemiosmotic mechanism that drives ATP synthesis since it relies on a higher concentration of protons to generate “proton motive force”. The amount of ATP created is directly proportional to the number of protons that are pumped across the inner mitochondrial membrane.
The electron transport chain involves a series of redox reactions that relies on protein complexes to transfer electrons from a donor molecule to an acceptor molecule. As a result of these reactions, the proton gradient is produced, enabling mechanical work to be converted into chemical energy, allowing ATP synthesis. The complexes are embedded in the inner mitochondrial membrane called the cristae in eukaryotes. Enclosed by the inner mitochondrial membrane is the matrix, which is where necessary enzymes such as pyruvate dehydrogenase and pyruvate carboxylase are located. The process can also be found in photosynthetic eukaryotes in the thylakoid membrane of chloroplasts and in prokaryotes, but with modifications.
By-products from other cycles and processes, like the citric acid cycle, amino acid oxidation, and fatty acid oxidation, are used in the electron transport chain. As seen in the overall redox reaction,
energy is released in an exothermic reaction when electrons are passed through the complexes three molecules of ATP are created. Phosphate located in the matrix is imported via the proton gradient, which is used to create more ATP. The process of generating more ATP via the phosphorylation of ADP is referred to oxidative phosphorylation since the energy of hydrogen oxygenation is used throughout the electron transport chain. The ATP generated from this reaction go on to power most cellular reactions necessary for life.
The Electron Transport Chain & Chemiosmosis
The electron transport chain is one of the many processes in cellular respiration that can be very confusing for students in both high school and college. Chemical processes such as this are very abstract to students they have a difficult time visualizing the various steps and, consequently, develop a less-than-complete understanding of these types of processes. Similarly, the process of chemiosmosis can be very challenging to teach and to visualize. Both of these processes are critical to cellular functioning, so it is critical that students leave an introductory biology course with a proficient understanding of these metabolic pathways.
Keys (these represent the electrons from NADH)
∘ An alternative object is a potato
Markers (these represent the hydrogen ions)
∘ Any object could be substituted here, such as marbles, bottle caps, etc.
Red and yellow 6 × 2 Lego pieces or something similar
∘ These represent ADP and inorganic phosphate for the purpose of synthesizing ATP
This demonstration works best in a room in which there are at least two rows of tables. The front row of tables represents the inner mitochondrial membrane (cristae), and the back row of tables represents the intermembrane space of the mitochondrion (see Figure 1). Students sitting at the front row of tables represent the various cytochromes and enzymes embedded in the inner mitochondrial membrane. In this simulation, the student on the far left side of the front row of tables is the first cytochrome. The student second from the far right of the front row of tables represents oxygen. The student on the far right of the front row represents ATP synthase. Students in the back row of tables represent the intermembrane space of the mitochondria. The teacher (with the keys) represents NADH. Providing nametags or some other indicator of the roles the students and the teacher are playing might be an additional help for students in visualizing the process.
Table and classroom setup.
Table and classroom setup.
Introduce the simulation by telling the students that they will be role-playing the electron transport chain and chemiosmosis. Start by giving each student in the front row of tables (except the one who represents oxygen) a marker or other object. These objects represent hydrogen ions (H + ) floating around in the matrix of the mitochondrion. In the following outline, statements and questions from the teacher are indicated by italics.
Stand next to the student on the far left of the front row of tables (the first cytochrome). Take out the keys, shake them, and tell the students that you are NADH and the keys are the electrons that NADH possesses.
Ask the students to identify where the electrons came from (the answer is glucose or pyruvate). Hand the keys to the first person in the front row. Ask them what happens to them and to NADH (the teacher) chemically when electrons are transferred (the student/cytochrome is reduced and the teacher/NADH is oxidized review oxidation and reduction if necessary).
At the same time, ask the first student to hand their marker (or other object) to the student behind them, who represents the intermembrane space of the mitochondrion.
Have the first student in the front row hand the keys to the next person and have them state who is oxidized and who is reduced. Explain that this is active transport, using the energy of the electrons being passed down the chain to pump H + ions through the inner mitochondrial membrane.
Have the second student pass their marker (or other object) to the student behind them. Remind the students that the markers represent hydrogen ions being transported across the cristae of the mitochondrion.
Continue doing this until all the students in the front row (except the two on the far right) have passed the keys and handed their markers to the students behind them.
Once the keys get to the student second from the end on the right (representing oxygen), explain to the students that the final electron acceptor is oxygen and that oxygen is reduced, along with hydrogen ions, to form water, a byproduct of cellular respiration.
Next, ask the students about the difference in the amount of hydrogen ions in the intermembrane space compared with the matrix, which is in front of the front row of tables.
Explain to them that there is an electrochemical gradient of hydrogen ions from the intermembrane space to the matrix that represent potential energy, and that based on the principle of diffusion, the hydrogen ions would normally want to diffuse across the cristae in order to achieve equilibrium.
In order to do so, the hydrogen ions must pass through ATP synthase. Have the students with markers line up behind the student who represents ATP synthase. One by one, have them hand their marker to the student representing ATP synthase.
Every time a student hands a marker to the student who represents ATP synthase, have the student representing ATP synthase take one red Lego block (which represents ADP) and attach it to one yellow Lego block (which represents an inorganic phosphate). This represents the synthesis of one ATP molecule.
Have the student who represents ATP synthase hand the markers (hydrogen ions) to the student who represents oxygen. This student will place the keys (electrons) and markers (hydrogen ions) in their hands, combining them to represent water.
Continue having students pass markers (hydrogen ions) to the student who represents ATP synthase until all the hydrogen ions have passed through. The level of detail that is discussed regarding ATP synthase function is up to the individual teacher.
All the above information on the processes of the electron transport chain and chemiosmosis comes from Campbell Biology (Reece et al., 2012).
A variation of this activity is to use a potato instead of keys to represent the electrons. In order to do this, obtain a raw potato and heat it in a microwave for 3–5 minutes until it becomes hot. Make sure that the potato is not too hot for the students to handle safely. Give the potato to the first person on the left of the front row of tables, and continue the demonstration as stated above. The potato will become cooler as it is passed from student to student, representing the stepwise loss of energy each time the electron is passed to a different cytochrome. Discuss with the class that the passage of electrons down a transport chain is necessary because the energy from the electrons in NADH cannot be transferred to oxygen in one reaction, because of the explosiveness of one reaction. The electron transport chain allows the stepwise transfer of energy from the electrons.
Assessment of this demonstration is really up to the individual teacher. A variety of assessments could be done, however. Students could complete a quiz on the two processes that were addressed, they could complete a write-to-learn activity that asks them to explain the two processes, questions could be asked on a test, or the students could be asked to act out and explain the processes without help from the teacher.
Lecture 3 - Glycolysis, Kreb's and Electron Transport
The citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle) or the Krebs cycle, (or rarely, the Szent-Györgyi-Krebs cycle) is a series of enzyme-catalysed chemical reactions of central importance in all living cells that use oxygen as part of cellular respiration. In eukaryotes, the citric acid cycle occurs in the matrix of the mitochondrion. The components and reactions of the citric acid cycle were established by seminal work from both Albert Szent-Györgyi and Hans Krebs.
In aerobic organisms, the citric acid cycle is part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to generate a form of usable energy. Other relevant reactions in the pathway include those in glycolysis and pyruvate oxidation before the citric acid cycle, and oxidative phosphorylation after it. In addition, it provides precursors for many compounds including some amino acids and is therefore functional even in cells performing fermentation.
Electron Transport Chain and Oxidative Phosphorylation
An electron transport chain couples a chemical reaction between an electron donor (such as NADH) and an electron acceptor (such as O2) to the transfer of H+ ions across a membrane, through a set of mediating biochemical reactions. These H+ ions are used to produce adenosine triphosphate (ATP), the main energy intermediate in living organisms, as they move back across the membrane. Electron transport chains are used for extracting energy from sunlight (photosynthesis) and from redox reactions such as the burning of sugars (respiration).
Electron transport chains in mitochondria
The cells of almost all eukaryotes (animals, plants, fungi, algae, protozoa – in other words, the living things except bacteria, archaea, and a few protists) contain intracellular organelles called mitochondria, which produce ATP. Energy sources such as glucose are initially metabolized in the cytoplasm. The products are imported into mitochondria. Mitochondria continue the process of catabolism using metabolic pathways including the Krebs cycle, fatty acid oxidation, and amino acid oxidation.
The end result of these pathways is the production of two kinds of energy-rich electron donors, NADH and succinate. Electrons from these donors are passed through an electron transport chain to oxygen, which is reduced to water. This is a multi-step redox process that occurs on the mitochondrial inner membrane. The enzymes that catalyze these reactions have the remarkable ability to simultaneously create a proton gradient across the membrane, producing a thermodynamically unlikely high-energy state with the potential to do work. Although electron transport occurs with great efficiency, a small percentage of electrons are prematurely leaked to oxygen, resulting in the formation of the toxic free-radical superoxide.
The similarity between intracellular mitochondria and free-living bacteria is striking. The known structural, functional, and DNA similarities between mitochondria and bacteria provide strong evidence that mitochondria evolved from intracellular prokaryotic symbionts that took up residence in primitive eukaryotic cells.
Oxidative phosphorylation is a metabolic pathway that uses energy released by the oxidation of nutrients to produce adenosine triphosphate (ATP). Although the many forms of life on earth use a range of different nutrients, almost all carry out oxidative phosphorylation to produce ATP, the molecule that supplies energy to metabolism. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis.
During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within mitochondria, whereas in prokaryotes, these proteins are located in the cells' inner membranes. These linked sets of enzymes are called electron transport chains. In eukaryotes, five main protein complexes are involved, whereas in prokaryotes many different enzymes are present, using a variety of electron donors and acceptors.
The energy released as electrons flow through this electron transport chain is used to transport protons across the inner mitochondrial membrane, in a process called chemiosmosis. This generates potential energy in the form of a pH gradient and an electrical potential across this membrane. This store of energy is tapped by allowing protons to flow back across the membrane and down this gradient, through a large enzyme called ATP synthase. This enzyme uses this energy to generate ATP from adenosine diphosphate (ADP), in a phosphorylation reaction. Unusually, the ATP synthase is driven by the proton flow which forces the rotation of a part of the enzyme—it is a rotary mechanical motor.
Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such as superoxide and hydrogen peroxide that lead to propagation of free radicals, damaging cells and contributing to disease and, possibly, aging. The enzymes carrying out this metabolic pathway are also the target of many drugs and poisons that inhibit their activities.
Adenosine-5'-triphosphate (ATP) is a multifunctional nucleotide that is most important as a "molecular currency" of intracellular energy transfer. In this role, ATP transports chemical energy within cells for metabolism. It is produced as an energy source during the processes of photosynthesis and cellular respiration and consumed by many enzymes and a multitude of cellular processes including biosynthetic reactions, motility and cell division. In signal transduction pathways, ATP is used as a substrate by kinases that phosphorylate proteins and lipids, as well as by adenylate cyclase, which uses ATP to produce the second messenger molecule cyclic AMP.
ATP is generated in the cell by energy-consuming processes and is broken down by energy-releasing processes. In this way ATP transfers energy between spatially-separate metabolic reactions. ATP is the main energy source for the majority of cellular functions. This includes the synthesis of macromolecules, including DNA, RNA, and proteins. ATP also plays a critical role in the transport of macromolecules across cell membranes, e.g. exocytosis and endocytosis.
ATP is critically involved in maintaining cell structure by facilitating assembly and disassembly of elements of the cytoskeleton. In a related process, ATP is required for the shortening of actin and myosin filament crossbridges required for muscle contraction. This latter process is one of the main energy requirements of animals and is essential for locomotion and respiration.
The structure of this molecule consists of a purine base (adenine) attached to the 1' carbon atom of a pentose sugar (ribose). Three phosphate groups are attached at the 5' carbon atom of the pentose sugar. ATP is also incorporated into nucleic acids by polymerases in the processes of DNA replication and transcription.
ATP is commonly referred to as a "high energy molecule" however by itself, this is incorrect. A mixture of ATP and ADP at equilibrium in water can do no useful work at all. Similarly, ATP does not contain "high-energy bonds," rather the "high-energy bonds" are between its products and water, and the bonds within ATP are notable simply for being of lower energy than the new bonds produced when ATP reacts with water. Any other unstable system of potentially reactive molecules would serve as a way of storing energy, if the cell maintained their concentration far from the equilibrium point of the reaction.
The amount of energy released from hydrolysis of ATP can be calculated from the changes in energy under non-natural conditions. The net change in heat energy (enthalpy) at standard temperature and pressure of the decomposition of ATP into hydrated ADP and hydrated inorganic phosphate is .5 kJ/mol, with a change in free energy of 3.4 kJ/mol. The energy released by cleaving either a phosphate (Pi) or pyrophosphate (PPi) unit from ATP, with all reactants and products at their standard states of 1 M concentration, are:
ATP + H2O → ADP(hydrated) + Pi(hydrated) + H+(hydrated) ΔG˚ = -30.54 kJ/mol (.3 kcal/mol)
ATP + H2O → AMP(hydrated) + PPi(hydrated) + H+(hydrated) ΔG˚ = -45.6 kJ/mol (.9 kcal/mol)
These values can be used to calculate the change in energy under physiological conditions and the cellular ATP/ADP ratio. The values given for the Gibbs free energy for this reaction are dependent on a number of factors, including overall ionic strength and the presence of alkaline earth metal ions such as Mg2+ and Ca2+. Under typical cellular conditions, ΔG is approximately kJ/mol ( kcal/mol).
The overall process of oxidizing glucose to carbon dioxide is known as cellular respiration and can produce up to 36 molecules of ATP from a single molecule of glucose. ATP can be produced by a number of distinct cellular processes the three main pathways used to generate energy in eukaryotic organisms are glycolysis and the citric acid cycle/oxidative phosphorylation, both components of cellular respiration and beta-oxidation. The majority of this ATP production by a non-photosynthetic aerobic eukaryote takes place in the mitochondria, which can make up nearly 25% of the total volume of a typical cell.
Metabolic flexibility of mitochondrial respiratory chain disorders predicted by computer modelling
Mitochondrial respiratory chain dysfunction causes a variety of life-threatening diseases affecting about 1 in 4300 adults. These diseases are genetically heterogeneous, but have the same outcome reduced activity of mitochondrial respiratory chain complexes causing decreased ATP production and potentially toxic accumulation of metabolites. Severity and tissue specificity of these effects varies between patients by unknown mechanisms and treatment options are limited. So far most research has focused on the complexes themselves, and the impact on overall cellular metabolism is largely unclear. To illustrate how computer modelling can be used to better understand the potential impact of these disorders and inspire new research directions and treatments, we simulated them using a computer model of human cardiomyocyte mitochondrial metabolism containing over 300 characterised reactions and transport steps with experimental parameters taken from the literature. Overall, simulations were consistent with patient symptoms, supporting their biological and medical significance. These simulations predicted: complex I deficiencies could be compensated using multiple pathways complex II deficiencies had less metabolic flexibility due to impacting both the TCA cycle and the respiratory chain and complex III and IV deficiencies caused greatest decreases in ATP production with metabolic consequences that parallel hypoxia. Our study demonstrates how results from computer models can be compared to a clinical phenotype and used as a tool for hypothesis generation for subsequent experimental testing. These simulations can enhance understanding of dysfunctional mitochondrial metabolism and suggest new avenues for research into treatment of mitochondrial disease and other areas of mitochondrial dysfunction.
Keywords: Electron transport chain Flux balance analysis Metabolism Mitochondrial disease.
Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
The complexes of the mitochondrial…
The complexes of the mitochondrial respiratory chain. The complexes of the respiratory chain…
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Summary of simulations on the effect of complex I deficiency on metabolism. As…
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Summary of simulations on the effect of complex II deficiency on metabolism. As…
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Biological Oxidation (With Diagram)
This article gives the answer to the question as to “How do the food we take in and the oxygen we respire, produce energy to continue the process of life”. The simplest answer is that the food we take in is oxidized by the enzymes present in the body.
During this process some reducing equivalents viz. NADH and FADH2 are produced which are electron rich in nature. These reducing equivalents donate their electrons to the oxygen we respire in, during which energy is released to produce adenosine triphosphate (ATP). ATP is known as the energy currency of the cell and it brings about the biological process of life. In order to understand the above process, we need to understand some basic terminologies.
Oxidation and reduction can be defined in three different ways as under:
i. Oxidation is ‘Addition of oxygen’ or ‘removal of hydrogen’ or ‘removal of electrons’.
ii. Reduction is ‘Removal of oxygen’ or ‘addition of hydrogen’ or ‘addition of electrons’.
Oxidizing agent or oxidant:
An electron acceptor is an oxidizing agent or oxidant.
Reducing agent or reductant:
An electron donor is a reducing agent or reductant.
The relative tendency of reductant to donate electrons as compared to hydrogen is termed as oxidation-reduction potential or ‘redox potential’ (E0).
The redox potential of hydrogen is taken as zero at pH 0 (-0.417), 25° C, in a solution of 1 molar concentration (1.0 atom of hydrogen).
i. A compound having a negative value of E0 is a better electron donor than hydrogen.
ii. A compound having a positive value of E0 is a poor electron donor than hydrogen.
The electrons flow from compounds with negative value of redox potential to those with positive values of redox potential, because there will be loss of energy and thus, the compound becomes stable.
Every chemical substance has a certain amount of energy built into it, which is the energy of the chemical bonds holding the atoms together. This is the free energy.
High energy compounds and energy rich bond (
Any bond, which on hydrolysis gives a minimum free energy of 7.4 Kcal/mol, is known as energy rich bond and the compound which has an energy rich bond is known as high energy compound. Ex. ATP, pyrophosphate, 1, 3-diphosphoglyceric acid, phosphoenol pyruvate, creatine phosphate and acetyl-CoA.
Adenosine triphosphate (ATP):
ATP is also known as the ‘energy currency’ of the living cell, because it transfers energy from energy yielding sources to the energy requiring cell processes. ATP has two pyrophosphate bonds. On hydrolysis of each of the terminal two phosphate groups there is release of more than 7.4 Kcal/mol of energy but the third bond yields only 3 Kcal/mol of energy, hence it is not a high energy bond. On hydrolysis ATP is converted to ADP and to AMP.
Biological oxidation is catalysed by enzymes which function in combination with coenzymes and/or electron carrier proteins.
Different enzymes associated with biological oxidation are:
These enzymes catalyse the removal of hydrogen from the substrate and add it to another substance, thus bringing about oxidation reduction reaction. Ex. Glyceraldehyde—3— Phosphate dehydrogenase.
These enzymes catalyse the removal of hydrogen from the substrate and add directly to the molecular oxygen. Ex. Cytochrome oxidases, tyrosinase, uricase.
These enzymes incorporate oxygen into the substrates.
Adds only one atom of oxygen to the substrate. These are also known as mixed function oxidases.
Adds both the atoms of oxygen to the substrate. Ex. Homogentisic acid di-oxygenase.
4. Aerobic dehydrogenases:
These enzymes remove hydrogen from the substrate and add it either directly to oxygen or any other artificial acceptors like methylene blue. The product formed is hydrogen peroxide.
5. Anaerobic dehydrogenases:
These enzymes use other substrates or substances to donate the hydrogen. They transfer hydrogen’s to some other hydrogen acceptor, but not directly to oxygen. Thus the hydrogen acceptors are NAD, FAD and FMN. Heme proteins like cytochromes also receive hydrogen’s. The cytochromes are ‘b’, ‘c1‘, ‘c’, ‘a’ and ‘a3‘.
6. Hydro peroxidases:
These enzymes have either hydrogen peroxide (H2O2) or organic peroxide as their substrate.
There are two types of hydro peroxidases:
Their prime function is destruction of H2O2.
Electron Transport Chain:
When electrons are transferred from the most electronegative system [(NADH or FADH2) (-0.32V)] to the most electropositive system (+0.82V) (Oxygen), there will be liberation of all the energy at one time in an explosive manner. But, if they are transferred in a step wise manner through some intermediate systems then there will be slow release of energy and it can be captured by the cell to synthesize energy rich compounds. During biological oxidation, electrons are transferred through electron transport proteins which are arranged in a specific chain to form the electron transport chain (ETC), which is situated in the inner mitochondrial membrane.
Respiratory Chain or ETC:
Transfer of electrons from substrate to molecular oxygen through a chain of electron carriers is called electron transport chain or respiratory chain. Mitochondria contains a series of catalysts forming the respiratory chain which are involved in the transfer of electrons and hydrogen and their final reaction is with oxygen to form water. The components of respiratory chain are arranged sequentially in the order of increasing redox potential.
Electrons flow through the chain in a stepwise manner from lower redox potential to higher redox potential. Some amount of energy is liberated with transfer of electron from one component to another. Whenever there is a release of 7.4 Kcal of energy or a little more, then ATP formation takes place there. NADH forms 3 ATPs whereas FADH2 forms only 2 as it enters ETC at the site beyond the first site of ATP formation.
The/three sites of ATP formation in the ETC or respiratory chain are:
1. Between NADH dehydrogenase (flavoprotein) and ubiquinone (coenzyme Q).
2. Between cytochrome-b and cytochrome-c1.
3. Between cytochrome-a and cytochrome-a3 (cytochromes oxidase).
The components of ETC, their redox potential and their sequence is:
Esterification of a phosphate through a high energy bond (7.4 Kcal) is known as phosphorylation. Combination of inorganic phosphate (Pi) with any other compound through high energy bond is known as phosphorylation. Or formation of ATP from ADP and phosphate or NTP from NDP and Pi is known as phosphorylation.
There are two types of phosphorylation:
1. Substrate level phosphorylation:
Formation of high energy phosphate bond at the level of a substrate without the involvement of the respiratory chain is known as substrate level phosphorylation. Ex. Phosphoenolpyruvate is converted to pyruvate by pyruvate kinase where ATP is formed from ADP.
2. Oxidative phosphorylation:
The enzymatic phosphorylation of ADP to ATP coupled with electron transport from a substrate to molecular oxygen is known as oxidative phosphorylation or respiratory chain phosphorylation.
Mechanism of Oxidative Phosphorylation:
There are three theories or hypothesis, explaining the formation of ATP through electron transport chain.
1. Chemical coupling hypothesis:
It states that a high energy compound is formed taking the energy liberated by electron transfer and this compound in turn phosphorylates ADP to ATP.
2. Conformational coupling hypothesis:
There are many proteins in the wall of inner mitochondrial membrane one of them is F0F1, ATPase, which is responsible for the ATP production. According to this hypothesis the energy liberated from ETC brings a conformational change in the proteins of the membrane and is then transferred to FQF1ATPase which thereby also gets a conformation change and hence becomes unstable. In order to attain stability it provides energy for ATP synthesis.
3. Chemiosmotic hypothesis:
It states that electron transport pumps H + from the mitochondrial matrix across the inner mitochondrial membrane to the outer aqueous phase, thereby the matrix becomes basic and the outer phase becomes acidic. Due to this osmotic difference (i.e. more acidic outside and more basic inside the mitochondrial matrix) the H influx (diffuse) into the matrix through a pore in the F0F1 ATPase which provides the energy for the ATP synthesis.
The number of inorganic phosphates esterified per atom of oxygen consumed is known as P/O ratio. For NADH it is 3 and FADH, it is 2.
Formation and Detoxification of H2O2:
During ETC, O2 accepts four electrons forming two H2O. If by chance O2 accepts only two electrons, the product formed is H2O2 and if it accepts only one electron then superoxide radical (:O2
) is formed. Both these damage the membrane structure by attacking the unsaturated fatty acids of the membranes.
Superoxide is detoxified as—
Cytochrome-a3 is also known as cytochrome oxidase. It has two molecules of heme with long hydrocarbon side chains. To the other end of the heme, two copper atoms are attached which can directly react with oxygen to donate four electrons.
Inhibitors of ETC:
Inhibitors of ETC are those which inhibit or stop the flow of electrons in the electron transport chain.
Some of the inhibitors of ETC are:
(a) At the first site of ATP formation, rotenone and barbital inhibit the flow of electrons
(b) At the second site antimycin-A and amytal inhibits the flow of electrons.
(c) At the third site cyanide (Cn – ), carbon monoxide (CO) and H2S gas inhibit.
Un-couplers of Oxidative Phosphorylation:
Un-couplers are those substances which prevent oxidative phosphorylation (formation of ATP) though ETC is normally operating. Due to the effect of un-couplers there is a continuous flow of electrons but there is no formation of ATP i.e. ETC is not coupled to the ATP formation, so the energy is dissipated as heat.
Some of the un-couplers are:
1. 2, 4-Dinitrophenol (DNP):
It transfers protons across the mitochondrial membrane thereby diverting its flow from F0F1 ATPase.
It transfers K 4 ions, disturbing the osmotic pressure.
It transfers Na + ions, across the membrane.
All the above three are known as ‘ionophores’ i.e. those which disrupt the membrane permeability to ions, thereby uncoupling phosphorylation with ETC.
It inhibits adenine nucleotide transport protein of the mitochondrial membrane which transport ATP in exchange of ADP.
Some of the Mechanisms/Applications of Un-couplers are:
1. The mechanism by which body heat is increased during fever is by uncoupling.
2. Increase in the heat of the penis during erection is due to uncoupling.
3. Reduction in fat (weight) of obese persons is by the mechanism of uncoupling (banned).
4. Newly born infants have special type of mitochondria called brown fat mitochondria which are highly porous containing more cytochromes. They help in release of more heat by uncoupling, thus helping in maintaining the body temperature in the infants as they do not have sub-cutaneous fat resulting in loss of more heat.
It involves using the toxic properties of chemical substances to kill, injure or incapacitate an enemy. The offensive use of living organisms (such as anthrax) is considered to be biological warfare rather than chemical warfare the use of non-living toxic products produced by living organisms, ex. toxins such as botulinum toxin, ricin, or saxitoxin is considered as chemical warfare. Chemical used in warfare is called a ‘chemical warfare agent (CWA)’.
About 70 different chemicals have been used or stockpiled as chemical warfare agents during the 20 th and 21 st century. These agents may be in liquid, gas or solid form. Liquid agents are generally designed to evaporate quickly such liquids are said to be volatile or have a high vapor pressure. Many chemical agents are made volatile so that they can be dispersed over a large region quickly.
Chemical warfare agents are divided into lethal and incapacitating categories. A substance is classified as incapacitating if less than 1/100 of the lethal dose causes incapacitation, ex. through nausea or visual problems.
Chemical warfare agents are organized into several categories according to the manner in which they affect the human body.
The names and number of categories vary slightly from source to source, but in general, different types of chemical warfare agents are-
Without oxygen, pyruvate (pyruvic acid) is not metabolized by cellular respiration but undergoes a process of fermentation. The pyruvate is not transported into the mitochondrion but remains in the cytoplasm, where it is converted to waste products that may be removed from the cell. This serves the purpose of oxidizing the electron
carriers so that they can perform glycolysis again and removing the excess pyruvate. Fermentation oxidizes NADH to NAD+ so it can be re-used in glycolysis. In the absence of oxygen, fermentation prevents the build-up of NADH in the cytoplasm and provides NAD+ for glycolysis. This waste product varies depending on the organism. In skeletal muscles, the waste product is lactic acid. This type of fermentation is called lactic acid fermentation. In strenuous exercise, when energy demands exceed energy supply, the respiratory chain cannot process all of the hydrogen atoms joined by NADH. During anaerobic glycolysis, NAD+ regenerates when pairs of hydrogen combine with pyruvate to form lactate. Lactate formation is catalyzed by lactate dehydrogenase in a reversible reaction. Lactate can also be used as an indirect precursor for liver glycogen. During recovery, when oxygen becomes available, NAD+ attaches to hydrogen from lactate to form ATP. In yeast, the waste products are ethanol and carbon dioxide. This type of fermentation is known as alcoholic or ethanol fermentation. The ATP generated in this process is made by substrate-level phosphorylation, which does not require oxygen.
Fermentation is less efficient at using the energy from glucose since only 2 ATP are produced per glucose, compared to the 38 ATP per glucose produced by aerobic respiration. This is because the waste products of fermentation still contain plenty of energy. Ethanol, for example, can be used in gasoline (petrol) solutions. Glycolytic ATP, however, is created more quickly. For prokaryotes to continue a rapid growth rate when they are shifted from an aerobic environment to an anaerobic environment, they must increase the rate of the glycolytic reactions. For multicellular organisms, during short bursts of strenuous activity, muscle cells use fermentation to supplement the ATP production from the slower aerobic respiration, so fermentation may be used by a cell even before the oxygen levels are depleted, as is the case in sports that do not require athletes to pace themselves, such as sprinting.
Alpha ketoglutarate is converted to the 4-carbon succinyl CoA. A molecule of CO2 is removed and NAD+ is reduced to NADH + H+ in the process.
CoA is removed from the succinyl CoA molecule and is replaced by a phosphate group. The phosphate group is then removed and attached to guanosine diphosphate (GDP) thereby forming guanosine triphosphate (GTP). Like ATP, GTP is an energy-yielding molecule and is used to generate ATP when it donates a phosphate group to ADP. The final product from the removal of CoA from succinyl CoA is succinate.
Biochemistry 08: the citric acid cycle and the electron transport chain
To understand this post, it’s useful to know the mitochondrial structure (see also Cell Biology 03). Here’s a rad CC BY Wikimedia Commons diagram by Kelvinsong & Sowlos:
citric acid cycle
Khan Academy’s introduction:
The citric acid cycle or Kreb’s cycle is the cell’s metabolic hub, as it not only generate energy from pyruvate (the product of glycolysis) but along the way also can consume and produce metabolites relevant to host of other processes. It’s called a “cycle” and not a “pathway” because it both begins and ends with oxaloacetate. It takes place inside the mitochondrial matrix.
Pyruvate from glycolysis does not enter directly into the citric acid cycle. First it passes through a “transition phase” where it is undergoes oxidative decarboxylation to CO2 at the hands of the pyruvate dehydrogenase complex, transferring acetyl groups to coenzyme A to yield acetyl-CoA. This can be considered as step 0 and is depicted at the top of the below diagram. Pyruvate dehydrogenase deficiency causes a neurodevelopmental disorder and can result from mutations in many of the different genes involved in the complex.
What’s not depicted in this diagram is there are a few ways to enter the CAC. Pyruvate can come either from glycolysis or amino acid catabolism. Amino acid catabolism or fatty acid catabolism can also yield acetyl-CoA directly.
As the pyruvate dehydrogenase complex is the chief entry point into the CAC, its proper regulation is key for controlling the rate of cellular energy production. It is tightly regulated by allosteric regulation by products of the CAC / electron transport chain:
|inhibited by||activated by|
tldr: when ample fuel is available, i.e. the [ATP]/[AMP] ratio and [NADH]/[NAD+] ratios are high, there’s no need to continue running the cycle.
Three other highly exergonic CAC steps are also checkpoints for regulation: citrate synthase, isocitrate dehydrogenase, and the alpha ketoglutarate dehydrogenase complex. Orthogonally, you can think of 4 main mechanisms by which the CAC is regulated:
- substrate availability. availability of oxaloacetate and acetyl-CoA as inputs.
- product inhibition. for instance NADH not only inhibits the pyruvate dehydrogenase complex (above table) but also inhibits other regulatory steps.
- allosteric activation. for instance ADP (whose accumulation is a sign of energy need) activates enzymes such as the pyruvate dehydrogenase complex (above table)
- competitive feedback inhibition. for instance, succinyl-CoA, an intermediate, competes with acteyl-CoA for the attention of citrate synthase.
It also makes sense to have glycolysis and CAC running at about the same rate (think just in time delivery), so there’s a mechanism for their co-regulation. Citrate, an intermediate in the CAC, inhibits PFK-1, a step in glycolysis.
The CAC is described as “amphibolic” because it is both catabolic and anabolic – it both produces and consumes intermediates relevant to a host of other pathways. Here’s a video describing these roads in and out of the CAC. The diagram at 0:06, which should be the thumbnail below, is a concise overview.
Pyruvate can actually feed into the cycle in two ways. As discussed above, it can be spent on creating acetyl-CoA. Think of that as submitting a job to the computing cluster. Pyruvate can also be converted to oxaloacetate by pyruvate carboxylase- think of that as adding a node to the computing cluster. In other words, while acetyl-CoA is just consumed in the CAC, oxaloacetate is consumed and then regenerated (the cycle starts and ends with it), and so the amount of oxaloacetate available can be limiting and thus determine the number of instances of the citric acid cycle that can run in parallel. Converting pyruvate to oxaloacetate and thus increasing the number of parallel CAC jobs is a way of increasing the rate at which intermediates are produced for other cellular processes. This is especially important when intermediates are being rapidly siphoned off for other processes, causing acetyl-CoA to accumulate while very few of the CAC cycles are actually running to completion. Under this circumstance, acetyl-CoA feeds back to activate pyruvate decarboxylase, thus causing more OAA to be generated and more CAC cycles to run in parallel.
That process is especially important in the liver and kidney because they perform gluconeogenesis. In this process, intermediates from other processes enter the CAC and then oxaloacetate is siphoned off to create glucose. Under these conditions, the CAC is not replenishing its own supply of OAA, and so pyruvate carboxylase activity is key. This activity is higher in the liver and kidney than elsewhere.
The CAC’s contribution to cellular ATP supplies is indirect. It produces NADH, FADH2, and GTP, which enter the electron transport chain to produce ATP. Here’s a diagram I made of how ATP is produced in glucose metabolism:
Note that this depicts a production of 32 ATP / glucose. The exact figure depends on things like the leakiness of membranes and such, see discussion on Wikipedia. The theoretical maximum is 38 ATP, some people think the actual figure is closer to 29 or 30. The above graphic assumes 2.5 ATP/NADH and 1.5 ATP/FADH2.
quick review of oxidative phosphorylation
Glycolysis and the CAC both oxidize fuels to CO2 in order to reduce NAD + to NADH and FAD to FADH2. In the electron transport chain, NADH and FADH2 are then exergonically reoxidated to release the energy which will be used to oxidatively phosphorylate ADP to ATP. In the electron transport chain, e- are transferred through multiple complexes to ultimately reduce O2 to H2O.
Remember that redox is the transfer of electrons. Q is ubiquinone, popularly known as CoQ10. QH2 is called ubiquinol.
A substance’s tendency to accept electrons is quantified as its reduction potential, denoted ε°’. Electrons flow spontaneously from the substance with the lower reduction potential to the substance with the higher reduction potential. For example, Q’s ε°’ is .045, and NADH’s is -.315, so electrons will flow from NADH to Q. You can add the half reactions together to get the reduction potential for the whole reaction:
|Q + 2H+ + 2e- ↔ QH2||ε°’ = .045 V|
|NADH ↔ NAD+ + 2H+ + 2e-||ε°’ = .315V|
|NADH + Q ↔ NAD+ + QH2||Δε°’ = .360 V|
Note that in the second line, both the equation and the sign of the reduction potential have been swapped.
Gibbs free energy and reduction potential are related as follows:
Where F = one Faraday, a constant equal to 96,485 J/(V·mol) and n = number of moles of electron transferred per mole of reactant. ΔG is large and negative when Δε is large and positive. Thus electrons flow from compounds with low ε to compounds with high ε. Their desire to do so is what drives ATP production in the electron transport chain.
electron transport chain
Khan Academy’s introduction:
The electron transport chain’s ultimate purpose is to create a proton gradient which is then used to drive ATP production. The mitochondrial outer membrane is porous, so the intermembrane space has the same ionic composition as the cytosol. The ETC creates a proton gradient across the inner membrane.
To do this, it transfers “reducing equivalents” from the cytosol into the matrix via the malate-aspartate shuttle system. Also the adenine nucleotide translocase moves ATP out of the matrix and brings ADP into the matrix. Pi used to create more ATP in the matrix is imported via an H + symporter which takes advantage of the proton gradient.
In the electron transfer chain, electrons flow from Complex I to Complex II to Q to Complex III to cytochrome c to Complex IV. This series of complexes trades electrons in a way that allows e - to migrate up to higher and higher reduction potential (i.e. the direction they want to go). The final step is reduction of O2 to H2O. The energy release at some steps pumps H + into the intermembrane space.
Complex II, which is succinate dehydrogenase, does not directly contribute to the proton gradient, but it is another source of electrons. It oxidizes succinate to fumarate, thus reducing FAD to FADH and then using FADH to reduce Q. It thus contributes electrons to the “quinone pool”.
Complex III conducts the Q cycle. Ubiquinone (Q) is reduced to the semiquinone radical (QH) which is reduced to ubiquinol (QH2). In the Q cycle, QH2 donates electrons to cytochrome b and ISP, and protons to the intermebrane space, thus becoming Q. ISP sends its newfound electron to cytochrome c1 which gives it to cytochrome c, which was the point of this entire convoluted operation. Q is then is re-reduced to QH2 by another mechanism within the Q cycle.
Note: the fact that a proton gradient can later be used to drive ATP synthesis is referred to as “proton motive force” or “the chemiosmotic theory”.
Once the proton gradient is created, F1F0 ATP synthase (sometimes called Complex V) is responsible for harnessing it to create ATP. It has an F1 component (composed of α, β and γ subunits) facing into the matrix and an F0 component (composed of a, b and c subunits) embedded in the inner membrane. Each of the 12 c subunits of the F0 complex has an H + binding site, and the cylinder of these units rotates 30° every time. The gamma subunit has only three possible positions, so it moves 120° once for every 4 protons. Each time it does this, it alters the conformation of the beta subunit.
ADP and Pi bind β in the loose (L) conformation. The γ rotation causes β to move to a tight (T) conformation, producing ATP. The γ then rotates another 120°, causing β to change to the open (O) conformation and releasing ATP. Thus you get 3 ATP per full turn
Uncouplers can allow the proton gradient to dissipate without producing ATP. This means that the energy of metabolism just gets dissipated as heat (e.g. via the uncoupling protein thermogenin, gene symbol = UCP1) rather than harnessed for ATP production. It’s an important mechanism for keeping body temperature up during hibernation in bears as well as in human infants (in brown adipose tissue). There are also small molecule uncouplers such as DNP, which was used as a diet pill for a few years.
Thermogenesis in infants and bears is regulated hormonally. The thermogenin channel is normally blocked open by ATP, ADP, GTP or GDP. Epinephrine signaling can turn on heat production. Epinephrine → GPCR → AC → cAMP → PKA → triacylglycerol lipase cleaves triacylglycerol into free fatty acids which will displace the nucleotides, blocking the channel open.
Brown fat was long believed to be unique to infants but has recently been shown to exist in adult mice and humans [reviewed in Lee 2013].
Both the ETC and ATP synthase are also popular targets for toxins. Rotenone inhibits Complex I (though then electrons can still enter the chain through succinate and start at Complex II), cyanide ions (CN - ) inhibit Complex IV and the antibiotic oligomycin inhibits F0.
In the lab, the ETC can be studied in isolated mitochondria with O 2 consumption as a marker of ETC progression. Adding ADP + Pi will allow the ETC to run very slowly once you add succinate it really takes off. If you add CN - , it then stops. ATP is pretty much in lockstep with O2 consumption through all this.
If you add oligomycin to block ATP synthase, this actually stops the ETC even though ATP synthase is downstream of the ETC. This is because without ATP synthase to relieve the proton gradient, the gradient becomes so steep that the ETC can no longer fight it.
A few final notes on the co-regulation of glycolysis, CAC and ETC:
- When ATP consumption increases, the ATP/ADP ratio decreases and this results in increases in all of the processes’ activities. ADP activates PFK-1, increasing glycolysis. ADP activates regulatory enzymes in the CAC. And the ETC also increases for unexplained reasons.
- When ATP production increases, the ATP/ADP ratio increases and this inhibits all of the processes. ATP inhibits PFK-1, slowing glycolysis ATP inhibits pyruvate dehydrogenase, slowing the CAC, and the ETC is also slowed.
- If the ETC or ATP synthase are inhibited, glycolysis speeds up to generate more ATP to compensate. But since the ETC is not running, NADH accumulates, increasing the NADH/NAD+ ratio. This slows down the CAC.
About Eric Vallabh Minikel
Eric Vallabh Minikel is on a lifelong quest to prevent prion disease. He is a scientist based at the Broad Institute of MIT and Harvard.
Lesson 2: Thermodynamics
After completing this lesson, you should be able to:
- Explain the basic principle of thermodynamics as it pertains to biochemical systems.
- Describe the two laws of thermodynamics.
- Differentiate between entropy and energy.
- Define Gibbs Free Energy and calculate the change in Gibbs Free Energy (&DeltaG).
- Demonstrate an understanding of the difference between endergonic, exergonic, and equilibrium reactions.
- Demonstrate an understanding of thermodynamic equations.
Readings and Activities
- Read &ldquoEntropy and Energy&rdquo and &ldquoGibbs Free Energy&rdquo (pages 28&ndash31 in the textbook).
- For additional information on metabolic strategies, watch the two video lectures.
(The links for these videos are also provided in Lesson 1 Energy Reactions, and on page 26 of the textbook.)
All living things are composed of biomolecules. To sustain life, the cells of a living body constantly form new molecules or break down existing ones. These continual changes of biomolecule structure (&ldquodynamics&rdquo) use energy or release energy, or both the process usually results in the production of heat (&ldquothermo‑&rdquo). The measurement of energy change is therefore known as &ldquothermodynamics.&rdquo
The first law of thermodynamics states that energy can be exchanged between physical systems as heat and work.
The second law of thermodynamics states that entropy, a measurement of the molecular disorder in a system, exists and that isolated systems increase in entropy.
The change in entropy of a system depends on the initial and final states. In an irreversible reaction, entropy would increase. In a reversible process, entropy is conserved.
Enthalpy is the amount of energy in a thermodynamic system and is equivalent to the total heat content of a system.
As mentioned in the text, living cells are not isolated systems and, to counter the tendency to disorder, a cell needs to use energy. Energy for the cell is often in the form of high‑energy phosphate molecules such as ATP, GTP, CTP, and UTP. ATP is the most common form of energy used by the cell. Hydrolysis of the phosphate bonds in ATP and other phosphate molecules releases energy.
Gibb&rsquos Free Energy is a measurement of thermodynamic potential. It is important to note that the maximum amount of work/energy from a closed system can only be realized in a completely reversible reaction. The equation is:
where &DeltaG is Gibb&rsquos Free Energy, H is enthalpy (joule), T is temperature (in kelvin), and S is entropy (joule/kelvin).
The change in Gibbs Free Energy can be used to determine whether a given chemical reaction can occur spontaneously. A negative &DeltaG indicates a spontaneous reaction, while a positive &DeltaG indicates a reaction cannot occur spontaneously.
At standard temperature and pressure, every system will attempt to achieve a minimum of free energy. &DeltaG is reduced by increased entropy (S) and excess heat (T).
Cells must work within the laws of thermodynamics, which sets limitations on their biochemical reactions. When you consider how cellular processes must interact and balance, it makes sense that the &DeltaG value is critical, because it determines whether or not a reaction will go forward.
The reaction is favourable (exergonic, spontaneous).
The reaction is at equilibrium.
The reaction is unfavourable (endergonic, non‑spontaneous).
The equation can be modified at pH 7 considering substrates and products in a reaction to determine &DeltaG:
where A is the substrate and B is the product and b and a are integers.
With multiple substrates and products (aA + cC ↔ bB + dD), it becomes:
R = gas constant (8.3144598 J mol &minus1 · K &minus1 )
T = absolute temperature (in Kelvin)
Q = reaction quotient (unitless)
&DeltaG°&prime is the Standard Gibbs Free Energy. This is the change in energy that occurs when all products and reactants are at standard conditions and pH = 7. This value is constant for a given reaction. This can be calculated using the equilibrium equation for a system at chemical equilibrium:
where K = equilibrium constant (also known as Keq)(unitless)
Temperature is a constant in biological reactions. Since &DeltaG°&prime is also a constant for a particular reaction, then &DeltaG is dependent on the ratio of [products]/[reactants]. If a reaction is in standard conditions where everything except protons is at 1 M (molar), the RT ln ([products]/[reactants]) term is zero, so the &DeltaG°&prime value will determine the direction of the reaction. This is why a negative &DeltaG°&prime indicates an energetically favourable reaction (releases energy) and a positive &DeltaG°&prime indicates an energetically unfavourable one (requires energy).
Increasing the ratio of [products]/[reactants] causes the value of the natural log (ln) to become more positive (less negative), so &DeltaG is more positive. Decreasing the ratio of products to reactants causes ln to become more negative (less positive) and &DeltaG to become more negative. In a closed system, &DeltaG will always move towards zero or equilibrium.
In biological systems, some reactions may be &ldquocoupled&rdquo to drive an unfavourable reaction. For example, the reaction of glucose with fructose to form sucrose has a &DeltaG value of +5.5 kcal/mole and will not occur spontaneously. However, the breakdown of ATP to form ADP and inorganic phosphate (Pi) has a &DeltaG value of &minus7.3 kcal/mole, coupling these reactions, so that glucose binds with ATP to form glucose‑phosphate and ADP creates an overall &DeltaG of &minus1.8 kcal/mole. The glucose‑phosphate can bond fructose, yielding sucrose and inorganic phosphate. Coupling reactions to alter the Gibbs Free Energy is a basic principle behind enzymatic action in biology.
&DeltaG is expressed in kcal or kcal/mole or joules/mole and &DeltaG°&prime is expressed in joules.
Some information in this commentary is from the Wikipedia pages Thermodynamics, Gibbs free energy, and Biological thermodynamics.
- Outline the first and second laws of thermodynamics.
- What is entropy? How does an increase in entropy affect the value of &DeltaG?
- What does a negative &DeltaG mean for a reaction? A positive &DeltaG value? What if the &DeltaG value is zero?
- In a biological system, how can reactions be coupled to turn an unfavourable reaction (positive &DeltaG) into a favourable one (negative &DeltaG)?
Calculate &DeltaG for the following reaction:
where the reaction occurs at 293 kelvin, the change in heat is 19,070 calories, and the change in entropy is 90 cal/k.
Is this reaction endergonic or exergonic?
Pyruvate + NADH &rlarr Lactate + NAD +
This reaction occurs at 298 K and the equilibrium concentrations of the above products and reactants are as follows:
pyruvate: 20 &muM
NADH: 30 &muM
Lactate: 60 &muM
NAD + : 10 &muM
R = 8.314 J · K &minus1
Is this reaction endergonic or exergonic?
If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at [email protected]
Cellular Respiration Lab
Cellular respiration is defined as the process in which cells harvest energy by catabolizing glucose for the production of adenosine triphosphate (ATP). The whole process of respiration is organized in three stages: glycolysis, the Kreb’s cycle, and the electron transport chain (ETC). Glycolysis is an anaerobic process that only yields two molecules of ATP however, the Kreb’s cycle and ETC require oxygen in order to yield cellular respiration’s total profit of thirty-six molecules of ATP. While the Kreb’s cycle cannot occur without the presence of oxygen, it does directly use oxygen within the cycle. The ETC, on the other hand, directly requires oxygen. Oxygen functions as the terminal electron acceptor and consequently forms water. If no oxygen is present to accept electrons then the electron transport chain will not function and no ATP produced by it.
If the rate of cellular respiration is compared between animal cells and plant cells, then animal cells will respire at a higher rate because the second law of thermodynamics stipulates that that producers (e.g. germinating beans) are only capable of harvesting 10% of solar energy, while consumers (e.g. pillbugs) are only able to harvest 10% of the energy obtained at the previous trophic level. Thus, consumers have a more immediate requirement of energy, making it necessary for consumers to respire at a faster rate.
Cellular respiration is the process by which cells harvest energy. It is also known as oxidative metabolism, as it is entirely dependent on oxygen in order to yield 36 molecules of ATP necessary to sustain eukaryotic life. If the rate of cellular respiration is compared between animal cells and plant cells, then the results will show that animal cells will respire at a higher rate. This can be inferred based on the Second Law of Thermodynamics, specifically the rule of energy flow and trophic levels. This experiment utilized pill bugs and germinating bean sprouts to measure the rate of respiration between animal cells and plant cells, respectively. A Vernier oxygen gas sensor was used to measure the consumption of oxygen gas in the atmosphere, in order to measure the rate of respiration. The results revealed that pill bugs respired at a rate of -0.00778 %/s, while the bean sprouts respired at a rate of -0.00027486 %/s. The hypothesis proposed was correct the pillbugs respired at a higher rate than the germinating bean sprout. This can be attributed to the second law of thermodynamics as well as the high requirement of energy and respiration due to energy transfer in the trophic levels differences in metabolic activity can play a role in the animals immediate requirement of energy.