Respiration revision notes:

Outline why living organisms need to respire.

Describe the structure of ATP.

State that ATP provides the immediate source or energy for biological processes.

What is respiration?

RESPIRATION: the process whereby energy stored in complex organic molecules (carbohydrates, fats and proteins) is used to make ATP. It occurs in living cells. A metabolic pathway.

ATP: Molecule (nucleotide derivative) which is found in all living cells and involved in energy transfer. It’s a phosphorylated nucleotide and is the universal energy currency. When hydrolysed it releases energy.

What is energy?

ENERGY: is the ability to do work

Energy exists as potential (stored) energy and kinetic energy Large organic molecules can contain chemical potential energy Energy cannot be created or destroyed but can be converted from one form to another It’s measured in joules or kilojoules It has many forms (e.g. sound, light, heat, electrical, chemical and atomic).

Why do we need it?

ANABOLIC: type of metabolism: biochemical reactions that syntheses large molecules from smaller molecules. This requires energy/ATP.

CATABOLIC: type of metabolism: biochemical reactions that produce small molecules by hydrolysis of larger molecules.

All living organisms need to drive their biological processes All the reactions that take place within organisms are known as metabolism Metabolic reactions that build large molecules are described as anabolic Reactions that break large molecules into smaller molecules are catabolic

Metabolic reactions that need energy include:

Active transport : moving ions and molecules across a membrane against a concentration gradient-Much of an organism’s energy is used for this- All cell membranes have sodium-potassium pumps and these maintain the resting potential. When this pump temporarily stops in neurone membranes, sodium ions enter the neurone and an action potential occurs

Secretion : Large molecules made in some cells are exported by exoctyosis Endocytosis: bulk movement of large molecules into cells Anabolism : Synthesis of large molecules from smaller ones:

-Proteins from amino acids-Steroids from cholesterol-Cellulose from B- glucose

Replication of DNA and synthesis or organelles before a cell divides Movement

-Movement of bacterial flagella

-Eukaryotic cilia-Undulipodia-Muscle contraction-Microtubule motors that move organelles inside cells

Activation of chemicals-Glucose is phosphorylated at the beginning of respiration so that it is more unstable and can be broken down to release energy

Some of the energy from catabolic reactions is released in the form of heat. This is useful as metabolic reactions are controlled by enzymes, so organisms need to maintain a suitable temperature that allows enzyme action to proceed at a speed that will sustain life.

Where does the energy come from?

PHOTOAUTOTROPHS: organisms that carry out photosynthesis to acquire energy.

HYDROLYSIS: The chemical breakdown of a compound due to reaction with water

Plants, some Protoctists and some bacteria are photoautotrophs They use sunlight energy in photosynthesis to make large, organic molecules that contain chemical potential

energy, which they and consumers and decomposers can use Respiration releases energy, which is used to phosphorylate (add inorganic phosphate to) ADP, making ATP This phosphorylation also transfers energy to the ATP molecule

The role of ATP:

ATP is a phosphorylated nucleotide High-energy intermediate compound Found in both eukaryotic an prokaryotic cells Easily broken down- the covalent bond linking the second and third phosphate groups is unstable, and is

easily broken by hydrolysis It can be hydrolysed to ADP and Pi releasing 20.5 kJ energy per mol This is an exergonic reaction, as energy is released Energy is immediately available to cells in small, manageable amounts that will not damage the cell or be

wasted Can’t pass out the cell, so cell always has an immediate energy supply Described as the universal energy currency Respiration occurs in many small steps The energy released at each stage joins ADP and Pi to make ATP ATP is continually being hydrolysed and resynthesised Small and soluble so can easily be transported around the cell Organisms store little ATP, despite it being the chemical energy that is used for all endergonic biological

activity Therefore, needs to be continually produced

Each molecule contains:

Adenosine (adenine and ribose sugar) 3 phosphate groups

Coenzymes:

Explain the importance of coenzymes in respiration, with reference to NAD and coenzyme A.

The stages of respiration:

Four main stages:

GLYCOLYSIS: Metabolic pathway. The first stage of respiration. It is anaerobic and occurs in the cytosol (cytoplasm). Although anaerobic, it involves oxidation as substrate molecules are dehydrogenated.

LINK REACTION: Stage of aerobic respiration that links Glycolysis with the Krebs cycle. In eukaryote cells it occurs in the mitochondrial matrix.

KREBS CYCLE: Third stage of respiration. It is aerobic and in eukaryotes in takes place in the matrix of the mitochondria.

OXIDATIVE PHOSPHORYLATION: The formation of ATP, in the presence of oxygen, by chemiosmosis.

1. Glycolysis: -Cytoplasm of all cells-Ancient biochemical pathway-Doesn’t require oxygen-Anaerobic & aerobic respiration-Glucose (6C) is broken down into pyruvate (3C)

2. The link reaction: -Matrix of the mitochondria-Pyruvate is dehydrogenated and decarboxylated and converted to acetate

3. Krebs cycle - Matrix of the mitochondria-Acetate is decarboxylated and dehydrogenated

4. Oxidative phosphorylation: -On the folded inner membrane of the mitochondria (cristae)-ADP is phosphorylated to ATP

The last three stages occur under aerobic conditions Under anaerobic conditions, pyruvate is converted to either ethanol or lactate

Why are coenzymes needed?

COENZYME: an organic non-protein molecule that binds temporarily with substrate to an enzyme active site. It is essential for enzyme activity. Aids the function of an enzyme by transferring a chemical group from one molecule to another. In respiration, many coenzymes remove hydrogen atoms from substrates.

During Glycolysis, the link reaction and the Krebs cycle, hydrogen atoms are removed from substrate molecules in oxidation reactions

These reactions are catalysed by dehydrogenase enzymes Enzymes are not very good at catalysing reduction or oxidation reactions so coenzymes are needed to help

them carry out the oxidation reactions of respiration The hydrogen atoms 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 membranes Here they will be involved in oxidative phosphorylation which produces a lot of ATP Delivery of the hydrogens to the cristae reoxidises the coenzymes so they can combine with more hydrogen

atoms from the first three stages

NAD:

NAD: Coenzyme involved in respiration. It removes hydrogen atoms from substrates. It becomes reduced NAD which carries hydrogen atoms (protons and electrons)

Organic, non-protein molecule that helps dehydrogenase enzymes to carry out oxidation reactions Nicotinamide adenine dinucleotide is made of 2 linked nucleotides Made in the body of nicotinamide (vitamin B3), ribose (5C) , adenine and 2 phosphoryl groups One nucleotide contains the nitrogenous adenine the other a nicotinamide ring that accepts hydrogen

atoms which can later be split into a H+ and an E- When a molecule of NAD has accepted 2 hydrogen atoms with their electrons, it is reduced When it loses electrons, it is oxidised NAD operates during Glycolysis, the link reaction and Krebs cycle and during the anaerobic ethanol and

lactate pathways

Coenzyme A:

COENZYME A: A coenzyme that carries acetate from the link reaction to Krebs cycle

Made from pantothenic acid (B-group vitamin), adenosine, three phosphoryl groups and cysteine (an amino acid)

Carries ethanoate (acetate) groups made from pyruvate during the link reaction onto Krebs cycle. It can also carry acetate groups that have been made from fatty acids or from some amino acids onto Krebs

cycle

Coenzyme FAD:

Flavine adenine dinucleotide Becomes reduced in the Krebs cycle, is tightly bound to a dehydrogenase enzyme that is embedded in the

inner membrane The H atoms accepted by FAD do not get pumped into the inert membrane space. Instead, they pass back

into the mitochondrial matrix

Glycolysis:

State that Glycolysis occurs in the cytoplasm of all cells.

Outline the process of Glycolysis.

State that in aerobic respiration, pyruvate is actively transported into mitochondria.

GLYCOLYSIS: Metabolic pathway. The first stage of respiration. It is anaerobic and occurs in the cytosol (cytoplasm). Although anaerobic, it involves oxidation as substrate molecules are dehydrogenated.

Cytoplasm of all cells Ancient biochemical pathway Prokaryotic and eukaryotic cells Best understood metabolic pathway Doesn’t require oxygen Anaerobic & aerobic respiration Glucose (6C) is broken down into 2 molecules of pyruvate (3C) Involves a sequence of 10 reactions each catalysed by different enzymes Involved coenzyme NAD

Four main stages:

1. Phosphorylation2. Splitting of hexose 1,6- biphosphate3. Oxidation of triose phosphate4. Conversion of triose phosphate to pyruvate

Phosphorylation:

HYDROLYSIS: The chemical breakdown of large molecules into smaller molecules by the addition of water

Glucose is a hexose sugar- its molecules are stable and need to be activated before they can be split into two

Glucose has to be given some activation energy which is provided by 2 ATP which break down to ADP and Pi-1 ATP is hydrolysed and the phosphate group released is attached to the glucose molecule at carbon 6-Glucose 6-phosphate is changed to fructose 6- phosphate, catalysed by the enzyme phospho-glucose isomerase-Another ATP is hydrolysed and the phosphate group released is attached to fructose 6-phosphate at carbon-1-This activated hexose sugar is now called fructose 1, 6-biphosphate-The energy from the hydrolysed ATP molecules activated the hexose sugar and prevents it from being transported out the cell

ATP used/glucose molecule: 2

ATP formed/glucose molecule: -

NAD reduced: -

Splitting of Fructose 1, 6- biphosphate

Each molecule of hexose 1,6- biphosphate is split into 2 molecules of triose phosphate (3C sugar molecules with one phosphate group attached)

Catalysed by the enzyme Fructose-biphosphate aldolase Splits fructose 1, 6- biphosphate (6C) into 2 triose phosphate molecules (3C) Also known as GALP (glyceraldehyde 3-phosphate)

Oxidation of triose phosphate:

SUBSTRATE LEVEL PHOSPHORYLATION: formation of ATP from ADP and Pi during Glycolysis and the Krebs cycle

Although anaerobic, it involves oxidation 2 hydrogen atoms (with their electrons) are removed from each triose phosphate molecule (the

substrate) This involves dehydrogenase enzymes These are aided by coenzyme NAD which is a hydrogen acceptor The dehydrogenase enzymes removes hydrogen atoms from the triose phosphate molecules NAD combines with the hydrogen atoms, becoming NADH (reduced) The removal of these hydrogen atoms, releases energy, which enables an additional phosphate group to

be added to the triose phosphate to form 1,3- biphosphate glycerate (GALP) ADP removes and joins to these additional phosphate groups to from ATP (phosphorylation) 2 molecules of ATP are formed (substrate level phosphorylation)

ATP used/glucose molecule: -

ATP formed/glucose molecule: 2

NAD reduced: 2

Conversion of triose phosphate to pyruvate:

This returns it to triose phosphate again 4 enzyme-catalysed reactions convert each triose phosphate molecule to a molecule of pyruvate (3C) In the process another 2 molecules of ADP are phosphorylated (an inorganic phosphate group, Pi) is added

to 2 molecules of ATP (by phosphate level phosphorylation)

Products of Glycolysis:

ATP used/glucose molecule: 2 which were used to start the process

ATP formed/glucose molecule: A net gain of 2 (2 were used, 4 were made)

NAD reduced: 2. these will carry hydrogen atoms to the inner mitochondrial membranes and be used to generate more ATP during oxidative phosphorylation

2 molecules of pyruvate: Mostly actively transported into the mitochondrial matrix for the next stage or respiration. Under anaerobic conditions it will be changed, in the cytoplasm, to lactate or ethanol

Regenerating NAD+ with or without oxygen:

The NADH produced during glycolysis must be oxidised back to NAD+ If not, NAD would run out and no more ATP would be made If oxygen is available, NAD is regenerated when NADH releases hydrogen into the mitochondria. The

hydrogen enters the electron transport chain and generates about 6 more molecules of ATP If oxygen is unavailable, NAD is regenerated by fermentation

The link reaction and the Krebs cycle:

Pyruvate produced during Glycolysis is actively transported across the inner and outer mitochondrial membranes to the matrix

It’s changed into a 2-carbon compound, acetate, during the link reaction Acetate is then oxidised during Krebs cycle

The link reaction: pyruvate to acetyl-Co-A: oxidative decarboxylation

Outline the link reaction, with reference to Decarboxylation of pyruvate to acetate and the reduction of NAD, and state that it takes palace in the mitochondrial matrix.

Explain that coenzyme A carries acetate from the link reaction to the Krebs cycle.

Outline the Krebs cycle, the roles of NAD and FAD, and substrate-level phosphorylation, and state that is takes place in the mitochondrial matrix.

LINK REACTION: Stage of aerobic respiration that links Glycolysis with the Krebs cycle. In eukaryote cells it occurs in the mitochondrial matrix. It converts pyruvate to acetate. NAD is reduced.

Decarboxylation and dehydrogenation of pyruvate to acetate are enzyme catalysed reactions.

Pyruvate dehydrogenase removes hydrogen atoms Pyruvate decarboxylase removes a carboxyl group, which eventually becomes carbon dioxide Coenzyme NAD accepts the hydrogen atoms Coenzyme A accepts acetate to become acetyl coenzyme A. CoA’s function is to carry acetate to the Krebs

cycle

2 Pyruvate + 2NAD+ + 2CoA = 2CO2 + 2NADH + 2 acetyl CoA

NAD+ is oxidised NAD 2 molecules of pyruvate are derived from each molecule of glucose No ATP is produced Each NADH will take a pair of hydrogen atoms to the inner mitochondrial membrane and they will be used

to make ATP during oxidative phosphorylation

(per 2 cycle) ATP: -

CO2: 2- released as a waste product

NAD reduced : 2 – to oxidative phosphorylation

FAD reduced: -

Acetate: 2. To Krebs cycle

The Krebs cycle:

KREBS CYCLE: Third stage of respiration. It is aerobic and in eukaryotes in takes place in the matrix of the mitochondria. It oxidises acetate to carbon dioxide. NAD and FAD are reduced. ATP is made by substrate-level phosphorylation.

Series of enzyme-catalysed reactions that oxidise the acetyl group of acetyl CoA to 2 molecules of CO2 Produces 1 molecules ATP by substrate-level phosphorylation Reduces 3 molecules of NAD and 1 molecule of FAD These reduced coenzymes have the potential to produce more ATP during oxidative phosphorylation

1. Formation of citrate: The 2 acetate is offloaded from CoA and diffuses from the cytoplasm into the mitochondria where it joins

with Oxaloacetate (4C) to from Citrate (6C) CoA goes back to the link reaction to be used again

2. Formation of a 5-C compound: Citrate is decarboxylated and dehydrogenated to form a 5C. (α-ketogluterate) The pair of H atoms is accepted by a molecule of NAD, which becomes reduced CO2 is released as waste gas

3. Regeneration of a 5-carbon compound: The 5C (α-ketogluterate) is decarboxylated and dehydrogenated to form a 4C (succinyl co-A) and another

molecule of NAD is reduced The 4C is changed into another 4C (Succinate). During this reaction, a molecule of ADP is phosphorylated to

produce a molecule of ATP (substrate level phosphorylation) The 2nd 4C is changed into another 4C (fumarate). A Pair of H atoms is removed and accepted by coenzyme

FAD, which is reduced The 3rd 4C is further dehydrogenated into another 4 (malate) which is dehydrogenated and regenerates

Oxaloacetate. Another molecules of NAD is reduced

ATP: 2- energy

CO2: 4- released as a waste product

NAD reduced : 6- to oxidative phosphorylation

FAD reduced: 2- to oxidative phosphorylation

Oxaloacetate: regenerated for next Krebs cycle

CoA: reused in the next link reaction

Although oxygen is not used, it’s still needed as it won’t occur without it. There is one turn of the Krebs cycle for each molecule of acetate, made from one molecule of pyruvate 2 turns of the cycle per glucose molecule Other food substrates can be respired besides glucose Fatty acids are broken down to acetates and can enter the Krebs cycle via CoA Amino acids can be deaminated and the rest of the molecule may enter Krebs cycle directly or be changed

to pyruvate or acetate, depending on the type of amino acid

Synthesising ATP by substrate level phosphorylation:

The redox reactions are exergonic They release enough energy to synthesis 1 molecule of ATP each turn of the cycle This synthesis is called substrate-level phosphorylation because the addition of Pi to ADP is coupled with

the exergonic breakdown of a high-energy substrate molecule Each molecule of glucose that enters glycolysis generates 2 ATP as this produces 2 molecules of acetyl-coA This doubles the production of glycolysis The release of hydrogen during the Krebs cycle provides the reducing power to generate more ATP

molecules in the next stage, the electron transport chain

Oxidative phosphorylation and chemiosmosis:

Outline the process of oxidative phosphorylation, with reference to the roles of the electron transport carriers, oxygen, and mitochondrial cristae.

Outline the process of chemiosmosis, with reference to the electron transport chain, proton gradients and ATP synthase.

State that oxygen is the final electron acceptor in aerobic respiration.

Explain that the theoretical yield of ATP per glucose molecule is rarely, if ever, achieved.

CHEMIOSMOSIS: the flow/diffusion of hydrogen ions through ATP synthase enzymes. The force of this flow allows the production of ATP. Occurs across the thylakoids membranes during the light dependent stage of photosynthesis. Also occurs across the inner mitochondrial membrane (cristae) during oxidative phosphorylation.

OXIDATIVE PHOSPHORYLATION: The formation of ATP by chemiosmosis by adding a phosphate group to ADP in the presence of oxygen which is the final proton acceptor.

Oxidative phosphorylation is the process where the energy carried by electrons from reduced coenzymes (NADH and FADH2) is used to make ATP

Involves 2 processes:-Electron transport chain-Chemiosmosis

Involves electron carriers embedded in the inner mitochondrial membranes These membranes are folded into cristae, increasing the surface areas for electron carriers and ATP

synthase enzymes NADH and FADH are reoxidised when they donate hydrogen atoms which are split into protons and

electrons, to the electron carriers The first electron carrier to accept electrons from NADH is a protein complex known as NADH-coenzyme Q

reductase (or NADH dehydrogenase) The protons go into solution in the matrix The main purpose of the Krebs cycle is to feed electrons into the next stage of aerobic respiration The hydrogen atoms carried by reduced NAD and FAD are the source of electrons

The electron transport chain:

ELECTRON ACCEPTORS: chemicals that accepts electrons from another compound. They are reduced while acting as oxidising agents.

CHEMIOSMOSIS: the flow/diffusion of hydrogen ions through ATP synthase enzymes. The force of this flow allows the production of ATP. Occurs across the thylakoids membranes during the light dependent stage of photosynthesis. Also occurs across the inner mitochondrial membrane during oxidative phosphorylation.

Hydrogen atoms are released from NADH and FADH2 as they are oxidised. The hydrogen atoms split into protons and electrons The electrons are passed along a chain of electron carriers in the electron transport chain, energy at each

carrier is released and used, by coenzymes associated with some of the electron carriers This energy is used by the electron carriers to pump protons from the mitochondrial matrix across the

intermembrane space The concentration of protons is now higher in the intermembrane space than in the matrix- this forms an

electrochemical gradient Potential energy builds up in the intermembranal space

The hydrogen ions cannot diffuse through the lipid part of the inner membrane but can diffuse through ion channels in it which are associated with the enzyme ATP synthase

Protons move down the electrochemical gradient, from the intermembrane space, into the mitochondrial matrix, via ATP synthase

This movement drives the synthesis of ATP from ADP and Pi The flow of H+ across a membrane, which generates ATP is called chemiosmosis The force of this flow drives the rotation of part of the enzyme and allows ADP and Pi to be joined to make

ATP In the matrix, at the end of the transport chain, the protons, electrons and oxygen (from the blood)

combine to form water Molecular oxygen, is the final electron acceptor

Oxidative phosphorylation:

OXIDATIVE PHOSPHORYLATION: The formation of ATP by chemiosmosis by adding a phosphate group to ADP in the presence of oxygen which is the final proton acceptor.

The formation of ATP by the addition of Pi to ADP in the presence of oxygen As protons flow through an ATP synthase enzyme, they drive the rotation part of the enzyme and join ADP

and Pi to form ATP The electrons are passed from the last electron carrier in the chain to molecular oxygen, which is the final

proton acceptor Hydrogen ions also join so that oxygen is reduced to water

4H+ + 4e- O2 = 2H20

ATP BEFORE OXIDATIVE PHOSPHORYLATION: 2 (from Glycolysis) + 2 (from Krebs cycle) = 4

ATP during oxidative phosphorylation:

More ATP will produced, where the NADH and FADH molecules are reoxidised The NADH and FADH will both provide electrons to the electron transport chain to be used in oxidative

phosphorylation NADH provides H+ for contributing to the build up of the proton gradient for chemiosmosis The hydrogens from FADH stay in the matrix but can combine with oxygen to form water 10 molecules of NADH can produce 26 molecules ATP in oxidative phosphorylation For each NADH that is reoxidised, 2.6 molecules of ATP can be made

Total ATP made

Should be 34 per glucose molecule respired/net 36 ATP NET as 2ATP used up in glycolysis FADH2 produces 2 ATP in oxidative phosphorylation NADH produces 2.6 ATP in oxidative phosphorylation FADH2 stays in the matrix, but donates electrons However, this is rarely achieved Protons leak across the inner mitochondrial membrane reducing the number of protons to generate the

proton motive force- some protons may leak into the matrix without passing through ATP synthase and without making ATP

Some ATP produced is used to actively transport substances into the mitochondria-Pyruvate-ADP and Pi

Some ATP is used for the shuttle to bring hydrogen from NADH made during Glycolysis, in the cytoplasm, to the mitochondria

Some of the NADH formed during the first 2 stages of respiration is used in other reaction in the cell instead of in oxidative phosphorylation

Anaerobic respiration in mammals and yeast:

Explain why anaerobic respiration produces a much lower yield of ATP than aerobic respiration.

Compare and contrast anaerobic respiration in mammals and in yeast.

ANAEROBIC RESPIRATION: The release of energy from substrates in the absence of oxygen

Oxygen acts as the final electron acceptor in oxidative phosphorylation Without oxygen, the electron transport chain cannot function Krebs cycle and link reaction stop NADH formed during the oxidation of glucose has to be reoxidised so that Glycolysis can keep happening

which increases the survival of the organism in adverse conditions

There are 2 anaerobic pathways:

1) Ethanol fermentation : Used by fungi such as yeast, and plant root cells use under waterlogged conditions2) Lactate fermentation : used by animals

Neither pathways produce ATP but 2 molecules of ATP are made in Glycolysis per molecule of glucose in substrate-level phosphorylation

Glycolysis produces 2 molecules of ATP, 2 molecules of NADH and 2 molecules of pyruvate Both take place in the cytoplasm

Lactate fermentation:

Occurs in mammalian muscle tissue during vigorous exercise, when the demand for ATP (for muscle contraction) is high and there is an oxygen deficit

NADH is reoxidised to NAD+, to transfer hydrogen to pyruvate to form lactate Pyruvate is the hydrogen acceptor- it accepts hydrogen atoms from NADH NAD is now reoxidised and is available to accept more hydrogen atoms from glucose in glycolysis Glycolysis can continue, generating enough ATP to sustain muscle contraction The enzyme lactate dehydrogenase catalyses the oxidation of NADH, and the reduction of pyruvate to

lactate The lactate is carried in the blood away from the muscles, to the liver When more oxygen becomes available, the lactate can be converted back into pyruvate, which may enter

the Krebs cycle via the link reaction, or it may be recycled to glucose and glycogen Muscle fatigue is not caused by the build up of lactate, but the resultant reduction in pH which will reduce

enzyme activity in the muscles

C6H12O6→2C3H 6O3 (lactate )

Alcoholic fermentation:

Under anaerobic conditions in yeast cells:

Each pyruvate molecule loses a CO2 molecule (decarboxylated) and becomes ethanal This reaction is catalysed by pyruvate decarboxylase (not present in animals) which has a coenzyme

(thiamine diphosphate) bound to it Ethanal accepts hydrogen atoms from NADH which becomes reoxidised as ethanal is reduced to ethanol

(catalysed by ethanol dehydrogenase) This production of ethanol also regenerates NAD The reoxidised NAD can now accept more hydrogen atoms from glucose, during glycolysis Yeast is a facultative anaerobe- it can survive without oxygen, but it is killed when the concentration of

ethanol builds up to around 15% Growth rate is faster under aerobic conditions At the beginning of the brewing process, yeast in grown under aerobic conditions and then placed in

anaerobic conditions to undergo alcoholic fermentation Anaerobes only partly break down food molecules, rather than fully breaking down to carbon dioxide

H

C6H12O6→2C2H5OH+2CO2¿¿

Structure and function of mitochondria:

Explain, with the aid of diagrams and electron micrographs, how the structure of mitochondria enables them to carry out their functions.

Ultrastrucutre:

MITOCHONDRIA: are organelles found in eukaryote cells. They are the sites of the link reaction, Krebs cycle and oxidative phosphorylation- the aerobic stages of respiration

First identified animal cells All have an outer and inner phospholipid membrane which make up the envelope The outer membrane is smooth and the inner is folded into cristae that gives the inner membrane a large

surface area The 2 membranes enclose and separate the two compartments within the mitochondrion Between the membranes is the intermembranal space The matrix is enclosed by the inner membrane The matrix is semi-rigid and gel-like, consisting of a mixture of lipids and proteins The matrix also contains looped mitochondrial DNA, mitochondrial ribosomes and enzymes Matrix contains all the reactants and enzymes needed for Krebs cycle to take place

Shape, size and distribution:

Rod shaped or thread-like Shape can change but most are 0.5-1.0 µm diameter and 2-5 µm long Athletes may have longer mitochondria in muscle tissue Metabolically active cells have more mitochondria which often have longer and more densely packed

cristae to house more densely packed electron transport chains and more ATP synthase enzymes Can be moved around the cells by the cytoskeleton (microtubules) In some cell types, the mitochondria are permanently positioned near a site of high ATP demand, such as at

the synaptic knob of nerve cells

Structure to function: Matrix

Matrix is where the link reaction and the Krebs cycle occur. It contains:

Enzymes that catalyse these reactions Molecules of coenzyme NAD Oxaloacetate- 4C that accepts acetate from the link reaction Mitochondrial DNA, which codes for some of the mitochondrial enzymes and other proteins Mitochondrial ribosomes where these proteins are assembled.

The outer membrane:

The phospholipid composition of the outer membrane is similar to the membranes around other organelles

Contains proteins, some of which form channels or carriers that allow the passage of molecules such as pyruvate

Other proteins in this membrane include enzymes

The inner membrane:

Different lipid composition from the outer membrane and is impermeable to most small ions including hydrogen ions

Folded into many cristae to give a large surface area Has embedded in it many electron carriers and ATP synthase enzymes

The electron carriers:

Protein complexes, arranged in electron transport chains Each electron carrier is an enzyme, each associated with a cofactor (non-protein groups). They are haem

groups and contain an iron atom The cofactors can accept and donate electrons because the iron atoms can become reduced (to Fe2+) by

accepting an electron and oxidised (to Fe3+) by donating an electron to the next electron carrier They are oxidoreductase enzymes as they are involved in oxidation and reduction reactions Some of the electron carriers have a coenzyme that pumps (using energy from the passage of e-) protons

from the matrix to the intermembranal space Because the inner membrane is impermeable to small ions, protons accumulate in the intermembranal

space building up a proton gradient- a source of potential energy

ATP synthase enzymes:

Large and protrude from the inner membrane into the matrix Known also as stalked particles Allow protons to pass through them

Chemiosmosis:

CHEMIOSMOSIS: the flow of hydrogen ions through ATP synthase enzymes. The force of this flow allows the production of ATP. Occurs across the thylakoids membranes during the light dependent stage of photosynthesis. Also occurs across the inner mitochondrial membrane during oxidative phosphorylation.

Protons flow down a proton gradient, through the ATP synthase enzymes, from the intermembranal space into the matrix

This flow is called chemiosmosis The force of this flow drives the rotation of part of the enzyme and allows ADP and Pi to be joined to make

ATP

Coenzyme FAD:

Flavine adenine dinucleotide Becomes reduced in the Krebs cycle, is tightly bound to a dehydrogenase enzyme that is embedded in the

inner membrane The H atoms accepted by FAD do not get pumped into the inert membrane space. Instead, they pass back

into the mitochondrial matrix

Evaluating the evidence for chemiosmosis:

Evaluate the experimental evidence for the theory of chemiosmosis.

CHEMIOSMOSIS: Is the diffusion of ions through a partially permeable membrane. It relates specifically to the flow of hydrogen ions (protons) across a membrane, which is coupled to the generation of ATP during respiration. In eukaryotic cells the membrane is the inner mitochondrial membrane and in prokaryotes it is the cell surface membrane, which may be invaginated to increase surface area.

Early evidence:

1940’s:

link between oxidation of sugars and the formation of ATP was made knew that reduced NAD linked metabolic pathways, such as Krebs cycle with the production of ATP did not know the biochemical mechanism which synthesises ATP Thought that the energy associated with NADH was first stored in a high-energy intermediate chemical

before being used to make ATP Didn’t understand the connection between the electron transport chain and ATP synthesis in respiration

1960’s:

Analysing mitochondria structure using electron microscopes and special staining techniques Identified inner and outer membranes, and the intermembranal space Observed folding (cristae) of the inner membrane covered on the inner surface with many small (9nm

diameter), mushroom shaped particles Chemiosmotic theory idea- energy lost from electrons moving down the electron transport chain creates a

proton gradient which is then used to synthesise ATP Now the most widely accepted theory

Peter Mitchell’s theory:

PROTON MOTIVE FORCE: Force produced as hydrogen ions flow, through ATP synthase channels, down their concentration gradient. The force causes ADP and Pi to combine and form ATP.

1961: Peter Mitchell realised that the build-up of H+ on one side of a membrane would be a source of potential energy and that the movement of ions across the membrane down an electrochemical gradient could provide the energy needed for formation of ATP form ADP and Pi (chemiosmosis theory)

The inner mitochondrial membrane is therefore an energy transducing membrane Thought that the energy released from the transfer of electrons along the electron transport chain was to

pump H+ from the matrix to the intermembranal space Thought these protons then flowed through protein channels, attached to enzymes The kinetic energy, or the force of this flow, the proton motive fore, drove the formation of ATP Initially, people were sceptical as it was very different from previous idea of a high energy intermediate

compound In 1978, there was a lot of supporting evidence Since then, has been established that the stalked particles are ATP synthase and how they function Also know that some of the enzyme have coenzymes associated that can use the energy released from

electron transport to pump H+ across the membrane, into the intermembrane space, where a proton or electrochemical gradient builds up

Experiment 1: low pH:

pH of the intermembrane space was found to be lower that the pH of the matrix a lower pH means the intermembrane space is more acidic- a higher concentration of H+ ions This observation shows that a proton gradient exists between the intermembrane space and the matrix of

the mitochondria

Experiment 2: low pH:

Artificial vesicles were created from phospholipid bilayers to present the inner mitochondrial membrane Proton pumps from bacteria (not from mitochondria, but have proton pumps which are similar to those in

mitochondria) and ATP synthase were added to the vesicle membranes The proton pumps are activated by light, so when light was shone into these vesicles, they started to pump

protons The pH inside the vesicle decreased- protons were being pumped into the vesicle from outside When ADP and Pi were added to the solution outside the vesicles, ATP was synthesised This artificial system shows that a proton gradient can be used to synthesise ATP (but doesn’t show that this

happens in mitochondria)

Experiment 3: mitochondria:

Mitochondria were put into a slightly alkaline solution (pH 8) They were left until the whole of each mitochondria (matrix and intermembrane space) reach pH8

When these mitochondria were given ADP and Pi, no ATP was produced

Mitochondria were transferred to a more acidic solution of pH 4 The outer membrane is permeable to protons- the protons moved into the intermembrane space creating a

proton gradient across the inner membrane

In the presence of ADP and Pi, ATP was produced. This shows that a proton gradient can be used to make ATP by mitochondria.

Experiment 4: uncouplers

Uncouplers are substances that destroy the proton gradient across the inner mitochondrial membrane An uncoupler was added to mitochondria, along with ADP and Pi

No ATP was made. This experiment shows that a proton gradient is required to synthesise ATP in mitochondria.

Evidence from other studies:

Some researchers isolated mitochondria by placing them in solutions of very low water potential so that the outer membrane is ruptured, releasing the contents of the intermembrane space

By further treating the mitoblasts (mitochondria split of their outer membranes) with strong detergent, they could rupture the inner membrane and release the contents of the matrix

This allowed them to identify where various enzymes are in the mitochondria, and to work out where the link reaction and Krebs cycle take place in the matrix, whilst the electron transfer chain enzymes are embedded in the inner mitochondrial membrane

Electron transfer in the mitoblasts did not produce any ATP so they concluded that the intermembrane space was involved

ATP was not made if the mushroom shaped parts were removed from the inner membrane of mitochondria

ATP was not made in the presence of oligomycin, an antibiotic now known to block the flow of protons through the ion channel part of the stalked particles

In intact mitochondria:

The potential difference across the inner membrane was -200mV, being more negative in the matrix side of the membrane than on the intermembrane space of the membrane

The pH of the intermembrane space was also lower than that of the matrix

Lag time:

Long time between making a discovery and being made a theory Other scientists conduct further research and experiments to gather more evidence More studies are replicated, with other scientists reaching same conclusions, the more reliable the evidence

is

Respiratory substrates:

Define the term respiratory substrate.

Explain the difference in relative energy values of carbohydrate, lipids and protein respiratory substrates.

Energy values of different respiratory substrates:

RESPIRATORY SUBSTRATE: an organic substance that can be used for respiration.

Majority of ATP made during respiration is produced during oxidative phosphorylation when hydrogen ions flow through channels associated with ATP synthase enzymes, on the inner mitochondrial membranes

The hydrogen ions and electrons that combine with oxygen to produce water The more protons the more ATP produced So, the more hydrogen atoms there are in a molecule of respiratory substrate, the more ATP can be

produced when that substrate is respired If there are more hydrogen atoms per mole of respiratory substrate, the more oxygen is needed to respire

that substrate as more electrons would be carried along electron transport chain

3 types (KJ/g):

Carbohydrate (glucose): 15.8 Lipid: 39.4 Protein: 17.0

Carbohydrate:

Cn(H2O)n+1 Glucose is the main respiratory substrate Some mammalian tissues can only use glucose for respiration

-Brain cells-Red blood cells

Animals store glucose as glycogen and plants store it as starch Both can be hydrolysed to glucose for respiration Other monosaccharides, such as fructose and galactose are changed to glucose for respiration Theoretical maximum energy yield for glucose in 2870KJ/mol It takes 30.6 KJ to produce 1 mol ATP Theoretically, 1 mole of glucose should produce nearly 94 mol ATP Actual yield is more towards 30 KJ/mol- an efficiency of about 32% The remaining energy is released as heat, which helps maintain a suitable body temperature, thus allowing

enzyme-controlled reactions to proceed

Protein:

Excess amino acids released after protein digestion, may be deaminated This involves the removal of the amine group that is converted to urea The rest of the molecule is converted into glycogen or ketoacid These can then be stored and respired to release energy When an organism is undergoing fasting, starvation or prolonged exercise, protein from muscle can be

hydrolysed to amino acids which can be respired Some can be converted to pyruvate, or to acetate and carried to the Krebs cycle Some enter the Krebs cycle directly 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

Lipids:

Important respiratory substrate for many tissues, especially muscles Triglycerides are hydrolysed by lipase to fatty acids + glycerol Glycerol can be converted to glucose, then respired, but fatty acids can’t

Fatty acids:

Fatty acids are long chain hydrocarbons with a carboxylic acid group In each molecule, there are many carbon atoms and even more hydrogen atoms These molecules are a source of many protons for oxidative phosphorylation so they produce a lot of ATP Each fatty acid is combined with CoA which requires energy from the hydrolysis of a molecule of ATP to

AMP (adenosine monophosphate) and 2Pi The fatty acid-CoA complex is transported into the mitochondrial matrix where it is broken down into 2-

carbon acetyl groups that are attached to CoA During this breakdown, by the β-oxidation pathway, NADH and FADH2 are formed The acetyl groups are released from the CoA and enter the Krebs cycle where 3 molecules of NADH, 1

molecule of FADH2 and one molecule of ATP (by substrate-level phosphorylation) are formed for each acetate

The large amount of NADH is reoxidised at the electron transport chain, during oxidative phosphorylation, producing large amounts of ATP by chemiosmosis

Fats are more highly reduced than sugars so more oxygen is required for their oxidation

Respiration experiments:

The volume of oxygen taken up or the volume of carbon dioxide produced indicates the rate of respiration A respirometer measures the rate of oxygen being taken up- the more oxygen taken up, the faster the rate

of respiration.

Respirometer:

Each tube contains potassium hydroxide which absorbs carbon dioxide The control tube is set up in exactly the same way as the test tube, but without the insects, to make sure the

results are only due to the insect respiring (e.g. contains glass beads which have the same mass as the insects)

The syringe is used to set the fluid in the manometer to a known level The respirometer is left for a set period of time. During that time, there’ll be a decrease in the volume of the air in the test tube, due to oxygen

consumption by the insects All the CO2 produced is absorbed by the potassium hydroxide The decrease in the volume of air will reduce the pressure in the tube and cause the coloured liquid in the

manometer to move towards the test tube The distance moved by the liquid in a given time is measured. This value can then be used to calculate the

volume of oxygen taken in by the woodlice per minute Any variables that could affect the results are controlled

-Temperature-Volume of KOH solution in each test tube-Breed of insect

To produce more reliable results the experiment is repeated and a mean volume of oxygen is calculated Could use water instead of KOH which will give an indication of much CO2 is produced Can then calculate RQ.

Respiratory quotient:

RESPIRATORY QUOTIENT: the ratio of the amount of carbon dioxide and oxygen exchanged in a given time

RQ= (volume∨molecules )Carbondioxide produced(volume∨molecules )Oxygenused

Glucose= 1.0 Protein=0.9 Fat= 0.7

If the internal radius of the manometer tube is known, the volume of gases can be calculated by using the equation:

Volume=πr 2h

R= internal radius H= distance moved by the liquid

ANABOLIC: type of metabolism: biochemical reactions that syntheses large molecules from smaller molecules. This requires energy/ATP.

ATP: Molecule (nucleotide derivative) which is found in all living cells and involved in energy transfer. It’s a phosphorylated nucleotide and is the universal energy currency. When hydrolysed it releases energy.

CATABOLIC: type of metabolism: biochemical reactions that produce small molecules by hydrolysis of larger molecules.

CHEMIOSMOSIS: Is the diffusion of ions through a partially permeable membrane. It relates specifically to the flow of hydrogen ions through ATP synthase enzymes. The force of this flow allows the production of ATP during respiration. In eukaryotic cells the membrane is the inner mitochondrial membrane and in prokaryotes it is the cell surface membrane, which may be invaginated to increase surface area.

COENZYME A: A coenzyme that carries acetate from the link reaction to Krebs cycle

COENZYME: an organic non-protein molecule that binds temporarily with substrate to an enzyme active site. It is essential for enzyme activity. Aids the function of an enzyme by transferring a chemical group from one molecule to another. In respiration, many coenzymes remove hydrogen atoms from substrates.

DEAMINATION : The removal of the amine group from an amino acid to produce ammonia.

ELECTRON ACCEPTORS: chemicals that accepts electrons from another compound. They are reduced while acting as oxidising agents.

ENERGY: is the ability to do work

GLYCOLYSIS: Metabolic pathway. The first stage of respiration. It is anaerobic and occurs in the cytosol (cytoplasm). Although anaerobic, it involves oxidation as substrate molecules are dehydrogenated.

HYDROLYSIS: The chemical breakdown of large molecules into smaller molecules by the addition of water

KREBS CYCLE: Third stage of respiration. It is aerobic and in eukaryotes in takes place in the matrix of the mitochondria. It oxidises acetate to carbon dioxide. NAD and FAD are reduced. ATP is made by substrate-level phosphorylation.

LINK REACTION: Stage of aerobic respiration that links Glycolysis with the Krebs cycle. In eukaryote cells it occurs in the mitochondrial matrix. It converts pyruvate to acetate. NAD is reduced.

MITOCHONDRIA: are organelles found in eukaryote cells. They are the sites of the link reaction, Krebs cycle and oxidative phosphorylation- the aerobic stages of respiration

NAD: Coenzyme involved in respiration. It removes hydrogen atoms from substrates. It becomes reduced NAD which carries hydrogen atoms (protons and electrons)

OXIDATION : Chemical reaction involving loss of electrons, gain of oxygen or loss of hydrogen atoms

OXIDATIVE PHOSPHORYLATION: The formation of ATP by chemiosmosis by adding a phosphate group to ADP in the presence of oxygen which is the final proton acceptor.

PHOSPHORYLATION : The addition of a phosphate group

PHOTOAUTOTROPHS: organisms that carry out photosynthesis to acquire energy.

PROTEIN: A macromolecule. A polymer of many amino acids joined by peptide bonds. May also be called a polypeptide.

PROTON MOTIVE FORCE: Force produced as hydrogen ions flow, through ATP synthase channels, down their concentration gradient. The force causes ADP and Pi to combine and form ATP.

REDUCTION : Chemical reaction involving the gain of electrons, gain of hydrogen atoms or loss of oxygen atoms.

RESPIRATION: the process whereby energy stored in complex organic molecules (carbohydrates, fats and proteins) is used to make ATP. It occurs in living cells. A metabolic pathway.

RESPIRATORY QUOTIENT: the ratio of the amount of carbon dioxide and oxygen exchanged in a given time

RQ= (volume∨molecules )Carbondioxide produced(volume∨molecules )Oxygenused

RESPIRATORY SUBSTRATE: any organic substance that can be used for respiration.

SUBSTRATE LEVEL PHOSPHORYLATION: formation of ATP from ADP and Pi during Glycolysis and the Krebs cycle

UREA: An excretory product formed from the breakdown of excess amino acids.