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
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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.
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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:
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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
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-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
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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:
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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:
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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
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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.
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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)
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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 EWhen 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
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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:
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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.
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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:
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Phosphorylation
Splitting of hexose 1,6- biphosphate
Oxidation of triose phosphate
Conversion of triose phosphate to pyruvate
Phosphorylation:
HYDROLYSIS: The chemical breakdown of large molecules into smaller molecules by the addition of water
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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
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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
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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:
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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:
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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:
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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.
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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
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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.
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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
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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
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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:
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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.
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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.
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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
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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.
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4H+
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
+ 4e-
O2
=
2H20
ATP BEFORE OXIDATIVE PHOSPHORYLATION: 2 (from Glycolysis) + 2 (from Krebs cycle) = 4
ATP during oxidative phosphorylation:
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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
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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
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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 conditions
2) Lactate fermentation: used by animals
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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:
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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
C6 H12 O6 → 2C3 H6 O3 (lactate)
Alcoholic fermentation:
Under anaerobic conditions in yeast cells:
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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
C6 H12 O6 → 2C2 H5 OH + 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
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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:
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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:
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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:
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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:
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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:
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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:
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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.
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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:
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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:
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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:
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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.
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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:
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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:
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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:
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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
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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
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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:
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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:
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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:
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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.
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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):
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Carbohydrate (glucose): 15.8
Lipid: 39.4
Protein: 17.0
Carbohydrate:
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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:
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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:
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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:
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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 2carbon 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
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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:
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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:
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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
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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 =
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(𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐫 𝐦𝐨𝐥𝐞𝐜𝐮𝐥𝐞𝐬)𝐂𝐚𝐫𝐛𝐨𝐧 𝐝𝐢𝐨𝐱𝐢𝐝𝐞 𝐩𝐫𝐨𝐝𝐮𝐜𝐞𝐝
(𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐫 𝐦𝐨𝐥𝐞𝐜𝐮𝐥𝐞𝐬)𝐎𝐱𝐲𝐠𝐞𝐧 𝐮𝐬𝐞𝐝
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 = 𝝅𝒓𝟐 𝒉
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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
(𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐫 𝐦𝐨𝐥𝐞𝐜𝐮𝐥𝐞𝐬)𝐂𝐚𝐫𝐛𝐨𝐧 𝐝𝐢𝐨𝐱𝐢𝐝𝐞 𝐩𝐫𝐨𝐝𝐮𝐜𝐞𝐝
𝐑𝐐 =
(𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐫 𝐦𝐨𝐥𝐞𝐜𝐮𝐥𝐞𝐬)𝐎𝐱𝐲𝐠𝐞𝐧 𝐮𝐬𝐞𝐝
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.