A2 Biology

INDEX PAGE
THE NEED TO RESPIRE
STRUCTURE OF ATP AND ATP USES
COENZYMES IN RESPIRATION:
NAD AND COENZYME A
GLYCOLYSIS
ACTIVE TRANSPORT OF PYRUVATE IN AER.
RESPIRATION
STRUCTURE AND FUNCTION OF
MITOCHONDRIA
LINK REACTION:
DECARBOXYLATION OF PYRUVATE TO
ACETATE
REDUCATION OF NAD
COMBINATION OF ACETATE AND COENZYME
A
KREBS CYCLE
OXIDATIVE PHOSPHORYLATION:
ELECTRON CARRIERS

CHEMIOSMOSIS:
ELECTRON TRANSPORT CHAIN
PROTON GRADIENTS
ATP-SYNTHASE
EXPERIMENTAL EVIDENCE FOR
CHEMIOSMOSIS
THEORETICAL MAXIMUM YIELD OF ATP
ANAEROBIC RESPIRATION:
LOWER ATP PRODUCTION
MAMMALS AND YEAST
RESPIRATORY SUBSTRATES
ENERGY LEVELS OF PROTEINS, CARBS AND
LIPIDS

START -- ADENOSINE TRIPHOSPHATE

ADENOSINE
TRIPHOSPHATE
HEINEMANN BIOLOGY 1.4.1

ATP AND RESPIRATION

Respiration is the usage of the energy stored in complex organic molecules such as
lipids, carbohydrates and proteins for ATP production. All living cells respire. Moving
molecules possess kinetic energy which can be transferred down a gradient, large
organic molecules will contain chemical potential energy as bonds can be broken and
made to release energy.
We know from earlier study that;
- energy cannot be created or destroyed, only ever transformed or transferred
- energy is measured in joules or kilojoules
- energy exists in various forms; chemical, kinetic, light, potential, gravitational
potential and so on
Energy is needed to allow biological processes to happen; our metabolism (all the
chemical reactions in our body) requires energy. Anabolic reactions build up large
molecules from smaller ones, catabolic reactions break them down and both types
together make up our metabolism.
Metabolic reactions include the active transport of molecules and ions against the
concentration gradient, like the transport of sodium/potassium across a cell membrane to
maintain resting potentials (all cells have these pumps). Secretion, like exocytosis
(release of large molecules from a cell) or the opposite; endocytosis, are both metabolic.
Anabolic processes like the building of proteins from amino acids, or the building of
cellulose from beta glucose are both metabolic. Cellular locomotion (flagella etc), DNA

ATP
Photoautotrophs like some protoctists, plants and some bacteria use the Suns light
energy to synthesise complex organic molecules that contain chemical potential energy.
They, the consumers and the decomposers all respire these molecules to release energy,
which is used to phosphorylate (add inorganic phosphate to) ADP to create ATP; this
transfers energy to the ATP molecule.
What is ATP and what makes it so damn good?
- ATP is described as a high-energy intermediate compound, RIBOSE
meaning that is stores a lot of chemical potential energy,
- it can be hydrolysed to release 30.6 kJ/mol of energy, so it
provides small manageable quantities of energy
for cells,
PHOSPHATE
- small packets of energy means little is wasted,
- ATP is formed of a phosphorylated nucleotide; adenosine, a
ribose sugar and three phosphate (phosphoryl) groups,
- it is described as the universal energy currency.

ADENINE

Respiration occurs in many small stages, with the energy released at each being used to
join ADP and Pi (phosphate). Youll go through about 20 - 25 kgs of it a day, though only
5g will be present at any one time as it is hydrolysed and re-synthesised all the time. The
hydrolysis of ATP is coupled with a synthesis, like the replication of DNA. This is so that
the energy can be used immediately for biological processes.

ATP AND RESPIRATION

HEINEMANN 1.4.1 QUESTIONS
1.Outline the energy transformations that occur in fireflies as they use their food as an
energy source for bioluminescence
The complex organic molecules in the fireflys food will contain stored chemical energy
that is released when these molecules are respired. This energy is then used to
phosphorylate ADP into ATP, which can be used in bioluminescent reactions.
2.ATP is a nucleic acid/nucleotide derivate, but is it derived from DNA or RNA?...
ATP will be derived from RNA as the sugar present is ribose and not deoxyribose.
3.Decide whether each of the following are anabolic or catabolic
a) synthesis of spindle microtubes during mitosis [ANABOLIC]
b) digestion of starch to maltose [CATABOLIC]
c) formation of insulin in the cells of the pancreas [ANABOLIC]
d) conversion of glycogen to glucose in the liver cells [CATABOLIC]
e) digestion of a pathogen inside a phagolysosome inside a macrophage [CATABOLIC]

START -- COENZYMES

COENZYMES; NAD
AND CoA
HEINEMANN BIOLOGY 1.4.2

OUTLINE OF RESPIRATION

LACTATE
FERMENTATION

LACTATE

ETHANOL
FERMENTATION

ETHANOL
+ CO2

ANAEROBIC
GLYCOLYSIS
GLUCOSE
PYRUVATE
AEROBIC
LINK REACTION
PYRUVATE ACETYL
CoA

KREBS
CYCLE

CARBON DIOXIDE

CARBON DIOXIDE

OXIDATIVE
PHOSPHORYLATION

COENZYMES
Glycolysis is an ancient biochemical mechanism that splits a sex carbon glucose sugar
into two molecules of pyruvate (3C). It happens in the cytoplasm of cells. In the link
reaction, a pyruvate is dehydrogenated and decarboxylated, then converted to acetate.
The Krebs cycle takes place in the matrix of mitochondria, where the acetate molecule is
dehydrogenated and decarboxylated. Oxidative phosphorylation takes place in the folded
inner membrane of mitochondria (cristae) where ADP is phosphorylated into ATP. The last
three of these processes cannot take place in the absence of oxygen and so can only
The above
stages of respiration all depend on the
occur
aerobically.
action of enzymes, the link reaction, Krebs cycle and
glycolysis all involve hydrogen atoms being removed
PANTOTHENIC ACID
from substrate molecules (oxidation). This is
facilitated by the action of dehydrogenase enzymes
but enzymes arent generally good at catalysing
PHOSPHATE
CYSTEINE
oxidation or reduction reactions so coenzymes such
as NAD combine with these enzymes. NAD carries
COENZYME A
hydrogen atoms (become reduced) from around the
RIBOSE
cell, to the cristae of the mitochondria where they
are involved in the process of oxidative
phosphorylation, producing a lot of ATP. When the
NAD molecule/other coenzyme deposits the H atoms
in the membrane, it becomes re-oxidised so that it
may recombine with H atoms from the first three
stages of respiration. CoA is made from pantothenic
acid, three phosphoryl groups, cysteine and a ribose

START -- MITOCHONDRION
ULTRASTRUCTURE

MITOCHONDRION
ULTRASTRUCTURE
HEINEMANN BIOLOGY 1.4.4

STRUCTURE OF A MITOCHONDRION
We all know that mitochondria are the bean-shaped organelle with squiggles in it when
we see it under a microscope, but it really is a complex and multi-functional piece of kit.
The organelle itself has two membranes; an inner and an outer, that together make up
the envelope. Sandwiched in by this envelope is the intermembrane space. The large gel
like inner of the organelle is called the matrix and is enclosed entirely by the inner
membrane, it contains proteins, lipids, enzymes, ribosomes and looped mitochondrion
DNA. The line-like projections visible under a light microscope are examples of the cristae
that are formed through the elongation of the inner membrane, as to create a larger
surface area.
In numbers, mitochondria are about 0.5 - 1.0 m wide and about 2 - 5 m long, though
can be up to 10 m. Cells that are going to be using a lot more energy, or even just sites
within a cell that are near areas of high ATP demand, will have many mitochondria. For
example, a synapse in a nerve cell has many mitochondria. The cells that are more
metabolically active will have not only more mitochondria, but denser ones as well. This
is so that their intermembrane space can house more electron transport chains and ATP
synthase. When we talk about these organelles being in a certain position, they have
been moved there by the microtubule motors of the cytoskeleton, but as mentioned
before, cells like mammalian liver cells will contain up to 2500 mitochondria (20% of the
cell volume) and so microtubule motors play less of a role here.

STRUCTURE - DIAGRAMMATICALLY

STRUCTURE TO FUNCTION
The matrix of a mitochondrion is well adapted for its function; it contains a rich broth of
all the necessary components for the Link Reaction and Krebs cycle. This means that is
contains; enzymes that catalyse each step, the coenzyme NAD, oxaloacetate which
accepts acetate from the Link Reaction, mitochondrial ribosomes (same as prokaryotic)
and also mitochondrial DNA that codes for proteins and enzymes.
The inner membrane is also very well adapted. It has a different composition to the outer
membrane and as such, is impermeable to small ions, like H ions. This impermeably
means that a proton gradient can be set up as a source of potential energy. The large
surface area of the folded cristae combined with the presence of numerous electron
carriers and ATP synthase enzymes means that this organelle is capable of producing a
lot of ATP.
When we say electron carriers what we mean are; a series of oxidoreductase enzymes
with a non-protein cofactor (in this case; haem) that allows the transport of electrons via
a chain mechanism. The haem (Fe) group can accept an electron to become reduced
and then donate it to the next carrier to become oxidised. Some of these enzymes also
have a coenzyme that will pump protons (H ions) into the intermembrane space using
the energy created from the passage of electrons, so that a proton gradient can be
established and maintained, as a source of potential energy. The ATP synthase enzymes
use the proton gradient to phosphorylate ADP into ATP. The large headpiece protrudes
into the matrix of the organelle, whilst the base piece sits in the inner membrane. The
force of the protons moving through the enzyme (via chemiosmosis) causes the head to

ATP SYNTHASE - DIAGRAMMATICALLY

MATRI
X

MATRI
X

STATO
R

STATO
R

INTERMEMBRANE
SPACE

INTERMEMBRANE
SPACE

START -- THE RESPIRATION PATHWAY

THE RESPIRATION
PATHWAY
HEINEMANN BIOLOGY 1.4.3, 1.4.5, 1.4.5,
1.4.6 AND 1.4.7

GLYCOLYSIS STEP-BY-STEP
Occurring in the cytoplasm of all living cells that respire (therefore eukaryotes and prokaryotes),
glycolysis consists of ten steps, each catalysed by a different enzyme. Well look at it as just
three stages
Stage 1: Phosphorylation
- One molecule of ATP is hydrolysed to release a phorphoryl group that then attaches to a
glucose molecule at carbon six. The glucose then becomes a fructose molecule (still with the
phosphoryl at carbon six) before becoming fructose-1,6-bisphosphate after attachment of
another phosphate. The energy released from the two hydrolysed molecules activates the
molecule, so that it is now called hexose-1,6-bisphosphate . This stage gas required the use of
two molecules of ATP.
Stage 2: Splitting the hexose, oxidising the triose
- Each molecule of hexose is now split into two triose phosphates, then each stripped of two
hydrogen atoms via dehydrogenase action. NAD (nicotinamide adenine dinucleotide) assists
the enzyme here , so two molecules of NAD are used and reduced. Two molecules of ATP are
also produced here in substrate-level phosphorylation.
Stage 3: Conversion of triose to pyruvate
- The triose molecules are then converted to pyruvate molecules (3C) and two further
molecules of ATP are produced in substrate-level phosphorylation.

GLYCOLYSIS SUMMARY
After glycolysis, two molecules of ATP have been produced (net) as two have been
hydrolysed in stage 1. Two molecules of reduced NAD are also produced which carry
hydrogen atoms via a shunt mechanism to the inner mitochondrial membranes for
oxidative phosphorylation. The two molecules of pyruvate that are produced at stage 4
carry on via active transport to the mitochondrial matrix where they are dehydrogenated
and decarboxylated.
Isomerases are enzymes that change the shape of a molecule without affecting the
proportion of atoms within said molecule. These would be involved in stage 1 of
glycolysis where the glucose-6-phosphate is converted into a fructose-6-phosphate. The
fact that nearly all living organisms have this metabolic pathway indicates that we have
all evolved from a common ancestor at some point, and so supports the theory of
evolution.
GLUCOSE 6C

GLUCOSE 6P

FRUCTOSE
1P

HEXOSE-1,6BISPHOSPHAT
E

GLYCOLYTIC PATHWAY

2 X TRIOSE
PHOSPHAT
E

2X
INTERMEDIA
TE
COMPOUND

2X
PYRUVATE

LINK REACTION/KREBS CYCLE

The function of the link reaction is to convert pyruvate into acetate for the Krebs cycle.
This happens via the action of the enzymes; pyruvate dehydrogenase and pyruvate
decarboxylase. The hyrdogen that is stripped from the pyruvate is accepted by NAD, so
that it becomes reduced (NADH) and the carboxyl groups removed eventually become
carbon dioxide. The acetate is carried to the next stage via CoA, which accepts the
acetate to become Acetyl CoA.
2Pyruvate + 2NAD+ +2CoA 2CO + 2NADH + 2Acetyl CoA
In the Krebs cycle, the acetate is offloaded from the CoA and combines with oxaloacetate
to form a 6C (six carbon) compound, Citrate. This compound is then dehydrogenated and
decarboxylated, producing carbon dioxide and and reduced NAD. The resultant 5C
compound is the dehydrogenated and decarboxylated again, giving off another carbon
dioxide and another reduced NAD and a 4C compound that then is changed into another
4C, phosphorylating ADP in the process. After this substrate-level phosphorylation, the 4C
compound is dehydrogenated (this time reducing FAD) and then again (reducing NAD) to
regenerate the oxaloacetate.
As glucose produces two molecules of pyruvate which are converted to two molecules of
acetate, each molecule of glucose sends two molecules of acetate round the cycle.

LINK REACTION/KREBS CYCLE

For each molecule of glucose used (two turns of the Krebs cycle), the following is
produced;
PRODUCT PER MOLECULE OF
LINK REACTION
KREBS CYCLE
GLUCOSE
Reduced NAD

Reduced FAD

Carbon dioxide

ATP

Although oxygen isnt used in either of these two processes, they wont occur
anaerobically. Fatty acids can enter the Krebs cycle via CoA, some amino acids can enter
directly or may need to be converted to pyruvate or acetate, but will always need to be
deaminated.

OXIDATIVE PHOSPHORYLATION
The final stage of respiration is the transport of electrons in the electron transport chain,
a process that occurs in the inner mitochondrial membrane. Reduced coenzymes NAD
and FAD present in the matrix are present in their reduced state due to a shunt
mechanism that transports the hydrogen across the inner membrane, which is otherwise
impermeable to them. NAD donates its hydrogen atoms to the first carrier in the chain,
complex I/NAD dehydrogenase, where theyre split into protons and electrons. The
protons enter the matrix, where they are pumped via associated coenzymes into the
inter-membrane space, where a proton/pH/electrochemical gradient builds up as a source
of potential energy. FAD inputs its hydrogen further down the chain than NAD as it has a
higher affinity for the hydrogen than NAD.
The protons in the IM space can only diffuse back into the matrix via channels associated
with ATP synthase enzymes; this flow is called chemiosmosis.
Oxidative phosphorylation is simply the phosphorylation of ADP in the presence of
oxygen. As the final electron acceptor in the chain, oxygen combines with four electrons
and four hydrogen ions to become reduced to form water. The movement of some of
these hydrogen ions through the ATPase drives the rotation part of the enzyme, joining
ADP and Pi.

NET/THEORETICAL ATP PRODUCTION

THE NUMBER OF MOLECULES MADE FROM ONE MOLECULE OF GLUCOSE
NAME OF MOLECULE

STAGE OF RESPIRATION
GLYCOLYSIS

LINK REACTION

KREBS CYCLE

Reduced NAD

Reduced FAD

So to summarise, we should theoretically have 30 molecules of ATP at the end of one full
cycle for one molecule of glucose; four from substrate-level phosphorylation in glycolysis
and the Krebs cycle, 26 from oxidative phosphorylation (NAD). The reduced NAD and FAD
have provided the electrons for the chain, and the protons both for reduction of oxygen
and the establishment of a pH gradient (FADs protons stay in the matrix) and can now
be reused.
Although this is the theoretical yield, we never actually get 30 ATP molecules from one
molecule of glucose. This is due to the fact that some protons will leak across the inner
membrane and so cannot be in the intermembrane space to provide the pH gradient that
drives the phosphorylation of ADP. Also, some ATP is used to transport pyruvate into the
matrix and some is used to shuttle hydrogen from reduced NAD (via shunt mechanism)
into the matrix.

EVIDENCE FOR CHEMIOSMOSIS

Before 1961, it was generally accepted that energy associated with reduced NAD was
stored as a high energy intermediate compound, though investigation showed no such
evidence. In 1961, Peter Mitchell recognised that H+ ions moving down an
electrochemical gradient could be a source of potential energy and could provide the
energy needed to combine ADP and Pi. This was his chemiosmosis theory.
The membrane was therefore postulated to be energy transducing, where the energy
released from electron transfer in the membrane is used to pump protons from the
matrix into the IM space, which then flowed through pores to drive formation of ATP.

START -- ANAEROBIC RESPIRATION

ANAEROBIC
RESPIRATION
HEINEMANN BIOLOGY 1.4.8

ANAEROBIC CONDITIONS
Without oxygen, the link reaction, Krebs cycle and electron transport chain cannot occur;
it is only the first stage of respiration, glycolysis, that is able to do function anaerobically.
Two molecules of ATP are produced per molecule of glucose during substrate-level
phosphorylation in this stage, and so it cannot be used for a sustained period of time as
it is hideously inefficient and primitive, but it can be enough during periods of high ATP
demand and oxygen deficit.
In order for glycolysis to keep providing ATP via s.l.p, the NAD that is reduced during the
process must be reoxidised, which is done differently depending upon whether youre a
yeast cell, or a mammal. Yeast use the process of alcohol/ethanol fermentation, whereas
we use lactate fermentation.

ANAEROBIC RESPIRATION IN YEAST

Alcoholic fermentation consists of two simple steps. The first is the decarboxylation of
pyruvate to produce ethanal, which is catalysed by a type of pyruvate decarboxylase
with a thiamine diphosphate coenzyme attached (not found in mammals). This ethanal is
then able to accept hydrogen atoms from the reduced NAD, becoming reduced itself to
form ethanol and reoxidising the NAD. This stage is catalysed by ethanol dehydrogenase.
As a facultative anaerobe, yeast is able to live under anaerobic conditions, but dies in
ethanol concentrations above 15%. Yeasts growth rate is faster under aerobic conditions
and so is grown as such at the beginning of the brewing process, but then respires
anaerobically when alcoholic fermentation occurs.

ANAEROBIC RESPIRATION IN MAMMALS

Lactate fermentation is a far more efficient and effective method of anaerobic respiration as it gives a
product that has direct further use. The process, like with yeast, starts off with pyruvate, but simply
involves its reduction to lactate. This reaction is catalysed by the enzyme lactate dehydrogenase, as
pyruvate directly accepts hydrogen atoms from the reduced NAD, reoxidising the NAD and becoming
lactate.
This lactate/lactic acid is then transported away in the blood to the liver, where it can either; be stored
for later conversion to pyruvate (when the oxygen deficit is dealt with) or it can be recycled into glucose
and glycogen. Muscle fatigue is not caused by a build up of lactate in the muscles, as protein buffers in
the blood plasma prevent small changes of pH from surfacing. It is, specifically, the reduction of pH in
the muscles that reduces enzyme activity, so in this case, the buffers will not have been enough to
restore the pH.

START -- RESPIRATORY SUBSTRATES

RESPIRATORY
SUBSTRATES
HEINEMANN BIOLOGY 1.4.9

DIFFERENT FORMS OF ENERGY

A respiratory substrate is an organic molecule that can be respired to release energy. It is
the ability to donate hydrogen that makes a molecule a good source of energy, as it is
these atoms that drive the ATP synthase enzymes via chemiosmosis. It follows, that a
molecule with a lot of hydrogen per mole of substrate will be a good source of energy,
but will also require more oxygen. This is because the hydrogen atoms and electrons
reduce molecular oxygen into water in the matrix.
The main forms of energy well consider are; carbohydrates, lipids and proteins.
Carbohydrates such as glucose are the main substrates for respiration, with some cells
i.e. brain cells and red blood cells, only being able to respire glucose. Animals store
carbohydrates as glycogen, plants store it as starch but both are easily hydrolysed to
form glucose again. Other monosaccharides such as galactose and fructose are
converted to glucose for respiration, which has a maximum energy yield of 2870 kJ/mol.
As it only takes 30.6 kJ to produce 1 mole of ATP, this means we should be generating 94
mol of ATP per molecule of glucose, but the process is only 32% efficient in terms of ATP
synthesis as a lot of energy is released as heat, which helps maintain body temperature
and suitable conditions for enzyme activity.

DIFFERENT FORMS - PROTEINS

PYRUVATE
THIS DIAGRAM SHOWS THE AMINO
ACIDS
AND THE POINTS AT WHICH THEY
ENTER THE
LINK/KREBS CYCLE

ACETYL CoA

THREONINE, GLYCINE,
SERINE, CYSTINE,
TRYPTOPHAN

LYSINE, TRYPTOPHAN,
LEUCINE, ISOLEUCINE

PHENYLALANINE,
TYROSINE

THE NUMBER OF HYDROGEN

ATOMS PER MOLE OF AMINO
ACID THAT REDUCE NAD AND
ARE THEN USED IN OXIDATIVE
PHOSPHORYLATION ARE
SLIGHTLY HIGHER THAN WITH
GLUCOSE AND
CARBOHYDRATES, SO THEY
GIVE FRACTIONALLY MORE
ENERGY

GLUTAMIC ACID,
PROLINE, HISTIDINE,
ARGININE

DIFFERENT FORMS - LIPIDS

RESPIRATORY SUBSTRATE

MEAN ENERGY VALUE (kJ

per G)

Carbohydrate

15.8

Lipid

39.4

Protein

17.0

Lipids are important respiratory substrates for many tissues, like muscle. Fats are
hydrolysed via the action of lipase into glycerol, which can be converted to glucose, and
fatty acids; which have to be combined with CoA and then broken down.
In this process, the fatty acid is first combined with CoA using energy from the hydrolysis
of ATP to AMP (and two phosphoryl groups). This complex is then transported the to the
mitochondrial matrix, where it is split into 2C acetyl groups attached to CoA. Then, via
the oxidation pathway, acetyl groups are released after having reduced many NAD and
FAD molecules. These acetyl groups enter the Krebs cycle and go on to produce a further
three NADH, one FADH and an ATP via substrate-level phosphorylation. A large amount
of NAD is reoxidised in the electron transport chain and so lipids produce a lot of ATP via
chemiosmosis.