B io Factsheet

www.curriculum-press.co.uk Number 223

Chemiosmosis
• Osmosis is the diffusion of water molecules across a selectively permeable membrane; this movement occurs down a water potential
gradient.
• Chemiosmosis is the diffusion of ions across a selectively permeable membrane, down a solute potential gradient.
• In particular, the term chemiosmosis refers to the movement of hydrogen ions (protons, H+) across a membrane:
• The existence of a proton gradient across the membrane is equivalent to a form of potential energy, and the movement of the protons
can be coupled (linked) to metabolic work in cells.

Fig 1 : Potential energy coupling to work

Synthesis of ATP in mitochondria

and chloroplasts

H+ H+
Active transport of, e.g. K+ by cells
H+
Energy input
(e.g. oxidation Transfer of DNA during bacterial conjugation
of food) H+ and genetic transformation
Proton gradient proton
across membrane flow Movement of bacterial flagella
= potential energy
Production of heat

Chemiosmosis and ATP synthesis The proton gradient is dissipated by allowing protons to pass
The production of ATP is endogonic (energy-requiring) (Fig 2). through a pore in the membrane, and the movement of protons
drives the production of ATP by the enzyme ATP synthase (Fig 3).
Fig 2. ATP synthesis
Fig 3. Simple version of proton gradient generation
_ _ _
O O O
_
O P O P O + O P OH + H+
O O Energy
O
ADP Phosphate (oxidation of food or light absorbed
by chlorophyll)
energy
ATP synthase
H+ H+
_ _ _ H+ H+ H+
O O O H+
_ H+
O P O P O P O H+ H+

O O O membrane is
ATP almost completely
PUMP
impermeable to
protons
The energy required to combine adenosine diphosphate and
phosphate can be supplied by a proton gradient: this proton gradient H+ H+
is set up by an electron transport chain which uses a series of
oxidation-reduction reactions to provide the energy to ‘pump’ Proton pumped across
protons across a membrane. The combination of the gradient of membrane generate PMF
proton concentration and the gradient of charge i.e. the complete (proton motive force)
electrochemical gradient provides the proton motive force (PMF).

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Chemiosmosis in mitochondria : oxidative phosphorylation

Peter Mitchell, a British biochemist, knew that there was a series of electron carriers in the inner mitochondrial membrane. These carriers
(a) Are arranged asymmetrically, so that they can separate protons from electrons
(b) Contain ions with variable oxidation states e.g. Feii/feiii which can accept electrons (become reduced) and then pass them on (become
re-oxidised). The presence of Fe and Cu ions means that these carriers are coloured: they are sometimes called cytochromes (cell
colours).
(c) Can eventually be oxidised by molecular oxygen
(d) Are situated close to proton channels and molecules of ATP synthase.

Mitchell also knew that the inner mitochondrial membrane was almost completely impermeable to protons.
He provided a detailed explanation of how electrons carried from the TCA cycle and from glycolysis could pass along this electron
transport chain to generate a proton gradient, and how this proton gradient could then be used to drive the phosphorylation of ADP to
ATP. Because this set of reactions required a series of oxidations and the creation of ATP from ADP, it is known as oxidative phosphorylation.

Fig 4. Mitchell’s chemiosmotic theory and oxidative phosphorylation

inner membrane
outer membrane ATP synthase protein structure
crista matrix intermembrane Inner
membrane
stator
proton
channel
innermembrane folded
into crista = larger surface
base
area
piece
Electron carrier enzyme head piece
each enzyme is associated 3 2e - stalk/axle
2e -
Intermembrane
with a cofactor that
space Matrix
contains an iron atom 2e-
Fe
3 2e-
2 Electrons passed 2e-
along (from carrier 2e - 2e-
enzyme to carrier Fe -
Fe ATP 2e-
+ ½O2 → H2O
H
enzyme) 2H+
H-
2
Hydrogen atoms from + 2e- 2e - 6
1 2H+ H- 5
Krebs cycle and Link Fe
reaction carried by H- H- ADP + Pi ATP synthase enzyme
NAD & FAD matrix -
H H- Proton channel
-
H 4

intermembrane space

inner membrane
Build up of protons in the intermembrane space
4 producing a proton gradient across the inner membrane
(from the matrix to intermembrane space)

1. For each molecule of glucose, 8 molecules of reduced NAD are formed (2 in the Link reaction and 6 in the Krebs cycle)
2. The hydrogen atoms on reduced NAD are split into protons (H+) and electrons. The electrons are passed through a chain
of electron carriers (enzymes and their cofactors)
3. As electrons flow along the electron transfer chain (ETC) energy is released and used by coenzymes to pump the protons
from the matrix of the mitochondria across the inner mitochondrial membrane and into the intermembrane space
4. Thus, a proton gradient is established across the inner membrane. This proton gradient represents potential energy
5. The protons are unable to diffuse back across the inner membrane as it is impermeable to them. However, they can diffuse
through proton channels in the membrane – this flow is chemiosmosis
6. These channels are associated with the enzyme ATP synthase. The protons cause part of this enzyme to rotate which
enables the enzyme to join ADP with inorganic phosphate to form ATP

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223. Chemiosmosis
How science works: Peter Mitchell & chemiosmosis
When Peter Mitchell began his work on ATP production he was
ridiculed. At that time (in the 1950’s), biochemists knew that electrons
passed along a chain of electron carriers in the inner mitochondrial
membrane, and they knew that ATP could be produced in the
mitochondrion if oxygen was available. However, they believed
that the energy from electron transfer was temporarily stored as a
stable ‘high-energy intermediate’, and that this high energy
compound was then used to phosphorylate ATP.
Mitchell found it very difficult to raise funding for his research,
but eventually some private support was found and he was able
to set up his own independent laboratory. The government
financial support in the UK, the USA and elsewhere continued to
be given to those who believed in the ‘high-energy intermediate’,
but no matter how much work was done no scientists ever found
this compound.
Mitchell continued with his work: he understood that the build-
up of protons on one side of a membrane represented potential
energy. He also knew that the movement of these protons down
an electrochemical gradient could provide the energy needed for
the phosphorylation of ADP. This was his chemiosmosis theory.
Mitchell postulated that the energy released from electron transfer
chain was used to pump protons across the inner membrane and
that they then returned via ion channels which had enzymes
attached. He isolated the proton pumps, showed that pH changes
across the inner mitochondrial membrane could be measured and
that pH changes could be used to generate ATP by mitochondria
even in the absence of ‘food’ to oxidise. Other scientists replicated
his work –and got the same results. Eventually, scientists began
to accept his evidence for the chemiosmotic hypothesis and he
was awarded the Nobel prize for Chemistry in 1978.
In the 17 years between Mitchell’s initial theory and his
acceptance of the Nobel prize, scientists around the world were
working to test his theory. Key pieces of evidence included:
1. The outer membranes of isolated mitochondria were ruptured
by placing them in solutions of very low water potential. The
inner membranes were ruptured using detergents. This
enabled the contents of the intermembrane space and matrix
to be isolated and identified. This allowed scientists to work
out that the link reaction and Krebs cycle take place in the
matrix and the ETC takes place in the inner membrane.
2. If the stalked particles in the inner membrane of mitochondria
were removed, no ATP was generated. Thus, they must be
involved.
3. There is a strong potential difference across the inner
membrane and matrix – it is more negative on the matrix side
– suggesting that protons were being pumped from the matrix
across the inner membrane into the intermembrane space
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Chemiosmosis in chloroplasts
• During the light-dependent stage of photosynthesis, photons
of light are used to ‘excite’ electrons in the chlorophyll
molecules of photosystem ii.
• These electrons are replaced by the photolysis of water,
providing electrons (e-) and protons (H+).
• The energy released as the excited electrons pass down the
energy gradient between photosystem ii and photosystem i
is used to pump protons against their concentration gradient
across the thylakoid membrane, from the stroma of the
chloroplast to the interior of the thylakoid.
• The proton gradient is dissipated – the protons leave the
interior of the thylakoid – through a complex of a proton pore
and ATP synthase.
• As the protons pass through the complex, the energy released
is used to synthesise ATP (as in the mitochondria).
• The conversion of ADP to ATP is phosphorylation, and since
the initial energy to provide the electrons comes from the
absorption of light this process is called photophosphorylation.
Testing chemiosmosis in chloroplasts
Chloroplasts can be isolated from plant cells by homogenisation
and differential centrifugation. If isolated chloroplasts are
suspended in a neutral medium and then illuminated, the medium
becomes alkaline (i.e. pH rises). This is what would be expected
if the light-dependent reaction in the chloroplasts is removing
protons from the medium and pumping them into the thylakoids
of the chloroplasts.
Fig 5. Isolated chloroplasts
Light
pH meter
8.3
Value on pH meter
rises as H+ removed
from medium Isolated chloroplasts in
isotonic medium
If isolated chloroplasts are suspended in an acid medium (i.e.
low pH) then the interior of the thylakoids will become acidic
i.e. have a high H+ concentration.
If these thylakoids are transferred to an alkaline medium (i.e.
High pH and low H+ concentration) they can convert ADP and
P to ATP even in the absence of light. This would be expected
if a gradient of protons can be linked to the phosphorylation of
ADP.
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Practice Questions Answers

1. ATP is produced in both chloroplasts and mitochondria. 1.
The table below compares the process of ATP production in
these organelles. Complete the table with a tick (9) in the ATP production in
appropriate box if the statement is true for ATP production in
each organelle and a cross (×) if the statement is incorrect. (5) Statement Chloroplast Mitochondrion

ATP production in Electrons are excited by 9 ✗

photons
Statement Chloroplast Mitochondrion Electrons pass through 9 9
carriers
Electrons are excited by
photons
Involves oxidative 9 ✗
photophosphorylation
Electrons pass through
carriers
ATP produced from ADP 9 9
and phosphate
Involves oxidative
photophosphorylation
Occurs in day and night ✗ 9

ATP produced from ADP

and phosphate 2. (a) diffusion is the movement of molecules down a concentra-
Occurs in day and night tion gradient;
active transport is the movement against a concentration
gradient;
2. (a) Distinguish between membrane transport by diffusion and involves the expenditure of energy/ATP;
membrane transport by active transport. involves the use of carriers; max 4
Chemiosmosis is the process in which energy released when a
substance moves along a gradient is used to synthesise ATP. (b) (i) oxidative phosphorylation; 1
The diagram below illustrates this mechanism.
(ii) proton pumps;
High H+ concentration because they move hydrogen ions = protons;2

H+ (iii) enables protons/hydrogen ions to diffuse back across

H+
the membrane;
ref to proton motive force/surplus of positive ions on
inside of membrane;
inner mitochondrial movement of protons back releases energy;
membrane which is harnessed by enzyme to convert ADP + iP to
ATP; max 3
electron transport ATPase
chain carriers
ADP + P ATP

(b) (i) What name is given to the chemical reaction that

synthesises ATP? (1)
(ii) What type of pumps are the electron chain carriers?
Explain why. (2)
(iii) What are the functions of ATPase? (3)

Acknowledgements:
This Factsheet was researched and written by Ron Pickering and Kevin Byrne
Curriculum Press, Bank House, 105 King Street, Wellington, Shropshire, TF1 1NU.
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