• 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.
Synthesis of ATP in mitochondria and chloroplasts
Active transport of, e.g. K + by cells
H + H +
H +
Energy input
(e.g. oxidation of food)
H +
Proton gradient across membrane
= potential energy proton flow
Transfer of DNA during bacterial conjugation and genetic transformation
Movement of bacterial flagella
Production of heat
The production of ATP is endogonic (energy-requiring) (Fig 2).
O
_
O P O
O
ADP
O
_
P O
_
O
+ O
O
_
P OH
O
Phosphate
+ H + energy
O
_
O P O
O
O
_
P O
O
ATP
O
_
P O
_
O
The energy required to combine adenosine diphosphate and phosphate can be supplied by a proton gradient: this proton gradient is set up by an electron transport chain which uses a series of oxidation-reduction reactions to provide the energy to ‘pump’ protons across a membrane. The combination of the gradient of proton concentration and the gradient of charge i.e. the complete electrochemical gradient provides the proton motive force (PMF).
The proton gradient is dissipated by allowing protons to pass through a pore in the membrane, and the movement of protons drives the production of ATP by the enzyme
ATP synthase (
Fig 3).
Energy
(oxidation of food or light absorbed by chlorophyll)
ATP synthase
H +
H +
PUMP
H +
H +
H + H +
H +
H + H + membrane is almost completely impermeable to protons
H + H +
Proton pumped across membrane generate PMF
(proton motive force)
1

223. Chemiosmosis Bio Factsheet www.curriculum-press.co.uk
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. Fe ii /fe iii 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
.
inner membrane outer membrane crista matrix intermembrane
ATP synthase protein structure
Inner membrane stator proton channel
Electron carrier enzyme each enzyme is associated with a cofactor that contains an iron atom
1
2
Electrons passed along (from carrier
2e enzyme to carrier enzyme)
Hydrogen atoms from
Krebs cycle and Link reaction carried by
NAD & FAD matrix
2
-
+ 2e -
2H + innermembrane folded into crista = larger surface area base piece
2e -
3
2e -
Fe
2e -
Fe Fe
2e -
H -
H -
2e -
Fe
H -
H -
5
H -
H -
H -
4
H intermembrane space
2e -
3
6
Intermembrane space
2e -
ATP
2e -
2H +
+ ½O
2
→
H
2
O head piece stalk/axle
Matrix
ADP + P i
ATP synthase enzyme
Proton channel inner membrane
4
Build up of protons in the intermembrane
space 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
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 buildup 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
Bio Factsheet www.curriculum-press.co.uk
• 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
.
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.
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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223. Chemiosmosis Bio Factsheet www.curriculum-press.co.uk
1. ATP is produced in both chloroplasts and mitochondria.
The table below compares the process of ATP production in these organelles. Complete the table with a tick (
9
) in the appropriate box if the statement is true for ATP production in each organelle and a cross (
×
) if the statement is incorrect. (5)
1.
ATP production in
Statement Chloroplast Mitochondrion
Statement
Electrons are excited by photons
Electrons pass through carriers
Involves oxidative photophosphorylation
ATP produced from ADP and phosphate
Occurs in day and night
H
+
High H + concentration
H +
ATP production in
Chloroplast Mitochondrion inner mitochondrial membrane
Electrons are excited by photons
Electrons pass through carriers
Involves oxidative photophosphorylation
ATP produced from ADP and phosphate
Occurs in day and night
9
9
9
9
✗
✗
9
✗
9
9
2. (a) Distinguish between membrane transport by diffusion and membrane transport by active transport.
Chemiosmosis is the process in which energy released when a substance moves along a gradient is used to synthesise ATP.
The diagram below illustrates this mechanism.
2. (a) diffusion is the movement of molecules down a concentration gradient; active transport is the movement against a concentration gradient; involves the expenditure of energy/ATP; involves the use of carriers; max 4
(b) (i) oxidative phosphorylation;
1
(ii) proton pumps; because they move hydrogen ions = protons;
2
(iii) enables protons/hydrogen ions to diffuse back across the membrane; ref to proton motive force/surplus of positive ions on inside of membrane; movement of protons back releases energy; which is harnessed by enzyme to convert ADP + iP to
ATP; max 3 electron transport chain carriers
ADP + P
ATPase
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.
Bio Factsheets may be copied free of charge by teaching staff or students, provided that their school is a registered subscriber. No part of these Factsheets may be reproduced, stored in a retrieval system, or transmitted, in any other form or by any other means, without the prior permission of the publisher. ISSN 1351-5136
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