Biological free energy and ATP

The previous lecture described how the processes of life can therefore be considered as the gathering, storage and manipulation of free energy. This lecture introduces the ways in which the main types of biological free energy are interconverted and describes the role of ATP and ATP-synthase (Slide 1).

2.1 A membrane-based mechanism

2.1.1 Chemiosmotic coupling

There is a very general and efficient mechanism by which cells convert energy from light and from food into immediately usable energy stored in ATP – this mechanism is based on membranes (see Slides 11-13 of Lecture 1 in this topic). The energy transduction mechanism is called chemiosmotic coupling – because it links the chemical synthesis of ATP to transport of protons through the membrane (Slide 2).

Slide 2: Images bottom left and bottom right copyright © 2002 from Molecular Biology of the Cell by Alberts et al. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.

A capacitor stores energy as it stores charge. Energy obtained from light harvesting and from respiration – oxidation of food – is used to pump protons across a membrane, and the energy released by allowing them back is used to synthesise ATP. For prokaryotes (cells without nuclei – bacteria and archaea) this is the plasma membrane that surrounds the cell. For eukaryotes (cells with nuclei – including plants, animals, fungi) ATP generation takes place in intracellular organelles – mitochondria and (in plants) chloroplasts – that contain folded membranes with very large areas. We will concentrate on eukaryotic systems for the rest of the lecture.

Free energy is handled by chains of coupled chemical reactions or processes, each of which passes free energy from one form to another. Many of the reactions are called ‘electron transport’ reactions, because they can be described in terms of the transfer of electrons between molecules with different electron affinity. In the lower part of Slide 2, free energy is represented by height from the bottom of the slide. Electrons gain free energy from absorption of light or chemical break-down of food, which transfers them to “high-energy” molecules such as NADH (see later, Slide 5) or the very similar NADPH


In mitochondria (Slide 2, lower left), and in the plasma membrane of most prokaryotes, degradation of carbohydrates (producing CO2) drives the formation of high-energy covalent bonds, for example in NADH, one of the products of the citric acid cycle shown in Slide 2, lower left. High-energy molecules like NADH transport electrons to a system of membrane-bound protein complexes that forms an electron transport chain. The free energy released as the electrons move to states with ever lower potential energy is used to pump protons across the membrane. At the end of the chain, the electrons are transferred to molecular oxygen which reacts with protons to produce water. The proton gradient created by the proton pumps (pmf) is used to drive a rotary molecular machine called ATP synthase (see later, Slides 6-9) which uses the energy released by discharging the capacitor to synthesise ATP.

For further discussion see the Link lecture ‘ATP Synthase’.


Chloroplasts (Slide 2, lower right) and some photosynthetic bacteria also have membrane-bound electron transport chains, but the free energy input that generates high-energy electrons is from light absorbed by molecules of chlorophyll, and the electrons are taken from water to generate oxygen. As well as pumping protons, the electron transport chain in chloroplasts is also used to synthesise carbohydrate from CO2 in the carbon fixation cycle (also known as the Calvin cycle), which is functionally very similar to the citric acid cycle but in reverse.

The chemistry driven by light in chloroplasts is thus almost the reverse of that in mitochondria. Most of the proteins and small molecules used in electron transport in chloroplasts are different from their counterparts in mitochondria– for example NADPH replaces NADH, they are the same except for an extra phosphate attached to NADPH. This ensures that respiration and photosynthesis can be independently controlled in cells that perform both processes.

Also see ‘Biological Energy’ Lectures 3 and 4.

2.1.2 Generating pmf in respiration

The mitochondrion (Slide 3) has an outer membrane which is permeable to ions and small molecules, and a highly folded inner membrane where ATP generation takes place. The inner membrane is impermeable to ions but contains transport proteins that make it selectively permeable to molecules used as a source of energy and waste products from their oxidation.

Slide 3 images courtesy of Daniel S Friend, taken from Molecular Biology of the Cell by Alberts et al. copyright 2002 Garland Science/Taylor & Francis Books, Inc.

The images on the right are obtained by electron tomography – they are reconstructed from a series of transmission electron micrographs of the same structure in different orientations. Slides 4-8 give a little more detail on electron transport reactions and their role in respiration

2.1.3 Chemical free energies in bioenergetics

Glucose is an important energy-storage molecule (Slide 4). (See also Biological Molecules Lecture 5 ‘Sugars, polysaccharides, lipids and fats’.) In animals, glucose is the ‘energy currency’ of the organism in the same way as ATP is the energy currency of the cell – stored as glycogen and transported in the blood to the organs/tissues where it is to be used by cells. Gradual release of the free energy of glucose in many steps allows it to be used for many processes before eventual release as heat. As with all biological free energy transduction, this is an isothermal process – molecular machines are not heat engines, and heat production (usually) wastes energy.

Slide 4 image bottom right copyright © 2002 from Molecular Biology of the Cell by Alberts et al. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.

Conversion of glucose to ATP in cells proceeds in three stages:

1. Glycolysis

2. Proton pumping

3. ATP synthesis

1 and 2 are electron-transport reactions. 2 and 3 together are called oxidative phosphorylation because phosphate is added to ADP and oxygen is used up. 3 is performed by a rotary molecular machine, ATP-synthase.

Other types of energy-storage molecule are:

Poly-saccharides: e.g. glycogen in the liver, stocks last about half a day

Fat: Long-term energy storage

Protein: In extreme conditions (starvation)

The initial stage of the breakdown of a molecule of glucose (glycolysis) generates two molecules of ATP, two molecules of NADH (another energy-storage compound) and two molecules of pyruvate. These reactions take place outside the mitochondria and, without mitochondria, pyruvate is a waste product. However, the mitochondria are able to produce about 30 molecules of ATP per molecule of glucose by further processing. In the inner space of the mitochondria (the ‘matrix’) pyruvate is broken down in the citric acid cycle to produce CO2, and the energy released is captured by making high-energy chemical bonds in ‘energy carrier’ molecules – for example, NADH. See Slide 5. Details of glycolysis and the citric acid cycle can be found in many textbooks, including those by Nelson and by Alberts et al listed above.

Slide 5 images adapted from Molecular Biology of the Cell by Alberts et al copyright © 2002. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.

NAD+ is nicotinamide adenine dinucleotide (there is no need to remember this – you can always look it up). It can carry two high-energy electrons together with a proton – being converted to NADH or reduced nicotinamide adenine dinucleotide. This is an example of a redox reaction – the substrate is oxidised (loses electrons) and NAD+ is reduced (gains electrons) [mnemonic: oil rig – oxidation is loss, reduction is gain].

The reduction of NAD+ by transfer of two electrons plus a proton (together these comprise a hydride ion, H-, drawn red and yellow in Slide 5) from another molecule in the citric acid cycle is shown in the first part of Slide 5, top left.

The next image to appear (right) shows the details of how the hydride ion is carried by NAD+, converting it to NADH. NAD+ has a more stable electronic configuration (the π electrons in the nicotinamide ring are delocalised) so NADH stores free energy, i.e. it carries “high energy” electrons.

When the above electron transfers are reversed, NADH gives its electrons and proton, along with the free energy, to another molecule (NADH is oxidized, the other molecule is reduced). These processes can be thought of as the storage of free energy in the form of high energy electrons in NADH, as illustrated in the last image to appear in Slide 5, lower left. The two different representations in this image of the electrons plus proton, either separate or combined into a hydride ion, are entirely equivalent. In the mitochondrion, NADH gives up its electrons to the beginning of the electron transport chain in the membrane (see later, Slide 7).

The free energy changes in redox reactions (those which can be understood in terms of electron transfer between molecules with different electron affinity) can be quantified in terms of the redox potentials of the constituent half-reactions. The redox potential of a half-reaction is the equivalent voltage at which electrons reside in the electron accepting molecule, relative to electrons in a hydrogen molecule, H2, which serves as a common reference.

Slide 6 shows how redox potentials can be measured directly. Electrons will pile onto the wire on the left and off from the wire on the right, until the voltmeter reading is equal to the redox potential of the half reaction in the left-hand solution (-0.315 V in this example). At this point, the chemical free energy reduction for an electron leaving the ‘high-energy’ NADH, minus the chemical free energy for an electron leaving the ‘neutral energy’ H2, is balanced by the electrostatic energy gain for the same electrons jumping onto the wire at a negative potential in the NADH beaker. By definition the wire in the H2 beaker is at zero volts, so there is no electrostatic energy associated with electrons jumping between that wire and H2 molecules. The salt bridge makes sure the two solutions are at the same potential.

Slide 6 image copyright © 2002 from Molecular Biology of the Cell by Alberts et al. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.

Standard redox potentials are tabulated and can be looked up. The free energy change for a full bioenergetic reaction can be calculated by adding together redox potentials in the constituent half-reactions – cancelling the reference H2 molecules – to give the redox potential for the full reaction. Multiplying this by the electronic charge transferred in the full reaction ( -e times the number of electrons transferred, where e is the unit charge) gives the standard free energy change. Actual free energy changes can then be calculated according to Equations 7-13 from Lecture 1 of this topic (see Lecture 1 Slide 5).

2.1.4 Electron and proton transport chains

A high-energy electron released by NADH is passed from molecule to molecule along a chain of more than 15 different electron carriers in a respiratory chain(Slide 7 shows a simplified schematic view of this process). Most of these carriers are metal ions bound within proteins – these membrane proteins have evolved such that the progress of the electron is driven by a progressive loss of free energy as it moves to carriers with ever increasing electron affinity.

Slide 7 image copyright © 2002 from Molecular Biology of the Cell by Alberts et al. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.

There are three large respiratory enzyme complexes in the respiratory chain (NADH dehydrogenase complex, cytochrome b-c1 complex and cytocrhome oxidase complex). The electron loses a significant fraction of its total change in energy within these complexes. This energy is used to pump protons across the membrane, storing energy by doing work against the proton motive force. Electrons are carried between these complexes by smaller carriers – ubiquinone (a small molecule) and cytochrome c (a small protein). Electron carriers, both mobile carriers and parts of larger complexes, bind to each other very specifically through non-covalent interactions at precisely matching docking surfaces, allowing electrons to tunnel over distances of the order of 2 nm from one bound ion to the next.

At the end of the chain electrons are passed to molecular oxygen, which has a very high electron affinity, to generate water. The net result of the whole complex process – glycolysis, citric acid cycle and respiratory chain – is that food is oxidised to CO2 and water, and the energy released is captured by pumping protons across the membrane against an electrochemical potential gradient. By ensuring that energy is lost gradually, this system ensures that most of the free energy change of the reaction is stored in the membrane capacitor and is available to drive useful processes such as chemical synthesis.

Protons are highly mobile in water – they can hop as defects through the network of hydrogen bonds. Protons can move in the same way along a chain of hydrogen bonds within a proton-pumping protein (Slide 8).

Slide 8 images copyright © 2002 from Molecular Biology of the Cell by Alberts et al. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.

Protons can participate in electron transport. When a molecule is reduced by acquiring an electron its negative charge can be neutralised by picking up a proton from water – the net result is addition of a hydrogen atom. Similarly, oxidation of a molecule can result in removal of a hydrogen atom as a proton passed to water and an electron transferred to the oxidising molecule.

One way to couple electron transport to proton pumping is to ensure that the proton is picked up from and returned to solution on different sides of the membrane.

2.2 Generating ATP from pmf

For more detail on the material in this section see the link lecture ‘ATP synthase’.

Most ATP is made using the free energy of pmf by a remarkable and relatively well-understood molecular machine called F1FO ATP-synthase. (Slide 9) (Note that the subscript is letter O not zero – a historical accident.)

This machine generates ATP from pmf by using two rotary motors back-to-back on a common shaft, one running on each free energy source. Under normal conditions the pmf motor (FO) generates more torque, so the ATP motor (F1) runs backwards as a generator. In the diagram on the right, the common rotor is shown in white, the common stator in blue.

The stator is anchored by transmembrane a-subunits which are connected by an arm (b-subunits) to three α- and three β-subunits that form the head in the matrix (inner space of the mitochondrion). The shaft or ‘stalk’, the γ subunit, rotates within the 3-fold symmetric α3β3 complex and is connected to a rotor formed from 9-15 c-subunits that span the membrane.

(Note: there is some confusion in the naming of the subunits – eukaryotic and prokaryotic ATP-synthases use slightly different subunit names. However, except for δ- and ε-, all subunits named here and shown in Slide 9 have the same name in eukaryotic and prokaryotic ATP-synthases.)

Slide 10 is an animated movie of ATP synthase in action. The FOF1 system runs at approximately 150 Hz. Rotation of the rotor is coupled to transport of protons through the membrane, into the inner space of the mitochondrion, down the electrochemical gradient established by respiration.

Slide 10: ATP synthase in action. (Movie © Medical Research Council, reproduced by kind permission of John Walker.)

Slide 11 shows the mechanism of ATP synthesis in F1. There are three catalytic sites at interfaces between α and β subunits. As the γ stalk rotates the α- and β-subunits are distorted, changing the conformation of the catalytic sites and providing the energy necessary for ATP synthesis. The movie is based on X-ray crystallographic structures of the components of the complex.

Slide 11: Catalytic cycle: ATP synthesis in F1. (Movie © Medical Research Council, reproduced by kind permission of John Walker.)

This motion can be simplified to a cycle of three states of each catalytic site:

‘open’ (empty) – low affinity for ATP or ADP;

‘loose binding’ – high affinity for ADP, Pi, which diffuse in and are bound

‘tight binding’ – high affinity for the product ATP – in this environment ATP synthesis releases free energy;

return to open, releasing the product. Each site performs the same cycle, 120° out of phase with the other sites.

During this cycle the free energy required for ATP synthesis is provided through the mechanical distortions driven by the FO motor.

With each rotation three molecules of ADP are reduced to ATP in F1, and it is reasonable to assume that one proton per c-subunit passes through the membrane in the 9-15 fold symmetric FO. This gives a non-integer, and variable, ratio of ATP molecules generated to protons used, which is tailored to the prevailing conditions in the particular organism, in order to ensure synthesis (rather than proton pumping by ATP hydrolysis) while wasting the minimum of energy as heat. N.B. Non-integer stoichiometry is possible because of the mechanism of mechanical coupling in ATP-synthase.

Bacterial F-ATPase is usually reversible (can run backwards, hydrolysing ATP and pumping protons), eukaryotic F-ATPase is not (it is specifically inhibited from hydrolysis).

The movies in Slides 10 and 11 are included by kind permission of John Walker. For more animations of ATP-synthase in action see:

In Slide 12 F1 has been separated from the FO proton motor and is being driven backwards by ATP hydrolysis. A fluorescently labelled actin filament has been attached to the stalk and is observed by fluorescence microscopy. This is direct experimental evidence that the F1 ATPase really is a rotary molecular machine.

Movie from slide 12: Direct observation of rotation of F1 ATPase. (Movie used by permission of Wolfgang Junge.)

This and similar single-molecule experiments have made F1-ATPase one of the best understood of all molecular machines. (Also see the lecture on ATP synthase.) In 1997 Paul D Boyer and John E Walker shared a Chemistry Nobel Prize for elucidating the structure and function of FOF1 ATP synthase.

The movie in Slide 12 is included by kind permission of Wolfgang Junge. For more movies and animations of ATP-synthase rotation see:

The conversion of free energy from glucose to ATP takes place in many small steps, as described in slides 4-12, and is very efficient. Compare Slide 13 to the schematic diagram in Slide 4. (Units of energy are kBT.)

2.3 Generating pmf in photosynthesis

Here we give a brief introduction to photosynthesis. Subsequent lectures in this topic will provide more detail.

2.3.1 Chloroplasts and mitochondria

The energy stored in the food that is broken down to generate ATP through the proton motive force ultimately originates from light. Photosynthesis is the process of generating food (organic carbon) from water and CO2. Light harvesting (Slide 14) is the first part of photosynthesis – it occurs in membranes within specialist organelles in plants (chloroplasts) and in the cell membranes of photosynthetic bacteria.

Slide 15 compares chloroplasts and mitochondria, and shows the chloroplasts membrane structure.

Slide 15 left-hand image courtesy of K. Plaskitt, both images from Alberts et al. Molecular Biology of the Cell, reproduced by permission of Garland Science/Taylor & Francis Books, Inc. © 2002.

Common features are:

the matrix/stroma is where ATP is accumulated and used to make other things;

ATP synthase is very abundant;

Protons are pumped out of the matrix/stroma by electron transport.

Differences are:

chloroplasts have an extra compartment, within the thylakoid membranes;

in chloroplasts light is used to pump protons into the thylakoids, in mitochondria respiration pumps protons into the inter-membrane space.

2.3.2 Photosynthesis

Chloroplasts, like mitochondria, have membrane-bound electron transport chains, but, as shown in Slide 16, the free energy input that generates high-energy electrons is from light absorbed by molecules of chlorophyll, and the electrons are taken from water to generate oxygen and are used to synthesise carbohydrate from CO2. Slide 16 should be compared to Slide 7, the equivalent electron transport chain in respiration.

Slide 16 image copyright © 2002 from Molecular Biology of the Cell by Alberts et al. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.

There are two photochemical reaction centres which contain many molecules of chlorophyll arranged to create efficient light harvesting antennae. Excitation of any of these molecules leads to a transfer of energy by Förster (or fluorescence) resonant energy transfer (FRET) to a reaction centre. FRET is a near-field, non-radiative process by which the oscillating dipole of the excited chromophore excites a neighbouring chromophore – the matrix element for this process falls off (as the dipole field) as r-3, so the transfer rate scales as r-6. (FRET is exploited in techniques that probe motion on a molecular level. See Molecular Machines Lecture 2 ‘Single Molecule Methods’.)

Light generates a neutral excitation – but in the first reaction centre (called photosystem II) this is separated into a high energy electron, which enters an electron transport chain, and a hole which is used up in the process of splitting water:

1 photon → e + h (hole)

4h + 2H2O → O2 + 4H+

The electron transport chain pumps protons across the membrane, as in the respiratory chain. In photosystem I each electron is given a further boost in energy from an absorbed photon so that it has enough potential energy to reduce a molecule of NADP+ to NADPH that, with ATP, can be used for the carbon fixation reactions that create carbohydrate from CO2 and water. NADP+ / NADPH is an energy/electron storage system that is identical to NAD+/NADH, except for the addition of a phosphate group on one of the ribose rings that allows enzymes to distinguish the two systems. Alternatively, electrons can be cycled through photosystem I and back through the proton pump (cytochrome b6-f complex) to pump more protons across the membrane and generate more ATP.

The electron-transport chains illustrated in Slides 7 and 16 can also be represented as cascades of electron free energy (see Slide 17), similar to the schematics of Slides 2 and 4.

Slide 17 left-hand image courtesy of K.N. Ferreira, both images from Alberts et al. 2008 Molecular Biology of the Cell, reproduced by permission of Garland Science/Taylor & Francis Books, Inc. © 2008.

In photosynthesis electrons are stripped from water and deposited eventually on NADPH, using the free energy input of two photons. (Some bacteria have a simpler photosynthetic electron transport chain that uses only one photon and cannot strip electrons from water). In respiration electrons pass from NADH to water, with no free energy input from light.