Reversed electron transport experiments lead to a realisation in the 1950s that there must be a common, non-phosphorylated high-energy intermediate between the respiratory chain carriers and the synthesis of ATP. The concept is illustrated below. The arrows show the normal direction of flow, but most of the reactions are freely reversible, and energy added to the system via one process can be utilised elsewhere. The high energy pool was originally thought to be a chemical compound, but nowadays we know that it is actually the pH and electrical gradients developed across the inner mitochondrial membrane.
It is possible to measure the gradients by studying the distribution of permeant molecules across the mitochondrial inner membrane. This requires a method for the rapid separation of mitochondria from their incubation medium, either by rapid ultrafiltration, or by centrifugation through a layer of silicone oil. An impermeable radioactive marker (commonly sucrose) is added to the incubation in order to correct for external medium contaminating the organelles.
Lipid soluble weak acids can be used to measure the pH differential. This method exploits the easy membrane permeability of the electrically neutral free acid, and the impermeability of its ionised salts. It is vital that the free acid crosses the membrane in the protonated form without net movement of electrical charge. At equilibrium the concentration of the free acid is the same everywhere, but the ratio of [free acid] to [ionised salt] differs in the two compartments. In the matrix space the amount of salt is proportional to the internal pH, but outside the mitochondria the ionised salt concentration depends on the external pH. If the internal and external salt concentrations can be measured then application of the Henderson Hasselbach equation yields the internal pH.
Measurement of the membrane potential () requires a permeant cation, e.g. a radioactive alkali metal plus valinomycin, or the lipid soluble tetraphenyl phosphonium. In this case it is important that the cation penetrates the membrane with net movement of charge, so that the cation distribution is influenced by the membrane potential. If the internal and external concentrations of a permeant cation can be measured, then the membrane potential can be calculated from the Nernst equation.
Some dyestuffs change their absorbtion or fluorescence properties when they are aligned in a strong electric field. This provides an alternative method for the continuous measurement of mitochondrial membrane potential by adding such compounds to a mitochondrial suspension. Suitable compounds include carbocyanines, phenosafranine and bisoxonols. Although it is very convenient, this technique must be calibrated using the older, single point methods.
The sum of the membrane potential and the pH gradient are together known as the proton motive force (PMF). This indicates the total potential energy stored in the transmembrane gradients, which is available to drive protons back into the matrix space, and provide the power for biologically useful processes. Using the correct sign for the pH gradient (negative!) and substituting numerical values for the gas constant, faraday constant and the absolute temperature, it follows that:
PMF (in millivolts) = - 60pH
at 37oC. The minus sign is confusing: the pH gradient is itself negative, so the two components are normally additive in their effects.
In mitochondria the electrical gradient, (150mV, inside negative) makes a larger contribution than the pH gradient (0.5pH units, inside alkaline). In chloroplasts pH is more important. The mitochondrial pH corresponds to a three-fold difference in hydrogen ion concentration between matrix and cytosol. For each transmembrane process, the pH and components may act either separately or together, depending on the enzyme structure and the balance of biological advantage. A proton movement would normally carry one positive charge, but other ions may move as well, so there is no need for the proton and charge counts to balance for any individual membrane transport process, although the totals must obviously balance overall.
The best current estimates for the proton and charge counts for the repiratory chain are shown in the section on redox carriers. For each pair of electrons to traverse the chain, NADH dehydrogenase moves 4 protons and 4 positive charges outwards across the inner membrane from matrix to cytosol, ubiquinol cytochrome c reductase moves 4 protons and 2 charges, while cytochrome oxidase moves 2 protons and 4 charges. The re-oxidation of NADH to form NAD and water is thus associated with the net export of 10 protons and 10 positve charges from the mitochondrial matrix to the cytosolic compartment.
ATP synthesis by the F1ATPase is thought to require 3 protons carrying 3 positive charges to re-enter the matrix space. In addition, 1 proton (but no charge) is needed for phosphate uptake and 1 charge (but no proton) for the ATP/ADP exchange with the cytosol (see the metabolite transport pages for details). Thus the overall requirement is 4 protons and 4 charges for each ATP delivered to the outside world. This implies that the P:O ratio is 2.5 for NAD-linked substrates, and 1.5 for succinate, which is rather lower than some older text-book values, but much closer to the actual experimental results!
The lipid bilayer in the inner membrane is only about 5nm thick and is therefore subject to enormous electrical forces:
150 x 10-3 volts across 5 x 10-9 metres = 30,000,000 volts / metre
The inner membrane has an unusual phospholipid composition which includes cardiolipin (diphosphatidylglycerol) and is particularly "leak proof" to small ions.
Most types of phospholipid will spontaneously form liposomes when agitated with water, and this has been exploited to re-constitute the respiratory chain from purified components.