Some learning objectives

Bioenergetics has traditionally been a difficult area for students, partly because of its inherent complexity, and partly because some of the theories were fiercely contested. This is less of problem today, but many people still find particular difficulty with the idea of an ion gradient as a convertible form of energy, with the resolution of the proton gradient into electrical and pH components, and with the notion that the free energy available from a chemical reaction depends on the distance from equilibrium. Make sure that you understand these key topics.

Read the bio-energetics chapters in your text-book, which should be the most recent edition. The relevant chapters from Molecular Cell Biology by Darnell et al and Molecular Biology of the Cell by Alberts et al are very good, as are Devlin, Stryer, Voet, Wood and other offerings, provided they are up to date. Revise the sections on bioenergetics, mitochondria, electron transport and oxidative phosphorylation, and the material on membrane transport systems. A good specialist account is Bioenergetics 2 by Nicholls & Ferguson (Academic Press, 1992). There are multiple copies in Leeds in the Medical School Library and in the Edward Boyle Library. Some of these copies are kept in the student loan or counter collections.

The key points are as follows:

1) Mitochondria are subcellular organelles containing the Krebs cycle, fat oxidation pathway and the respiratory chain, which produce almost all of the 70kg of ATP used each day in the human body. They can be purified from tissue homogenates by differential centrifugation, using non-penetrant isotonic media to provide osmotic support. A few tissues (e.g. red blood cells, eye lens) have no mitochondria, and cannot respire aerobically.

2) Mitochondrial respiration can be measured with an oxygen electrode. Similar equipment is used in blood gas analysers, pollution monitoring and industrial process control. How does it work, and what are the principal sources of error?

3) Understand precisely what is meant by the respiratory control index, P:O ratio and uncoupling. Calculate P:O ratios for succinate, and for glutamate + malate using intact, coupled mitochondria.

4) Several of the mitochondrial components undergo spectral changes on oxidation and reduction. These observations suggest that a series of redox carriers (arranged approximately in the order of their oxidation/reduction potentials) transport reducing equivalents (electrons or hydrogen atoms) from substrates to oxygen. This sequence of carriers is known as the respiratory chain.

5) Understand the use of specific inhibitors to delineate biochemical mechanisms. Appreciate the differences between respiratory chain inhibitors, phosphorylation inhibitors and transport inhibitors using cyanide, antimycin, rotenone, TTFA, oligomycin, mersalyl and atractyloside as examples.

6) Energy captured by the respiratory chain is not converted directly into ATP. There is instead a temporary "clearing house" known as the high energy pool. Its chemical nature was a mystery for many years, but now we know that it is an electrochemical gradient for hydrogen ions across the inner mitochondrial membrane. In human mitochondria this ion gradient or proton motive force has a large electrical component and a small pH component.

7) Understand the operation of the major transmembrane ion pumps and the ATP synthetase.

8) Lipid bilayers are exceedingly impermeable to small ions, including protons and hydroxyl ions. Transmembrane ion gradients are another form of energy, interconvertible with chemical, electrical and mechanical energy. The chemiosmotic mechanism is universal in living organisms and is also exploited by chloroplasts for photosynthesis, and by bacteria.

9) Understand the mechanism of ionophores, the distinction between electroneutral and electrical carrier mechanisms, and the importance of trans-membrane ion and potential gradients. Know the meaning of symports, antiports and electrogenic uniports. Understand where the Nernst equation applies.

10) Appreciate the use of light scattering as a crude measure of particle size and the use of passive swelling in isotonic ammonium salt solutions to demonstrate the principal transport sytems. Study the interactions between the carriers and explain the effect of phosphate on malate uptake. Why does passive swelling in sodium (but not potassium) phosphate indicate the existence of a sodium/proton anti-porter?

11) Understand why the oxidation products from individual substrates are constrained by the transport systems in the inner mitochondrial membrane, and by the availability of other co-substrates, such as acetyl-CoA.

12) The electroneutral uptake systems for small anions and cations produce relatively modest concentration gradients since they exploit only the pH component from the proton motive force, but the electrical adenine nucleotide carrier produces a large gradient for ATP since it can exploit the much larger electrical differential across the mitochondrial inner membrane.

13) The Gibbs free energy G available from a physical process or chemical reaction depends on the distance away from equilibrium. This means that ATP has a higher free energy in the cytosol than it does in the mitochondria, and this is important for many cellular activities.

14) Many metabolites are unevenly distributed across the mitochondrial membrane. Energy driven aspartate / glutamate exchange is important for keeping the cytosol highly oxidising, and preventing the aerobic synthesis of lactic acid.

15) Mitochondria probably evolved from symbiotic bacteria. They still contain a small amount of DNA, which codes for 70S (bacterial type) ribosomes, transfer RNAs and thirteen polypeptides. These are mostly the intensely hydrophobic cores of the major proton pumps. Understand the mechanism whereby hundreds of other mitochondrial proteins are imported from the cytosol. Mitochondrial DNA appears to be maternally inherited.

16) Mitochondria are involved in many disease processes, although some defects may not be detected as a result of heteroplasmy, or because the affected embryos are not viable. There is good evidence for auto-immune effects, and the mitochondrial permeability transition is thought to play an important part in apoptosis.

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