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A-Level Biology Sixth-Form Tasters

Supercharged cells: Dr John Illingworth

Aerobic metabolism didn't appear overnight, but evolved gradually over several billion years as primitive fermentative cells adapted to the exhaustion of raw materials, and the appearance of oxygen in the earth's atmosphere.

Oxygen was the world's greatest ecological disaster. It is an immensely toxic gas, that readily generates free radicals which damage sensitive biological molecules. Even today, our bodies depend on delicate control mechanisms to deliver oxygen to our tissues in carefully regulated amounts. When free oxygen first appeared in the atmosphere, millions of primitive species either became extinct, or were confined to anaerobic niches where they were protected from its harmful effects.

This new threat was created by the blue-green algae, and subsequently by the green plants. These photosynthetic organisms progressively oxidised the earth's crust around two billion years ago. Bacteria were the first group to deal with this problem. Over hundreds of millions of years, as more and more powerful oxidants appeared in the biosphere, bacteria gradually evolved the respiratory chain, starting from NAD and NADPH at the reducing end, and creating new enzyme systems one by one until finally they could handle molecular oxygen: the ultimate killer molecule.

Our kind of cells were a bit slower off the mark. Our distant ancestors probably ate bacteria for a living. They were phagocytic cells that swallowed and digested bacteria in cytosolic vacuoles, just like our macrophages do today. Eventually some of these predators formed symbiotic partnerships with their prey, and eukaryotic cell lines were created. These were hugely successful team efforts. Our bacterial endosymbionts evolved into mitochondria that dealt with oxygen efficiently and manufactured ATP, while the larger host cell provided the physical bulk and swallowing capacity that bacteria could never emulate.

The detailed structure of eukaryotic cells still reflects their distant evolutionary origin. Each mitochondrion is still wrapped in its own membranous bag. In addition to the vital electron transport chain, mitochondria still have bacterial-type ribosomes for making proteins, and their own DNA in their own circular bacterial-type chromosome. These structures, however, are a pale shadows of their former selves. Most of the mitochondrial genome has been taken over by the cell nucleus, and mitochondria today are utterly dependent on manufactured proteins and lipids imported from the cytosol.

Energy metabolism is subject to ceaseless selection pressure. If you can swim 1% faster or further than the competition, then your progeny will inherit the earth, at least until something better comes along. If you examine the fine details of our energy-yielding pathways, then you find that every possible opportunity has been taken to increase the efficiency of the process, and to maximise the delivery of ATP. We have also evolved pollution control systems that minimise the production of toxic by-products.

The parallels between the molecular motors in living organisms, and the mechanical systems in modern vehicles are remarkable, although the components differ ten million times in size. Among the cellular machinery we can already identify emission control systems, fuel pumps, heaters, batteries, turbines, electric motors, drive shafts, gearboxes and universal joints. The major difference is that the cellular systems are self-maintaining and achieve performance levels that Ferrari can only dream of. Truly, we can speak of "supercharged cells".

In the very limited time available I can only provide a few selected examples. Rest assured that the remainder of this equipment is just as polished, and there will be many opportunities to study it in detail during your university course.

  1. Voltage gradients
  2. Active export of ATP
  3. Importing fuel supplies
  4. Gearboxes and universal joints
  5. Interior heaters and pollution control

This electron microscope picture of a chick embryo mitochondrion is used by kind permission of Professor Ruth Bellairs, Department of Anatomy and Developmental Biology, University College, Gower Street, London WC1E 6BT.

Mitochondria are usually about one thousandth of a millimetre in diameter. The outer membrane is full of holes and leaks like a collander, but the inner membrane is really tough, and is among the least permeable structures in the living world.

The Krebs cycle and related reactions take place in the mitochondrial matrix space, but carbohydrate metabolism takes place in the cytosol, outside the inner membrane barrier. Most respiratory chain enzymes are plugged right through the inner membrane, and protrude into both compartments. In metabolically active tissues the inner membrane is thrown into numerous folds, called cristae, which maximise its surface area.

1. Trans-membrane gradients:

You have already heard how ATP is manufactured by exploiting the pH and electrical gradients across the inner mitochondrial membrane to drive the rotary motion of the ATP synthase. Now I want you to consider exactly how big these transmembrane gradients must be.

   

We can measure these gradients by studying the distribution of lipid-soluble weak acids and charged ions across the inner membrane. For mammalian mitochondria the gradients are about 0.5 pH units, inside alkaline, and 150mV, inside negative. Just for comparison, an ordinary torch battery delivers about 1500mV, so with only one tenth of a torch battery to play with mitochondria don't sound anything special.

Mitochondrial motors are ten million times smaller than car engines. These modest voltages are truly amazing on a molecular scale. The insulating part of the inner mitochondrial membrane is only two molecules deep, and the central oily lipid layer that provides the insulation is approximately 5 nanometres thick. So the voltage gradient experienced by the inner membrane proteins and lipids is

150 * 10-3 volts / 5 * 10-9 metres = 30 * 10+6 volts / metre

The insulation on these tiny molecular motors is built to withstand 30 million volts / metre. This exceeds the specification for high voltage transformer oil, as used in power stations. Mitochondria achieve this formidable performance despite being continuously immersed in a warm, wet conductive salt solution. This requires some extra special insulation:

A small number of boys suffer from Barth syndrome. They have mutations on their single X chromosome in the gene coding for "taffazin", an enzyme involved in the biosynthesis of cardiolipin. Their mitochondria are clearly abnormal, and they cannot maintain normal rates of ATP production. These patients develop a life-threatening cardiomyopathy and muscle weakness. Girls have two X chromosomes, so they have a spare copy of the instructions and they do not suffer from this disease.

2. The export of ATP:

The catalytic centres of the rotary motor ATP synthase are on the inside of the mitochondrial inner membrane, but most of the cellular ATP is required outside the mitochondria, in the cytosol. This is a potential problem, because there is an electrical gradient of 30 million volts per metre across the inner mitochondrial membrane, and it isn't totally straightforward moving material from one side to the other.

A simple hole in the membrane would leak like a sieve, and make it impossible to maintain the required insulation. Instead the inner membrane contains a special protein called the adenine nucleotide translocator (ANT) that is responsible for ATP transport. This transport protein is the most active enzyme in animal cells. As well as exporting ATP it simultaneously imports ADP for recycling by the mitochondria. This swapping system is a very common arrangement for transmembrane proteins, and is called an antiporter.

However, ATP has an extra phosphate group compared with ADP, so it has an extra negative charge. The enzyme normally swaps internal ATP with 4 negative charges for external ADP with only 3 negative charges. These electrical charges don't balance, so the enzyme feels the full force from the 30 million volts per metre across the inner mitochondrial membrane. The net effect is that the adenine nucleotide translocator actively pumps ATP out of the mitochondria, while sucking up ADP from the cytosol. Contrary to expectation, there is much more ATP in the cytosol, where it is used than there is in the mitochondria where it is produced.

The breakdown of ATP into ADP is further from equilibrium in the cytosol than it is in the mitochondria. Therefore cytosolic ATP has a larger free energy of hydrolysis, or delta G, as predicted by the Gibbs equation:

The very high cytosolic ATP:ADP means that ATP is actually worth more in the cytosol, and delivers a bigger clout when it is converted into ADP. It is indeed fortunate that this should be the case, because many of the processes that we associate with eukaryotic cells, such as muscle contraction, beating cilia and cell division will only work with "supercharged" ATP.

At first sight the high voltage across the inner mitochondrial membrane looks like a problem, but evolution has exploited this situation, and instead turned it into an advantage. Eukaryotic ells go to a lot of trouble to maintain this advantage, and have even developed an elaborate shuttle system to rapidly deliver high potential ATP to the interior of contracting muscle fibres. They are always seeking that extra ounce of performance which makes the difference between eating and being eaten.

3. Importing fuel supplies:

Eukaryotic cells also exploit the mitochondrial inner membrane potential to drive the uptake of raw materials, as well as the export of finished goods. Consider, for example, the re-oxidation of cytosolic NADH (generated during aerobic glycolysis) by the mitochondrial respiratory chain. The substrate (NADH) is initially on the "wrong" side of the inner membrane, but the malate / aspartate cycle turns this problem to advantage and swiftly delivers it to the other side.

Eukaryotic cells use these pumps and shuttles to maintain distinct environments in different parts of each cell. The interior of the mitochondria forms a relatively reducing environment, whereas in the cytosol oxidising conditions prevail. This is very fortunate, because it allows the glycolytic pathway to work efficiently in the cytosol, without compromising the smooth operation of the Krebs cycle in the mitochondrial matrix space.

4. Gearboxes and universal joints:

We have already encountered one mechanical enzyme today, where an electric motor in the F0 baseplate uses the rigid driveshaft formed by the gamma subunits to power the mechanical system in the F1 head groups that makes ATP. This is by no means the only rotary system found in living cells. Several species of bacteria swim by rotating their flagella, and they even have a reverse gear!

Electron microscope picture of a Salmonella bacterium with several flagella

Some bacteria sprout flagella all over their surface, and you might imagine problems if some were pointing in the wrong direction. Not a bit. The flagella have a corkscrew shape, and they naturally organise themselves into a thick bundle if they are rotated in the correct sense. It doesn't matter if they are initially pointing in random directions, because every single one, where it emerges from the bacterial membrane, is equipped with a flexible universal joint.

The elaborate machinery at the base of each flagellum has been studied in the electron microscope. There are fluid seals and bearings where the drive shaft passes through the cell membranes. These closely resemble the corresponding structures on a motor car or boat. The motive power is derived from a series a small power units, anchored in the cell membrane and distributed around the periphery of a substantial "ring gear" on the inner end of each drive shaft.

Bacteria are very small, and on their scale of being water is not the runny liquid that comes out of our taps. Scaled down to bacterial dimensions, water is an extremely viscous fluid, and movement for bacteria is very much like swimming in treacle. Bacterial flagellar motors are obviously related to the F0 motor in mitochondria, in that they exploit the electrochemical energy stored in a transmembrane ion gradient to drive rotation. But one molecular motor on its own could not deliver sufficient force to spin a much larger flagellum at the required speed. We need a gearbox fitted with multiple drive units, and that is exactly what bacteria have evolved.

Bacteria haven't just invented an efficient motor, they can steer by switching them on and off. The entire bacterial power train is cleverly integrated into a much more complex system that can sense the composition of the environment, vary the direction of rotation, and use this to avoid toxic chemicals and actively seek out food.

5. Interior heaters and pollution control:

Normally mitochondria are about 50% efficient, with roughly half the energy available from our food being captured in the form of ATP. The remaining energy is lost as heat. This compares favourably with power stations and similar engineering feats. However there are circumstances when it is desirable to increase the heat output in order to keep warm.

New-born infants are small, wet and naked, so they rapidly lose heat to their surroundings. For most of human evolution they have not been delivered in a warm maternity ward. Babies are very good at keeping warm, because they are equipped with specialised mitochondria in their brown adipose tissue (BAT) located beneath their shoulder blades and along their backs. BAT has an excellent blood supply and is very rich in mitochondria, which are responsible for its unusual colour. Like all other kinds of adipose tissue, BAT responds to noradrenalin from the sympathetic nervous system by breaking down fat, but instead of exporting this to the rest of the body, these fats are oxidised in situ without the generation of ATP.

This is accomplished by an "uncoupling protein" called UCP1, which effectively catalyses a short circuit of protons across the inner mitochondrial membrane. Instead of generating ATP, all the energy available from the proton gradient is dissipated as heat.

It is a marvellous way of keeping warm. Our bodies can turn this heating system on and off, according to the needs of the moment. There is short term regulation via the nervous system and long term adaptation via thyroid hormone. In adults BAT plays a minor role, but if you went to work beyond the arctic circle you could soon switch it all back on again.

Scientists were surprised to discover recently that our bodies contain several other kinds of uncoupling protein UCP2, UCP3 and so forth, that have little to do with heat production. They seem to be associated with tissues that obtain much of their energy from fats, which generate a unique chemical feedstock for the respiratory chain. It appears that when these fat-fuelled mitochondria are only working slowly, they generate toxic hydroxyl and superoxide free radicals from the oxygen delivered in the blood.

This is what SHOULD happen: O2 + 4H+ + 4e- = 2H2O

but if the supply of electrons is inadequate, there is an increased risk of these side reactions:

O2 + 3H+ + 3e- = H2O + HO.   hydroxyl free radical, unpaired electron but no charge

O2 + e- = O2-   superoxide free radical with unpaired electron

Free radicals are extremely dangerous things to have around. They are highly reactive chemically and damage DNA leading to mutations and cancer. They also attack proteins and lipids, destroying their functionality and flooding the cell with toxic by-products.

Fortunately free radical production can be minimised by increasing the overall rate of electron transport via the respiratory chain. This appears to be function of UCP2 and UCP3, and in these proteins we may have stumbled on the first of nature's pollution control systems.

6. Conclusions:

Nowadays there is great interest in nanotechnology, building miniature machines using the construction techniques developed for the silicon chip. These devices however, are not necessarily new, since we find that many of the components like pumps, heaters, batteries, turbines, electric motors, gearwheels, drive shafts and universal joints have already been developed by living organisms. Many of these inventions have been in continuous use for the last two thousand million years.