MB ChB Year 1: Nutrition and Energy
This lecture includes several challenging concepts that defeated some of the best brains in science for almost 50 years. For this reason these notes are more detailed than the other lectures, not because we want you to study the topic in tremendous detail, but because we want you to understand what is taking place. Please don't try to rote learn complex chemical structures: it is easier to teach using accurate diagrams, but this doesn't mean that you have to learn every tiny detail. We will point out any structual features that you really ought to know. Look for the parallels with other areas, especially nerve, muscle and red cell function which provided much of the inspiration that enabled this problem to be solved.
Mitochondria can be prepared from tissue homogenates by centrifuging for a few minutes at about 10,000g. It is important to chelate calcium ions and to provide osmotic support. The preparation is pale pink, showing the presence of haem proteins and flavoproteins within the organelles. The spectrum has three strong haem absorption bands labeled a, b and c.
The haem compounds were called cytochromes, because they give much of the colour to isolated cells. Here is the spectrum of cytochrome c. The absorption pattern for all three cytochromes changes markedly when these molecules are oxidised and reduced. This was a great assistance to early investigators.
Redox potentials indicate whether a compound is a good oxidising or a good reducing agent. Biological values range from about -400mV (NADH) to +800mV (oxygen). Most cytochromes have redox potentials towards the oxygen end of the scale.
In addition to the cytochromes a, b and c, mitochondria also contain copper, non-haem iron proteins, flavoproteins and ubiquinone, all of which participate in the transport of electrons from substrates to oxygen. Mitochondria, bacteria and plant chloroplasts have many features in common, and seem to be constructed to a similar plan.
Non-haem iron proteins (also called iron-sulphur proteins) contain one or more iron atoms complexed with multiple cysteine (amino acid) residues in the protein. There are dozens of different varieties and they occur very widely in all living organisms: bacteria, plants and animals. Some non-haem iron proteins are very complicated. Their redox potentials depend greatly on the protein structure, but in general they have fairly negative values, comparable with NAD and FAD, towards the substrate end of the redox scale.
Ubiquinone is also very widely distributed in nature. It is the ubiquitous quinone. Plant chloroplasts contain a similar compound called plastoquinone. Quinones have redox potentials around zero volts - this is about the middle of the redox scale.
The long isoprene side chain attached to the quinone functional group makes these compounds almost insoluble in water. They are confined to the phospholipid bilayer of the mitochondrial inner membrane. All their reactions take place in a non-aqueous environment.
Intact mitochondria incubated with respiratory substrates and inorganic phosphate take up oxygen very slowly in the absence of ADP.
Mitochondrial respiration can be studied in oxygraph experiments. An oxygraph is a small thermostatted chamber fitted with an adjustable stopper and an oxygen electrode. The experimenter can make small additions through the stopper with a syringe.
"Clarke type" oxygen electrodes are widely used in medicine for blood gas analysers and foetal monitors. They can measure gas mixtures or dissolved oxygen in liquids, but it is necessary to control the temperature and the stirring speed. Oxygen diffuses through a thin plastic membrane and is reduced to water at a platinum cathode. The circuit is completed by the gradual oxidation of a silver anode to form silver chloride. Clarke electrodes pass a minute electrical current that is directly proportional to the oxygen concentration, so the amount of oxygen left in the chamber can be continuously recorded. The details are shown in the diagram below. Please don't feel that you have to learn all these details, but it may be of interest to know how it works.
Addition of ADP to mitochondria incubated with substrates and phosphate causes a rapid burst of respiration, which continues until all the ADP has been converted into ATP. This is called coupled respiration: oxygen consumption is coupled to the manufacture of ATP
Even when they are provided with inorganic phosphate and an oxidisable substrate, intact mitochondria respire relatively slowly in the absence of ADP. However the addition of ADP initiates a sudden burst of vigorous respiration [DO2 on the diagram below] and the ADP is rapidly phosphorylated to produce ATP. When all the ADP has been used up, the respiration rate returns towards it original value, although it is usually slightly faster than before.
This result astonished and delighted the early students of mitchondria, since the organelles achieve a remarkable chemical feat: the progress of a favourable chemical reaction (the oxidation of substrates) is constrained, and coupled to the performance of an unfavourable chemical reaction (manufacture of ATP) that would not occur spontaneously on its own. If the mitochondria are mechanically damaged then this coupling is easily lost.
The ADP effect on respiration can be repeated until all the oxygen has gone. The amount of extra oxygen consumed during each burst is proportional to the amount of ADP added. The P:O ratio is about 2.5 for NAD-linked substrates, or 1.5 for succinate.
P:O ratios are the number of moles of ADP converted into ATP per atom of oxygen reduced to water. Succinate isn't such a good reducing agent as pyruvate and the other Krebs cycle intermediates, so there is less energy released when it is oxidised. A lower yield of ATP seems reasonable in the circumstances and suggests that succinate joins the oxidation system after the first tranche of ATP has been creamed off.
For historical reasons, the active rate in the presence of ADP is often called "state 3 respiration" and the slower rate when all the ADP has been phosphorylated is referred to as "state 4". The ratio state 3 : state 4 is called the respiratory control index. This ratio varies with the substrate and the experimental details however it should be at least 5 if the isolated mitochondria are in reasonable condition. It is normally much larger than 5 for the undamaged mitochondria inside living cells, but it also varies in vivo.
A small group of compounds called uncoupling agents cause unrestrained oxygen uptake in the absence of ADP. None of the energy released during oxidation is captured, it is all dissipated as heat. Mechanical or other damage to mitochondria also causes uncoupling.
Uncoupled respiration proceeds at maximum speed until all the oxygen has gone. The original uncoupling agent was the compound dinitrophenol, which at one stage was marketed as a slimming pill until several people had been killed by it. A related uncoupling agent dinitro ortho cresol (DNOC) is still used as an insecticide today. Both the above compounds are relatively poor uncouplers, but there is a group of carbonyl cyanide phenylhydrazones that are effective at micromolar concentrations.
Many compounds inhibit mitochondrial respiration. There are associated spectral changes as mitochondrial respiratory components become oxidised or reduced. The precise pattern of inhibition can be very revealing and differs from one compound to the next.
Investigators soon realised that electrons flowed from Krebs cycle substrates to oxygen by jumping from one respiratory carrier to another, and that the various components must be arranged in a more or less linear sequence. In general, those carriers which are good reducing agents are found at the substrate end of the chain, while the best oxidising agents are found at the oxygen end of the chain. The fact that many carriers changed colour on oxidation and reduction was a considerable help. In general, addition of an inhibitor caused reduction of all the "upstream" components on the substrate side of the block, and oxidation of all the "downstream" components on the oxygen side. With several inhibitors available, it is easy to deduce how the carriers are arranged.
Important inhibitors are cyanide, antimycin, rotenone and TTFA, all of which block different locations in the electron transport chain. This allowed the sequence of carriers to be determined. This group are not affected by uncoupling agents.
Cyanide blocks respiration with all substrates, whether ADP is added or not. Most respiratory components show their fully reduced spectra, except for haem a which looks odd. This suggests that cyanide inhibits haem a, very near the oxygen end of a respiratory chain.
Cytochrome a is also known as "cytochrome oxidase" because it reacts with oxygen. It has a couple of copper atoms in the protein structure as well as two haems. It is inhibited by cyanide and by carbon monoxide - this is why these substances are poisonous. All the cytochromes function towards the 'oxygen' end of the electron transport chain.
Antimycin A blocks respiration with all substrates, EXCEPT for the artificial combination of ascorbate + TMPD (tetramethyl phenylenediamine) which passes electrons via cytochrome c. Cytochromes a and c show their oxidised spectra, but all the other components are reduced. This suggests that antimycin blocks on the substrate side of cytochrome c.
Ubiquinone, or coenzyme Q (CoQ) functions near the middle of the respiratory chain. It is reduced to ubiquinol without any change in its visible absorption spectrum, so it was discovered some time after the brightly coloured components.
Rotenone (an organic insecticide isolated from powdered derris roots) blocks respiration with all NAD-linked substrates, but leaves succinate oxidation intact. All the cytochromes show their oxidised spectra, suggesting that rotenone blocks near the substrate end of the chain.
More powerful reducing agents are needed to carry electrons at the 'substrate' end of the chain: flavin adenine dinucleotide (FAD) in the case of succinate, and flavin mononucleotide (FMN) for most of the other substrates that are oxidised using NAD and NADH. [These are usually called NAD-linked substrates.] The flavins were discovered at an early stage, because like the cytochromes they obligingly change colour when they are oxidised and reduced.
TTFA (thenoyl trifluoroacetone, an industrial chemical) blocks succinate oxidation, but leaves the oxidation of NAD-linked substrates intact. This indicates that the chain branches into separate channels on the substrate side of the antimycin block at cytochrome b.
None of the above inhibitors are affected by uncoupling agents, because they all act directly on the electron transport chain.
Most of the respiratory chain carriers are organised into four big multi-enzyme complexes that are embedded in the mitochondrial inner membrane. They penetrate the membrane like rivets and stick out on both sides of the lipid sheet into the aqueous phase. There is more protein than lipid in this membrane, and it may help to visualise the system as a rubblestone wall, where the irregular blocks of stone represent the proteins, and the lipids are like the mortar which seals the gaps. As far as we can tell these complexes are not tethered, but drift around slowly in the membrane, like rafts of pack-ice grinding against their neighbours in a three-dimensional sea. It would be difficult for such large objects to interact efficiently with each other for electron transfer, however ubiquinone and cytochrome c are two small, highly mobile carriers that ferry electrons from one complex to the next.
Complex 1: or NADH dehydrogenase is one of the largest macromolecular assemblies in the cell. It is bigger than a ribosome, and transfers electrons from NADH in the matrix space to ubiquinone (CoQ) dissolved in the membrane lipids. The products are ubiquinol which remains within the inner membrane, and NAD which is re-used for further rounds of mitochondrial substrate oxidation within the matrix space. Complex 1 contains FMN and at least four different kinds of non-haem iron. It can be inhibited by the natural product rotenone, which is isolated from ground-up derris roots and is much in vogue as an 'organic' garden insecticide.
Complex 2: or succinate dehydrogenase transfers electrons from succinate to ubiquinone. It is one of the eight enzymes that catalyse the Krebs cycle, as well as being physically part of the electron transport chain. The products are fumarate which is released into the matrix space, and ubiquinol which remains in the membrane. Complex 2 is blocked by TTFA. It contains FAD and another four varieties of non-haem iron.
In addition, several other major flavoproteins such as acyl-CoA dehydrogenase (the first step in fatty acyl CoA oxidation) and minor pathways such as glycerol phosphate oxidase also feed electrons directly or indirectly into ubiquinone.
Complex 3: or cytochrome c reductase transfers electrons from ubquinol within the membrane to cytochrome c, located in the inter-membrane space. By this point the respiratory chain has traversed the full thickness of the mitochondrial inner membrane, but in the next stage it will double back on itself to finish where it started in the matrix space. Complex 3 can be inhibited by the antibiotic antimycin A. It contains non-haem iron, two kinds of cytochrome b, and a high molecular weight version of cytochrome c, called cytochrome c1.
Complex 4: or cytochrome oxidase transfers electrons from cytochrome c in the inter-membrane space to oxygen, forming water in the matrix space. This means that electrons must re-traverse the inner membrane. The functional unit in complex 4 contains two molecules of haem and two atoms of copper, so in total it can hold up to four electrons, and accomplish the complete reduction of one oxygen molecule to water in a single step. It is important that this should happen cleanly: partial reduction of oxygen generates dangerous molecules such as superoxide radicals and hydrogen peroxide which are highly toxic to cells.
Oligomycin blocks the normal burst of respiration in response to ADP, but in this case uncoupling agents can still stimulate respiration. This suggests that oligomycin blocks ATP synthase, which is separate from the electron transport chain.
The inhibitors fall into two classes: those like cyanide are effective under all circumstances, while others such as oligomycin are relieved by uncoupling agents. In this second case (oligomycin + uncoupler) respiration proceeds rapidly, but without the manufacture of ATP. This gave rise to the concept of two independent systems that are normally coupled together: a respiration system that transfers electrons from substrates to oxygen, and a phosphorylation system that makes ATP. Providing that these two processes are tightly coupled together it makes no difference where an inhibitor acts, but if you break the connection with an uncoupling agent, only respiratory inhibitors will be effective.
A bicycle provides a simple analogy. When the equipment is intact, operating the pedals turns the rear wheel, and if you stuck a spanner through the spokes, or through the crank wheel, matters would come to a rapid halt. However, if you uncoupled pedalling from movement by removing the bicycle chain, then a spanner through the spokes would no longer have any effect on the operation of the crank, which would turn uselessly until the rider was exhausted.
In mitochondria blocked with antimycin at cytochrome b, energy from ATP hydrolysis can drive reverse electron transport, pumping electrons backwards up the chain, from succinate, which is a poor reducing agent, to good reducing agents like NADH. This showed that there was a common respiratory intermediate (coenzyme Q) between succinate and NADH on the substrate side of cytochrome b.
Reverse electron transport is sensitive to oligomycin when driven by ATP. However it can also be driven by energy captured from cytochrome c oxidation, in which case ATP is not needed and oligomycin has no effect. Either way, the process is sensitive to uncouplers. Therefore a common high energy intermediate, sensitive to uncouplers, exists between the respiratory chain and the manufacture of ATP.
Attempts to find a chemical intermediate were unsuccessful, and Peter Mitchell suggested that it was a proton and electrical gradient across the mitochondrial inner membrane.
The mitochondrial chemiosmotic coupling mechanism was first proposed by Peter Mitchell, who was awarded the Nobel Prize for Chemistry in 1978. Mitchell did much of his research in a converted farmhouse on Bodmin Moor with his colleague, Jennifer Moyle. They found it difficult to obtain funding for research equipment, and they were sometimes dependent on "hand me downs" from workers within the university system. Mitchell published his most significant paper privately after it was rejected by conventional scientific journals. Mitchell's theory did not merely explain the workings of mitochondria, it also accounted for bacterial energy metabolism and photosynthesis in green plants. Three major problems solved at the same time! Not bad for an amateur scientist working in a farmhouse on Bodmin Moor.
The respiratory carriers are organised into multi-enzyme complexes which are plugged right through the mitochondrial inner membrane, drifting like protein icebergs in a lipid sea. As electrons jump from one carrier to the next, these complexes pump positively-charged protons across the inner membrane from the mitochondrial matrix into the intermembrane space.
cytosol / intermembrane space
mitochondrial matrix space
Mitchell's theory has undergone considerable enhancement since it was first proposed in 1961. In its current form, respiratory complexes 1, 3 and 4 (but NOT complex 2) pump protons from the mitochondrial matrix into the intermembrane space as electrons flow from NADH to oxygen. There is another proton pump called the energy-linked transhydrogenase, which need not concern us here. Protons are acidic and positively charged, and these pumps create a pH differential of about 0.5 pH units and a voltage gradient of about 150mV across the inner mitochondrial membrane, with the inside alkaline and negatively charged.
This H+ pumping creates both pH and electrical gradients across the inner membrane, which have been measured. The pH component is small, ~0.5pH units, inside alkaline. The electrical gradient is enormous: more than 150mV (inside –ve) or 30,000,000 volts per metre across the phospholipid bilayer between the mitochondrial matrix and the intermembrane space. The inner membrane lipids are among the best electrical insulators known.
We can calculate these pH and potential gradients by studying the distribution of lipophilic drugs and synthetic permeant cations across the inner membrane. The concepts are identical to those encountered in the ADME of drugs and electrophysiology: the Henderson - Hasselbach and the Nernst equations apply. This is, however, an artificial situation engineered with radioactive tracers in order to measure these gradients. In contrast to the plasmalemma, which is often permeable to potassium and chloride ions, under normal circumstances there are no permeant ions distributed in equilibrium with the mitochondrial membrane potential. The voltage across the plasmalemma has a fundamentally different origin from the voltage across the inner mitochondial membrane. The potential at the plasmalemma arises from the differing sodium and potassium concentrations inside and outside the cell, and the differing passive membrane permeabilities for these two ions. The mitochondrial membrane potential arises because the enzymes in the respiratory chain have actively transported positively charged protons from the matrix into the inter-membrane space.
As well as supplying energy for the manufacture of ATP, these ionic gradients can drive the movement of charged molecules across the inner membrane. Cells separately exploit the pH differential to move inorganic phosphate and Krebs cycle acids across the inner membrane, and the voltage gradient to export ATP for ADP. A few compounds, such as glutamate and aspartate use both components. Cations can also be transported in this way.
Re-visit the material in Bioenergetics  for a discussion of these ion transport systems. Their existence and likely properties were predicted by Mitchell, and their subsequent discovery was an early indication that his theories were correct.
The immediate success of Mitchell's theory was to explain the action of uncouplers, which had previously appeared to be a rather disparate group of chemicals, without any common theme. It became apparent that they were all lipid-soluble weak acids, that shared the addional feature of a delocalised negative charge on the uncoupler anion which allowed both the ionised and the protonated forms to enter the hydrophobic lipid phase.
This is particularly obvious for the potent uncoupler meta chloro carbonylcyanide phenylhydrazone [CCCP]. This molecule can diffuse from the intermembrane space towards the matrix space in the protonated form, helping to eliminate the pH differential as it does so. Being uncharged, it is unaffected by the voltage difference between the two compartments. On arrival it releases its proton to become a CCCP lipid-soluble anion which immediately experiences an electrical field of 30 million volts per metre directed towards the outside world. The anion re-crosses the membrane rather quickly! As it does so, it helps to discharge the electrical gradient. Each CCCP molecule can repeat this trick thousands of times per second, leading to the complete dissipation of both the electrical and the pH gradients in a very short space of time.
In the absence of ADP, transmembrane gradients increase to the point where further proton expulsion becomes impossible and electron transport grinds to a halt. Uncoupling agents carry protons back across the membrane, collapsing the gradients and allowing respiration to restart without manufacturing any ATP.
150mV may not sound very much, since it is only one tenth of the voltage available from a single torch battery. It is important to see it in molecular terms. The inner membrane lipid is only about 5 nanometres thick, so that the voltage gradient experienced by each of these proton pumps is about 30 million volts per metre. Pumping protons is hard work. When respiration grinds to a halt in the absence of ADP, it is because the respiratory complexes cannot physically force any more protons from the depleted matrix compartment into the overcrowded cytosol and intermembrane space. The proton gradient stores chemical energy which is ultimately used to drive the synthesis of ATP.
ATP synthase is a large enzyme which can be visualised in the electron microscope as stalked particles attached to the inner face of the inner membrane. Each head group (F1) has a stalk and a protein base piece (F0) extending right across the inner membrane, which allows protons to return in a controlled fashion to the matrix space.
ATP synthase is also known as the F1/F0 ATPase because it is a reversible enzyme and was first identified through its ATPase activity. The F1 head group has 3 α and 3 β subunits in a hexagonal arrangement. Although there are six subunits, there are only three active centres in each head group. Part of the F0 component is able to rotate within the membrane. It is connected to the g subunit, which is a miniature protein drive shaft threaded through the middle of the complex. The drive shaft is not straight, but has a bulge in it, so as it turns it distorts the structure of the F1 subunit. In addition to the drive shaft, there is a protein bracket (coloured green on the cartoon) which prevents the head groups rotating.
Protein subunits within each F0 base piece are forced to rotate, like a water wheel or turbine, as protons re-enter the matrix space. Driven by the huge transmembrane gradient, these molecular motors generate a considerable turning force, which is applied via delicate protein drive shafts to the F1 head groups bordering the matrix space.
Each F1 head group has three active centres, which work 120 degrees out of phase. ADP and phosphate bind to one active centre, forming ATP, which binds extremely tightly to the protein. The energy needed for ATP formation comes from the tight binding to the active site. Rotation of the drive shaft induces a conformational change in the protein and forces the reluctant enzyme to release its product to the outside world.
While one of the three active centres is binding ADP, the second is forming ATP and the third is being forced to let go. Each complete rotation of the drive shaft requires 9 protons to re-enter the matrix space, and leads to the release of 3 molecules of ATP. The tiny protein bracket holds the F1 head groups steady, and prevents them spinning uselessly on the drive shaft instead of making ATP.
The cartoon on the right turns quite slowly to illustrate the mechanism, but in life the g subunit that forms the drive shaft (denoted by the black triangle) rotates about 100 times per second. Billions of these machines turn ceaselessly within our cells, in order to synthesise the 50-100kg of ATP that the average person uses each day. All the major living species (plants, animals and bacteria) are dependent on this protein. As far as we can tell, it is the most active enzyme in the universe.
Despite its considerable intricacy, the F1/F0 ATPase (or ATP synthase, as it is sometimes called) was one of the first enzymes to be perfected by living cells. It has scarcely changed in the last 2,000 million years, and the enzyme in E. coli is not so very different from the enzyme in humans. It used to be said that evolution had never invented the wheel, and it was a surprise to discover that we have been using them all the time.
It is not sufficient to manufacture ATP in the matrix space, since it must finally be exported to the cytosol and ADP recovered in exchange. Mitochondria also accumulate phosphate from the surrounding medium to support the production of ATP. These activities are catalysed by the inner membrane transport proteins described in the previous lecture. The metabolite carriers are driven by the transmembrane proton gradient, in addition to the synthesis of ATP.
The charge imbalance associated with the adenine nucleotide carrier leads to a large difference in the ATP/ADP ratio between matrix space and cytosol. Mitochondria scavenge almost all the ADP from the cytosol, and exploit the huge membrane potential to expel their ATP against a concentration gradient. ATP:ADP is high in the cytosol and low in the mitochondria. ATP is effectively "worth more" in the cytosol, because the ATP:ADP couple is maintained further away from equilibrium.
Overall, three protons re-enter the matrix to lever each ATP from the F1 enzyme, and another is required to drive phosphate uptake and ATP/ADP exchange. Ten protons are exported for each pair of electrons that traverse the chain, giving an overall P:O ratio of 2.5 for NADH.
There is a final twist to this account. Since each proton bears a positive charge, you might imagine that the number of protons and the number of positive charges must always balance for each of these numerous proton pumps. It ain't necessarily so. When four protons cross the membrane in complex 3, two electrons travel with them, heading for cytochrome c. This pump therefore carries four protons but only two charges. Conversely, cytochrome oxidase pumps only two protons out of the matrix space, but two electrons move the other way into the matrix space, so this carrier exports two protons but four positive charges. The phosphate carrier is an electroneutral porter and its hydroxyl counter ion costs the equivalent of one proton, but zero charges. Conversely the adenine nucleotide carrier is an electrical carrier that transports no protons but one electrical charge.
We are now in a position to draw up a detailed proton balance sheet for the manufacture of each ATP:
It is apparent that each pair of electrons that travels from NADH to oxygen promotes the export of ten protons, and that four protons must re-enter the matrix space to deliver each finished ATP. Thus each NADH will support the manufacture of 2.5 ATP molecules, as is actually observed. Succinate, however, whose electrons bypass complex 1, promotes the export of only six protons, sufficient for only 1.5 ATP molecules, again as actually observed.
The reaction is freely reversible, so ATP hydrolysis can spin the drive shafts and turbines backwards, pumping protons from the matrix into the inter-membrane space. This may have been its original function, to create the proton and electrical gradients necessary for substrate uptake by anaerobic bacteria before there was any oxygen to breathe.
The F1/F0 ATPase acts as proton pump when it hydrolyses ATP, and drives 3 protons from the matrix space to the intermembrane space for each ATP that was split. The respiratory chain pumps, however, can out-perform the F1/F0 ATPase, and they create such a powerful proton gradient across the inner mitochondrial membrane that they normally drive the ATPase in the opposite direction, forcing it to synthesise ATP instead of breaking it down.
The precise reaction mechanisms of the respiratory chain pumps have still to be established, but we are now certain that the F1/F0 ATPase operates as a rotary proton pump. The amino acid sequences and X-ray structures were solved by John Walker's group in Cambridge, confirming an earlier theory by Paul Boyer about the way the enzyme worked. This resulted in the award of a second Nobel prize in 1997. In the same year Noji et al in Japan devised an ingenious method to visualise the enzyme while it was working. Click here to see the moving parts rotate.
As green plants oxidised the biosphere 2500 million years ago, respiratory chain components evolved one by one, to exploit the better oxidants that were gradually becoming available.
Sequential development enables us to understand how such a complex system could evolve. The early atmosphere had no oxygen and simple fermentative organisms used proton gradients to scavenge substrates from their environment. Very modest gradients are initially sufficient for this purpose, but as the food supplies were exhausted, there was selection pressure for bigger gradients, and better pumping systems. The F1/F0 ATPase evolved in this environment. An alternative redox based proton pump, possibly involving the energy linked transhydrogenase, may also have been in use.
After photosynthesis started about 3500 million years ago, it probably took about 1000 million years to oxidise all the metallic iron and metal sulphides on the earth's surface. Only then would free oxygen appear in the environment. In the meantime, other electron acceptors such as sulphate ions may have been employed, as they are still used by anaerobic bacteria today. As better oxidants became available there was plenty of time for each respiratory complex to be added one by one.
ATP synthase is almost identical in plants, animals and bacteria. It evolved very early in our planet’s history, was quickly perfected and has changed very little since. Every living thing has depended on it for over 3000 million years: it is among the oldest, the most important and the most active enzymes in the universe.
This is an ancient, strictly aerobic process that makes 95% of our ATP.
There is cooperation and division of labour between cellular compartments (e.g. cytosol / peroxisomes / mitochondria).
Complex molecules are broken down to simpler compounds en route to the mitochondria (e.g. in peroxisomes).
A wide range of precursors eventually funnel into the Krebs cycle.
The Krebs cycle takes place within the mitochondrial matrix space.
The Krebs cycle mostly converts NAD+ into NADH (75% of total output).
The respiratory chain uses oxygen to recycle NADH back to NAD+.
Respiratory chain complexes are plugged through the mitochondrial inner membrane.
Respiratory chain complexes 1, 3 and 4 obligately pump protons from the matrix space.
There is a large “proton gradient” across the mitochondrial inner membrane.
The interior of the mitochondria is alkaline and negatively charged.
There is a huge inner membrane potential (maximum 180mV = 36 million volts / metre).
ATP synthase is also plugged through the inner mitochondrial membrane.
Protons re-enter the matrix space by spinning the central core of ATP synthase.
The rotating core forces ATP synthase to release the new ATP it has made.
The inner membrane potential also helps to pump ATP out into the cytosol.
ATP is “worth more” in the cytosol because it is further from equilibrium.
The mitochondrial proton gradient pumps other molecules as well as ATP.
These pumps maintain distinct conditions within each cellular compartment.
Mitochondrial compartmentation is destroyed during the apoptotic cascade.
Mitochondria have their own "bacterial type" DNA, RNA and ribosomes.
Mitochondria probably began as symbiotic bacteria 2 billion years ago.
Only 13 protein-coding genes remain in the human mitochondrial genome.
Our mitochondrial genes code for intensely hydrophobic membrane proteins.
All the other mitochondrial genes have been moved to the cell nucleus.
Mitochondrial genes show maternal inheritance because eggs are much bigger than sperm.
Mitochondrial mutations often show heteroplasmy.
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