by Nick Lane
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correspond to each other. This is in fact the case: the evolutionary trees of the respiratory genes that have been analysed so far do broadly correspond to the reference tree constructed from ribosomal RNA, implying that lateral gene transfer did not occur between bacteria and archaea.
The second line of evidence relates to more recent metabolic innovations such as photosynthesis. LUCA, it seems, could not photosynthesize.
No form of photosynthesis based on chlorophyll is found in any archaea.
A completely different form of photosynthesis, based on a pigment called bacteriorhodopsin, similar to the photoreceptor pigments in our eyes, is practised by the so-called halobacteria, archaea that live in high-salt conditions. This mode of photosynthesis is not found in any bacteria.
These disparate forms of photosynthesis presumably evolved independently in bacterial and archaeal lineages some time after the age of LUCA, and subsequently remained tied to their respective domains. If a metabolic innovation as important as photosynthesis did not cross from one domain to another, there is no reason to think that other forms of respiration would have done so. We should certainly be wary of postulating that respiratory genes crossed domains unless we have evidence that they did so; and the evidence from evolutionary trees suggests that they did not.
If we accept that lateral gene transfer between archaea and bacteria has been extremely rare, then the 16 respiratory genes must have been present in LUCA, and were later vertically inherited by different lines of bacteria and archaea. As these genes code for proteins involved in generating energy from a variety of compounds, including nitrate, nitrite, sulphate and sulphite, LUCA must have been a metabolically sophisticated organism. One gene in particular, however, shares a striking sequence similarity in archaea and bacteria, and it is this that Castresana and Saraste have used to paint an unexpected portrait of LUCA.
This gene codes for a metabolic enzyme called cytochrome oxidase, which transfers electrons to oxygen, to produce water, in the final step of aerobic respiration. If cytochrome oxidase was present in LUCA, then the logical, if ostensibly nonsensical, conclusion is that aerobic respiration evolved before photosynthesis. LUCA could breathe before there was any free oxygen. As Castresana and Saraste put it, no doubt relishing every word, “This evidence, that aerobic respiration may have evolved before oxygen was released to the atmosphere by photosynthetic organisms, is contrary to the textbook viewpoint.”
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The way in which cytochrome oxidase reduces oxygen is a marvel of nanoscale engineering. It receives electrons derived from the oxidation of glucose. It presses four electrons in turn, along with protons, onto an oxygen molecule to produce two molecules of water. This reaction is exactly the opposite of the water-splitting reaction of photosynthesis.
O2 + 4 e– + 4H+ 씮 2H2O
This is the single most important reaction in aerobic respiration — in effect, the combination of oxygen with hydrogen. Most people will remember the explosiveness of the reaction from school chemistry lessons. Like all reactions of oxygen, it must take place one electron at a time. The task of cytochrome oxidase is therefore a tightrope walk between harvesting the large amount of energy that can be released from this reaction, while preventing the escape of reactive free radicals. This is achieved with almost miraculous precision. In modern mitochondria, virtually no free radicals escape from cytochrome oxidase (almost all escape from other proteins in the mitochondrial chains of electron-transporting proteins). In fact, the ability of cytochrome oxidase to soak up any oxygen in the cell and transform it into water, without releasing toxic intermediates, makes it an antioxidant without equal. The fact that it also enables four times as much energy to be produced by a cell from a single molecule of glucose, compared with any other form of respiration, can almost be regarded as a bonus.
For many years, the antioxidant effect of cytochrome oxidase was cited as an explanation for its original evolution. It was logical to assume that the enzyme had first evolved in response to rising oxygen levels following the evolution of photosynthesis, and was only later pressed into service as a respiratory enzyme. This story was supported by the existence of a second (evolutionarily unrelated) form of cytochrome oxidase in some proteobacteria, including Escherichia coli and Azotobacter vinelandii. The alternative enzyme is 100 times less selective for oxygen (it does not discriminate between oxygen and similar molecules such as nitric oxide, NO) but works much faster, consuming excess oxygen very rapidly. Even more tellingly, the oxidase is only switched on when bacteria wander into high-oxygen environments, where it functions as a kind of vacuum cleaner, sucking up oxygen, or any similar molecules, that stray into its path.
We therefore have two different types of cytochrome oxidase, the activities of which vary according to the amount of oxygen in the
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vicinity. This does look very much like a defence against oxygen. The revisionist hypothesis of Castresana and Saraste thus poses a riddle. If a cytochrome oxidase was indeed present in LUCA, this protein could not have evolved in response to rising oxygen levels more than a billion years later; so why on earth did it evolve? In Chapter 7 we saw that lakes and sheltered ocean basins on the early Earth were probably under oxidative stress from ultraviolet radiation, which split water to produce oxygen free radicals and hydrogen peroxide. Antioxidant enzymes such as superoxide dismutase (SOD) are found in all three domains of life, and may well have been present in LUCA. Could cytochrome oxidase have evolved in the same way as an antioxidant defence against ultraviolet radiation, rather than rising oxygen levels?
The answer is not yet certain, but seems likely to be no. If it had evolved as an antioxidant to protect against ultraviolet radiation, then its respiratory function, where it harvests energy from the transfer of electrons to oxygen rather than simply mopping up oxygen gas, would have evolved later and independently in several different archaeal and bacterial lineages. If this had been the case, we would expect the detailed mechanism of energy-harvesting to vary in different lineages, whereas in fact it seems to be similar, suggesting descent from a common mechanism in a common ancestor.4 But if we dismiss a primordial antioxidant role for cytochrome oxidase, we are left with the rather stark alternative: that the enzyme evolved for its present purpose — to generate energy by the transfer of electrons to oxygen. Is this any more credible? We have seen that oxidative stress is feasible without oxygen, but can we really have oxygen respiration when no oxygen was present? As President Clinton might have said, that depends on exactly what you mean by ‘no oxygen’.
4 This is a testable hypothesis, but it has not been fully answered at the time of writing.
All true cytochrome oxidases generate energy from oxygen by pumping protons across a membrane. The proton gradient is then tapped to generate ATP, the energy currency of the cell. The generation of a proton gradient and its conversion into ATP is known as chemi-osmosis, and the process unifies energy generation in most forms of respiration as well as photosynthesis — another example of the fundamental unity of life. If it turns out that the cytochrome oxidases in archaea and bacteria pump protons by the same mechanism, this will be good evidence that their respiratory function had already evolved in a common ancestor. On the other hand, if the detailed mechanism of energy generation is different in archaea and bacteria, this would imply that cytochrome oxidase had evolved in LUCA for a different purpose — as an antioxidant, for example, or for denitrification (conversion of nitrates into nitrogen) — and had later adapted to oxygen metabolism independently in different lineages. Evidence to date suggests that cytochrome oxidases do pump protons in the same way in different lineages, and so had probably evolved for that purpose in LUCA.
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The term ‘anoxia’, meaning ‘no oxygen’, is surprisingly slippery and has different connotations for geologists, zoologists and micr
obiologists.
Geologists call an environment ‘aerobic’ (oxygenated) if the oxygen content exceeds about 18 per cent of present atmospheric levels, and
‘dysaerobic’ if it falls below 18 per cent. Levels lower than about 1 per cent are considered to be ‘azoic’ or ‘anoxic’. Zoologists talk about ‘normoxic’
and ‘hypoxic’ conditions, where hypoxia refers to oxygen levels that limit the rate of respiration — usually less than about 50 per cent of present atmospheric levels. The ground is even more shifting for microbiologists.
The so-called ‘Pasteur point’, at which some microbes switch from aerobic respiration to fermentation, is usually less than about 1 per cent of present atmospheric levels of oxygen. Some microbes, though, are affected by very low levels of oxygen, often much less than 0.1 per cent of present atmospheric levels. Such low oxygen levels — essentially anoxic to geologists — could have been attained on the ancient Earth by the splitting of water, particularly in shallow seas.
Remarkably, some microbes living today are able to use oxygen even at such low levels. Some species of proteobacteria, for example, are symbiotic and live inside nodules in the roots of leguminous plants. Here, in exchange for shelter and protection, they convert nitrogen from the air into the nitrates required for plant growth. The enzyme that catalyses this reaction is called nitrogenase and, crucially, its function is blocked by oxygen, even at very low levels. Leguminous plants and nitrogen-fixing bacteria are specially adapted to keep oxygen levels in the root nodules to a minimum. For example, the bacteria surround themselves with a thick capsule of mucus to restrict the entry of oxygen. If this fails, they activate an enzyme that consumes oxygen rapidly without generating any energy.
Leguminous plants also produce an oxygen-binding protein related to haemoglobin, called leghaemoglobin, which regulates the free oxygen concentration. Together, these adaptations maintain oxygen levels within the bacteria at less than 0.01 per cent of atmospheric oxygen, a concentration that does not inhibit the nitrogenase enzyme.
The remarkable fact is that, despite these adaptations to minimize oxygen levels, some of these nitrogen-fixing bacteria, such as Bradyrhizobium japonicum, are still aerobic. They possess a form of cytochrome oxidase known as FixN, which has an extremely high affinity for oxygen.
This enzyme is distantly related to mitochondrial cytochrome oxidase and probably evolved from a common ancestor. There is some evidence that the FixN oxidase is coupled functionally with leghaemoglobin,
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which only releases its bound oxygen when intracellular oxygen levels are very low. Thus, the system as a whole works as follows. Low oxygen levels are maintained by a variety of mechanisms, and any oxygen that slips through this net is bound by leghaemoglobin. At very low oxygen levels (below 0.01 per cent), leghaemoglobin relinquishes its oxygen to the FixN
oxidase, which uses it to produce energy in the form of ATP. Altogether, the system has balance and poise — it is concerned with the regulation of oxygen rather than its elimination.
The root nodule system is an extreme example of metabolism at low oxygen levels, but in conceptual terms it is very close to what actually happens inside ourselves. Our obvious dependency on oxygen conceals the fact that individual cells within internal organs are not at all adapted to bathe in an oxygen bath. The development of multicellular organisms can even be considered an antioxidant response, which has the effect of lowering oxygen levels inside individual cells. Our elegant circulatory system, which is usually presented as a means of distributing oxygen to individual cells, can be seen equally as a means of restricting, or at least regulating, oxygen delivery to the correct amount.
It’s worth dwelling on this point for a moment. Consider the following. The atmospheric pressure of dry air at sea level is about 760 mm of mercury (mmHg). Of this overall pressure, 78 per cent is contributed by nitrogen and 21 per cent by oxygen. The oxygen in the air therefore exerts a barometric pressure of about 160 mmHg. Within the lungs, oxygen is taken up by haemoglobin, which is packed tightly in the red blood cells circulating through the capillaries. In these capillaries, haemoglobin is usually 95 per cent saturated with oxygen. The pressure exerted by oxygen is about 100 mmHg. As the blood is transported around the body, haemoglobin gives up its oxygen and so the oxygen pressure begins to fall. As blood leaves the heart, the oxygen pressure has already fallen to about 85 mmHg; in the arterioles it falls further to about 70 mmHg; and in the capillary networks in our organs to about 50 mmHg. Here the saturation of haemoglobin is about 60 to 70 per cent. Oxygen dissociates from haemoglobin and diffuses into the individual cells from the capillaries down a concentration gradient. This gradient is maintained by the continuous removal of oxygen by respiration. In most cells, the oxygen pressure is approximately 1–10 mmHg. In the final stage, oxygen is sucked into the mitochondria, where active respiration lowers levels even further. The oxygen pressure inside the mitochondria is typically less than 0.5 mmHg. In percentage terms, this is less than 0.3 per cent of
Last Ancestor in an Age Before Oxygen • 167
atmospheric oxygen levels, or 0.07 per cent of total atmospheric pressure.
This is a surprisingly low figure, and not so far above the supposedly
‘anoxic’ conditions of the early Earth. Might it be that mitochondria have succeeded in preserving a ghost of times past?
There is a second point of comparison between the root nodules of leguminous plants and animal respiration, and that is the function of haemoglobin and its cousins (including the muscle protein myoglobin).
After the twists of this chapter, it may no longer come as a surprise to learn of the discovery of a haemoglobin-like protein — complete with tell-tale sequence similarities — in the archaeon Halobacterium salinarum, a finding reported in Nature in February 2000 by Shaobin Hou and his colleagues at the University of Honolulu in Hawaii. The great antiquity of haemoglobins and myoglobins is not in itself a surprise; a haemoglobin-like sequence has been found in bacteria. The significance of Hou’s finding is that haemoglobin-like molecules may have been present in LUCA.
Why should haemoglobin, the oxygen-transporting protein in the blood stream of animals, be found in LUCA, or for that matter in any other single-celled organism? The answer is clear enough if we change our perspective: we should not think of haemoglobin as an oxygen transporter, but rather as a molecule that regulates oxygen storage and supply.
This is essentially how leghaemoglobin works in the root-nodule system
— it maintains intracellular oxygen at a low level, releasing its own oxygen on demand. This is also how myoglobin works, the protein responsible for the red colour of muscles. Myoglobin is similar in structure to one of the subunits of haemoglobin, and has a greater affinity for oxygen than haemoglobin. This means that myoglobin in muscles can draw in oxygen from the blood stream and store it in the muscles until needed. Whales and other diving mammals have very large muscular stores of myoglobin, which can pool large quantities of oxygen, allowing them to remain under water without breathing for an hour at a time. The level of free oxygen in their muscles remains constant and low.
The same system is at work in single cells. Their haemoglobin-like proteins store, and then release, oxygen to maintain a constant and low intracellular concentration suitable for respiration. This regulatory role is emphasized by the findings of Hou and his colleagues. The haemoglobin-like molecule that they discovered in Halobacterium salinarum functions as an oxygen sensor, which enables the archaeon to sense oxygen levels and to migrate towards zones with ideal oxygen concentrations. Some bacteria also employ oxygen sensors in this way. The common denomina-
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tor in all these settings is the ability of haemoglobin-like molecules to maintain intracellular oxygen at an appropriate level.
From this point of view, the arguments of Castresana and Saraste, that LUCA could breathe oxygen, begin to seem reasonable. She wouldn’t have
needed much oxygen — a barely detectable amount — and she could probably store her own to use when required. If this is true, then many descendants of LUCA presumably lost the ability to use oxygen to generate energy as they adapted to specialized niches. Others lost the ability to metabolize sulphites or nitrites. The eukaryotes must have evolved from a lineage that had lost the genes for most respiratory proteins, including cytochrome oxidase, before regaining some of them from the purple bacteria that ultimately evolved into mitochondria. The unrecognizable relics of these defunct eukaryotic genes might still make up a part of our junk DNA.5 But the most startling point is that LUCA herself could use oxygen to generate energy nearly 4 billion years ago. She clearly knew how to cope with oxygen, and probably made use of a haemoglobin-like protein and antioxidant enzymes such as SOD to protect herself. The notion that antioxidants appeared later as a desperate response to rising oxygen levels in some kind of oxygen holocaust, is falsified once again, this time from a genetic point of view.
The story we have prised from the genes is inevitably speculative, but is backed up by intriguing and coherent evidence. The conclusion that LUCA was metabolically versatile solves a number of paradoxes that cannot easily be explained by the old textbook version of events — in particular, the evolution of photosynthesis, and the antiquity of haemoglobin and aerobic respiration. If the outlines of the story are valid, then conventional wisdom has been turned on its head at almost every point.