by Nick Lane
The chemiosmotic force is a fundamental property of life, perhaps more ancient than DNA, RNA, and proteins. In the beginning, naturally chemiosmotic cells might have formed from microscopic bubbles of iron-sulphur minerals, that coalesced in the mixing zone of fluids seeping up from deep in the crust, and the oceans above. Such mineral cells share some properties with living cells, and their formation needs no more than the oxidizing power of the sun—there is no call for complicated evolutionary innovations before the origin of hereditary replication through DNA. Chemiosmotic cells conduct electrons across their surface, and the current draws protons over the membrane to generate an electric charge across the membrane—a force field around the cell. This membrane charge links the spatial dimensions of the cell to the very fabric of life. All life, from the simplest bacteria to humans, still generates its energy by pumping protons across membranes, then harnessing the gradient to tasks such as motility, ATP production, heat production and the absorption of essential molecules. The few exceptions merely go to prove this general rule.
In cells today, electrons are conducted by the specialized proteins of the respiratory chains, which use the current to pump protons across the membrane. The electrons are derived from food, and pass down the respiratory chains to react with oxygen, or other molecules serving exactly the same purpose. All organisms need to control the flow of electrons down the respiratory chains. Too fast a flow fritters away energy wastefully, while too slow a flow can’t match demand. The respiratory chains behave like slightly cracked drainpipes—a clear flow presents no problems, but any blockage, either at the outflow or somewhere in the middle, is likely to spring a leak through the cracks. If blocked, the chains leak electrons, and these react to form free radicals. There are just a handful of possible reasons for electron flow to block, and only a few ways to restore the flow, yet the balance between power-generation, on one hand, and free-radical formation on the other—the same problem I faced in my kidneys—has written some of the most important, if unsung, rules in biology.
First among the reasons for a blockage of electron flow is some kind of defect in the physical integrity of the respiratory chains. The chains are assembled from a large number of protein subunits, which form into large functional complexes. In eukaryotic cells, genes in the nucleus encode most of the subunits, and genes in the mitochondria encode a small number. The continued existence of mitochondrial genes in all cells containing mitochondria is a paradox, for there are many good reasons to transfer them all to the nucleus, and no obvious physical reasons why this could not have been achieved, at least in some species. The most likely reason for their persistence is a selective advantage to retaining them in the mitochondria, and this advantage seems to be related to energy generation. So, for example, an insufficient number of complexes in the second part of the respiratory chains would block electron flow, leading to a backlog of electrons in the earlier part, and free-radical leakage. In principle, the mitochondria could detect the free-radical leakage, and correct the problem by signalling to the genes to make good the deficit—to produce more complexes for the second part of the chains.
The outcome depends on the location of the genes. If the genes are in the nucleus, the cell has no means of distinguishing between different mitochondria, some of which need new complexes, and many of which don’t: none are satisfied by the bureaucratic one-size-fits-all response of the nucleus. The cell loses control over energy generation, a grave penalty. Only if a small contingent of genes is retained in each mitochondrion, to code for the core protein subunits of the respiratory chains, can energy generation be controlled in a large number of mitochondria simultaneously. The additional subunits, encoded in the nucleus, fit themselves around the core mitochondrial subunits, using them as a beacon and a scaffold for construction.
The consequences of this system are profound. Bacteria pump protons across their external cell membrane, and so their size is limited by geometrical constraints: energy production slopes off with a falling surface-area-to-volume ratio. In contrast, eukaryotes internalize energy generation in mitochondria, and this frees them from the constraints facing bacteria. The difference explains why bacteria remained morphologically simple cells, while the eukaryotes were able to grow to tens of thousands of times the size, accumulated thousands of times more DNA, and developed true multicellular complexity, surely the greatest watersheds in all of life. But why did bacteria never succeed in internalizing their own energy generation? Because only endosymbiosis—a mutual, stable collaboration between partners living one inside another—is able to leave the right contingent of genes in place; and endosymbiosis is not common in bacteria. The precise concatenation of circumstances that forged the eukaryotic cell seems to have happened just once in the entire history of life on earth.
Mitochondria inverted the world of bacteria. Once cells had the ability to control energy generation across a large area of internal membranes, they could grow as large as they liked, within limits set by the distribution networks. Not only could they grow larger, but they also had good reason to do so—energetic efficiency improves with larger size in cells and multicellular organisms, just as it does in human societies, following the economies of scale. There is immediate payback for larger size—lower net production costs. The tendency of eukaryotic cells to become larger and more complex can be explained by this simple fact. The link between size and complexity is an unexpected one. Large cells almost always have a large nucleus, which ensures balanced growth through the cell cycle. But large nuclei are packed with more DNA, which provides the raw material for more genes, and so greater complexity. Unlike bacteria, which were obliged to remain small, and to jettison superfluous genes at the first opportunity, the eukaryotes became battleships—large, complicated cells with lots of DNA and genes, and as much energy as needed (and no longer any need for a cell wall). These traits made a new way of life possible, predation, in which the prey is engulfed and digested internally, a step that the bacteria never took. Without mitochondria, nature would never have been red in tooth and claw.
If the complex eukaryotic cell could only be formed by endosymbiosis, the consequences of two cells living together in mutual dependency were equally significant. Metabolic harmony may have been the rule, but there were important exceptions, and these too are attributable to the dynamics of the respiratory chain. The second reason for a block in electron flow is a lack of demand. If there is no consumption of ATP, then electron flow ceases. ATP is needed for replication of cells and DNA, and for protein and lipid synthesis—indeed, most housekeeping tasks. But demand is greatest when cells divide. Then the entire fabric of the cell must be duplicated. The dream of every living cell is to become two cells, and this applies as much to the erstwhile free-living mitochondria as to the host cells in the eukaryotic merger. If the host cell becomes genetically damaged, so that it can’t divide, then the mitochondria are trapped inside their crippled host, for they are no longer able to survive independently. And if the host cell can’t divide, it has little use for ATP. Electron flow slows down, and the chains become blocked and leak free radicals. This time the problem can’t be resolved by building new respiratory complexes, so the mitochondria electrocute their hosts from inside with a burst of free radicals.
This simple scenario lies at the roots of two major developments in life—sex, and the origin of multicellular individuals, in which all cells in the body share a common purpose and dance to the same tune.
Sex is an enigma. Various explanations have been put forward, but none explains the primal urge of eukaryotic cells to fuse together, as do the sperm and the egg, despite the costs and dangers of doing so. Bacteria don’t fuse together in this way, even though they do routinely recombine genes by lateral gene transfer, which apparently serves a similar purpose to sex. Bacteria and simple eukaryotes are often stimulated to recombine genes by various forms of physical stress, all of which involve free-radical formation. A burst of free radicals can be sufficient to induce a rudi
mentary form of sex, and in organisms like the green algae, Volvox, the free-radical signal for sex can come from the respiratory chains. In the early eukaryotic cells, mitochondria might have manipulated their hosts to fuse together and recombine their genes whenever the hosts were genetically damaged, and unable to divide by themselves. The host cell benefits, because the recombination of genes can fix or mask genetic damage, while the mitochondria themselves gain access to pastures new without killing their existing host, essential for their safe passage.
Sex may have benefited both the mitochondria and their hosts in single-celled organisms, but it no longer did in multicellular individuals. The gratuitous fusion of cells is a liability when the cells belong to an organized body, in which all constituent cells must share a common purpose. Now the same free-radical signal for sex betrays genetic damage to the host cell, which pays the penalty of death. This mechanism seems to be at the root of apoptosis, or programmed cell suicide, which is necessary for policing the integrity of multicellular individuals. Without the death penalty for cellular insurrection, multicellular colonies could never have developed the unity of purpose characteristic of the true individual—they would have been torn asunder by the selfish wars of cancer. Today, apoptosis is controlled by the mitochondria, using the same signals and machinery that they had once used to plead for sex. Much of this machinery was originally brought to the eukaryotic merger by the mitochondria. While the regulation of apoptosis is now, of course, far more complicated, at its heart the critical signal is still a burst of free radicals from a blocked respiratory chain, leading to depolarization of the mitochondrial inner membrane, and the release of cytochrome c and other ‘death’ proteins into the cell. Even today, it takes no more: injecting damaged mitochondria into a healthy cell is enough for that cell to kill itself.
There are ways of modulating electron flow down the respiratory chains, so these dire penalties don’t happen whenever electron flow temporarily comes to a halt. The most important is to uncouple the chains (so the passage of electrons is not tied to the formation of ATP). Uncoupling is usually achieved by making the membrane more permeable to protons, so their passage back across the membrane is not strictly through an ATPase (the enzyme ‘motor’ that is responsible for generating ATP). The effect is akin to the overflow channels in a hydroelectric dam, which prevent flooding at times of low demand. The continuous circulation of protons allows a continuous passage of electrons down the respiratory chains, without regard for ‘need’, and this prevents the accumulation of electrons in the respiratory chains and so restricts free-radical leakage. But the dissipation of the proton gradient necessarily generates heat, and this too has been put to good use over evolution. In most mitochondria, about a quarter of the proton-motive force is dissipated as heat. When enough mitochondria are assembled, as in the tissues of mammals and birds, the heat generated is sufficient to maintain a high internal temperature, regardless of the external temperature. The origin of endothermy, or true warm-bloodedness, in birds and mammals can be ascribed to such dissipation of the proton gradient, which later made possible the colonization of the temperate and frigid regions, as well as an active nightlife. It released our ancestors from the tyranny of circumstance.
The balance between heat generation and ATP production still affects our health in surprising ways. Uncoupling of the respiratory chain is restricted in the tropics, because too much internal heat production would be detrimental in a hot climate: we could very easily overheat and die. However, this means that the ‘overflow channels’ are partially sealed off, so more free radicals are generated at rest, especially on a high-fat diet. This makes Africans eating a fatty western diet more vulnerable to conditions such as heart disease and diabetes, which are linked with free-radical damage. Conversely, the Inuit, who have a low incidence of such diseases, dissipate the proton gradient to generate extra internal heat in the frozen north. Accordingly, they have a relatively low free-radical leakage at rest and are less vulnerable to degenerative diseases. On the other hand, dissipating energy as heat is counterproductive in sperm, which depend on the energetic efficiency of a small number of mitochondria to power their swimming. This gives the Arctic peoples a potentially higher risk of male infertility.
In all of these circumstances, free radicals are the signal for change. The respiratory chains act like a thermostat: if free-radical leakage rises, one of several mechanisms cuts in to lower their level again, then switches itself off, just as the fluctuations in temperature switch the boiler on and off in a thermostat. In the case of the respiratory chains, free radicals are almost certainly detected in concert with other indicators of the overall ‘health status’ of the cell, such as ATP levels. So a rise in free-radical leakage set against falling ATP levels within one mitochondrion is the signal to build new subunits for the respiratory chains; if ATP levels are high, free radicals are the signal for greater uncoupling, or perhaps for sex in unicellular eukaryotes; and a sustained, uncorrectable rise in free-radical leakage set against falling cellular ATP levels is the signal for cell death in multicellular individuals. In each case, fluctuations in free-radical leakage are as indispensable to the feed-back loop as are the temperature fluctuations to a thermostat: free radicals are vital to life, and attempting to get rid of them, for example using antioxidants, is folly. This simple fact has forced two other major innovations on life: the origin of two sexes, and the decline and fall of organisms into ageing and death.
Free radicals are reactive and cause damage and mutations, especially to the adjacent mitochondrial DNA. In lower eukaryotes, like yeast, mitochondrial DNA acquires mutations approximately 100 000 times faster than nuclear genes. Yeasts can sustain such a high rate because they don’t depend on mitochondria to generate energy. The mutation rate is far lower in the higher eukaryotes, such as humans, because we do depend on our mitochondria. Mutations in mitochondrial DNA cause serious diseases, and tend to be eliminated by natural selection. Even so, the long-term evolution rate of mitochondrial genes, over thousands or millions of years, is between 10 and 20 times faster than nuclear genes. What’s more, the nuclear genes are reshuffled every generation to give a new hand of genes. These disparate patterns set up a serious strain. The subunits of the respiratory chain are encoded by both nuclear and mitochondrial genes, and to function properly must interact with nanoscopic precision: any changes in gene sequence might change the structure or function of the subunits, and could potentially block electron flow. The only way to guarantee efficient energy generation is to match a single set of mitochondrial genes with a single set of nuclear genes in a cell, and test-drive the combination. If it crashes, the combination is eliminated; if it drives well, the cell is selected as a feasible progenitor for the next generation. But how does a cell select a single set of mitochondrial genes to test against one set of nuclear genes? Simple: it inherits its mitochondria from just one of the two parents. As a result, one parent specializes to pass on the mitochondria, in the large egg cell, whereas the other parent specializes to pass on no mitochondria—which is why sperm are so small, and why their handful of mitochondria are usually destroyed. Thus, the origin and deepest biological distinction between the two sexes, indeed the main reason for having two sexes at all, rather than none or an infinite number, relates to the passage of mitochondria from one generation to the next.
A similar problem occurs during adult life. This is the basis of ageing and the related degenerative diseases that all too often eclipse our twilight years. Mitochondria accumulate mutations through use, especially in active tissues, and these gradually undermine the metabolic capacity of the tissue. Ultimately, cells can only boost their failing energy supply by producing more mitochondria. As the supply of mint mitochondria dries up, cells are obliged to clone genetically damaged mitochondria. Cells that amplify seriously damaged mitochondria face an energy crisis and take the honourable exit—they commit apoptosis. Because damaged cells are eliminated, mitochondrial mutations don’t build up in agei
ng tissues, but the tissue itself gradually loses mass and function, and the remaining healthy cells are under a greater pressure to meet their demands. Any additional stresses, such as nuclear gene mutations, smoking, infections, and so on, are more likely to push cells over the threshold into apoptosis.
Mitochondria calibrate the overall risk of apoptosis, which rises with age. A genetic defect that causes little stress to a young cell causes far more stress to an old cell, simply because the old cell is by now closer to its apoptotic threshold. Age, however, is not measured in years, but in free-radical leakage. Species that leak free radicals quickly, such as rats, live for a few years and succumb to age-related diseases within this brief timeframe. Species that leak free radicals slowly, like birds, may live ten times as long and succumb to degenerative diseases over this long timeframe, although they often die of other causes (such as crash landings) before these diseases set in. Critically, birds (and bats) live longer without sacrificing their ‘pace of life’—their metabolic rate is similar to mammals that live but a tenth as long. The same mutations in nuclear genes cause the same age-related diseases in different species, but the rate at which they progress varies by orders of magnitude—and tallies with the underlying rate of free-radical leakage. It follows that the best way to cure, or at least postpone, the diseases of old age is to restrict free-radical leakage from the respiratory chains. This approach has the potential to cure all diseases of old age at once, rather than trying to tackle each independently, a tack that has so far failed to deliver a really meaningful clinical breakthrough, and is perhaps destined never to do so.
In sum, mitochondria have shaped our lives, and the world we inhabit, in ways that defy belief. All these evolutionary innovations stem from a handful of rules guiding the passage of electrons down the respiratory chains. Remarkably, we can elucidate all this after two billion years of intimate adaptations. We can do so because, despite their changes, mitochondria have retained distinctive imprints of their heritage. These clues have enabled us to trace the outlines of the story that we have followed in this book. The story is grander, more monumental, than any researcher could ever have guessed until recently. It is not the story of an unusual symbiosis, nor the tale of biological power, the industrial revolution of life. No, it is the story of life itself, not merely on Earth, but anywhere in the universe, for the morals of this story relate to the operating system that governs the evolution of all forms of complex life.