by Nick Lane
The explanatory power of proton power
Mitchell’s hypothesis neatly solved the nagging difficulties that dogged the older theories. It explained why a membrane was necessary, and why it had to be intact—a leaky membrane would allow a drizzle of protons back through, and so dissipate the proton-motive force as heat. A porous dam is no use to anyone.
It also explained how the mysterious uncoupling agents worked. Recall that ‘uncoupling’ refers to the loss of correspondence between glucose oxidation and ATP production, like a bike that loses its chain—the energy put into peddling is no longer connected to a useful function. Uncoupling agents all disconnect the energy input from the output, but otherwise seemed to have little else in common. Mitchell showed that they did have something in common—they are weak acids, which dissolve in the lipids of the membrane. Because weak acids can either bind or release protons, according to the acidity of their surroundings, they can shuttle protons across the membrane. In alkaline or weakly acidic conditions they lose a proton and gain a negative charge. Drawn by the electric charge they cross to the positive, acidic side of the membrane. Then, being a weak acid in strongly acidic conditions, they pick up a proton again. This neutralizes the electrical charge, so they become subject to the concentration gradient again. The weak acid traverses the membrane to the less acidic side, whereupon it loses the proton and once more becomes subject to the electrical tug. This kind of cycling can only happen if the uncoupling agent dissolves in the membrane regardless of whether it has bound a proton or not; and it was this subtle requirement that confounded earlier attempts at an explanation. (Some weak acids are soluble in lipids, but only when they have bound to a proton or vice versa; when they have released their proton they are no longer soluble in lipids, and so can’t cross back over the membrane; they therefore can’t uncouple respiration.)
Even more fundamentally, the chemiosmotic hypothesis explained the voodoo ‘action at a distance’ that seemed to beg a high-energy intermediate, the elusive squiggle. Protons pumped across a membrane in one spot generate a force that acts equally anywhere on the surface of the membrane, just as the pressure of water behind a dam depends on the overall volume of water, not on the location of the pump. So protons are pumped over the membrane in one place, but can return through an ATPase anywhere else in the membrane with a force that depends on the overall proton pressure. In other words, there was no chemical intermediate, but the proton-motive force itself acted as an intermediate—the energy released by respiration was conserved as the reservoir of protons. This also explained how a non-integer number of electrons could generate ATP—although a fixed number of protons are pumped across the membrane for every electron transported, some of the protons leak back through the dam, whereas others are tapped off for other purposes: they are not used to power the ATPase (we’ll return to this in the next section).
Perhaps most importantly, the chemiosmotic theory made a number of explicit predictions, which could be tested. Over the following decade, Mitchell, working in the refurbished Glynn House with his life-long research colleague Jennifer Moyle, and others, proved that mitochondria do indeed generate a pH gradient as well as an electrical charge (of about 150 millivolts) across the inner membrane. This voltage might not sound like a lot (it’s only about a tenth of that available from a torch battery) but we need to think of it in molecular terms. The membrane is barely 5 nm (10–9 m) thick, so the voltage experienced from one side to the other is in the order of 30 million volts per metre—a similar voltage to a bolt of lightning, and a thousand times the capacity of normal household wiring. Mitchell and Moyle went on to show that a sudden rise in oxygen levels elicited a transient rise in the number of protons pumped over the membrane; they showed that respiratory ‘uncouplers’ really did work by shuttling protons back across the membrane; and they showed that the proton-motive force did indeed power the ATPase. They also demonstrated that proton pumping is coupled to the passage of electrons down the respiratory chain, and slows or even stops if any raw materials (hydrogen atoms, oxygen, ADP, or phosphate) run short.
By then, Mitchell and Moyle were not the only experimentalists working on chemiosmotics. Racker himself helped to convince the field by showing that if the respiratory complexes were isolated and then added to artificial lipid vesicles, they could still produce a proton gradient. But perhaps the one experiment that did more than any other to convince researchers, or botanists at least, of the veracity of the theory was carried out by André Jagendorf and Ernest Uribe at Cornell University in 1966. Jagendorf’s initial reaction to the chemiosmotic hypothesis had been hostile. He wrote: ‘I had heard Peter Mitchell talk about chemiosmosis at a bioenergetics meeting in Sweden. His words went into one of my ears and out the other, leaving me feeling annoyed they had allowed such a ridiculous and incomprehensible speaker in.’ But his own experiments convinced him otherwise.
Working with chloroplast membranes, Jagendorf and Uribe suspended the membranes in acid, at pH 4, and gave the acid time to equilibrate across the membrane. Then they injected an alkali, at pH 8, into the preparation, creating a pH difference of 4 units across the membrane. They found that large amounts of ATP were created by this process, without the need for light or any other energy source: ATP synthesis was powered by the proton difference alone. Notice that I’m talking about photosynthetic membranes here. A striking feature of Mitchell’s theory is that it reconciles quite distinct modes of energy production that seem to be unrelated, like photosynthesis and respiration—both produce ATP via a proton-motive force across a membrane.
By the mid 1970s, most of the field had come round to Mitchell’s point of view—Mitchell even maintained a chart showing the dates when his rivals ‘converted’, to their fury—even though many molecular details still needed to be worked out, and remained controversial. Mitchell was sole recipient of the Nobel Prize for chemistry in 1978, another source of acrimony, although I believe his conceptual leap justified it. He had been through a personally traumatic decade, fighting poor health as well as a hostile bioenergetic establishment, but lived to see the conversion of his fiercest critics. In thanking them for their intellectual generosity in his Nobel lecture, Mitchell quoted the great physicist Max Planck—‘a new scientific idea does not triumph by convincing its opponents, but rather because its opponents eventually die.’ To have falsified this pessimistic dictum, said Mitchell, was a ‘singularly happy achievement.’
Since 1978, researchers have whittled away at the detailed mechanisms of electron transport, proton pumping, and ATP formation. The crowning glory was John Walker’s determination of the structure of the ATPase in atomic detail, for which he shared the Nobel Prize for chemistry in 1997 with Paul Boyer, who had suggested the basic mechanism many years earlier. (This was broadly similar in principle, but differed in detail, from the mechanism favoured by Mitchell.) The ATPase is a marvellous example of nature’s nano-technology: it works as a rotary motor, and as such is the smallest known machine, constructed from tiny moving protein parts. It has two main components, a drive shaft, which is plugged straight through the membrane from one side to the other, and a rotating head, which is attached to the drive shaft, resembling a mushroom head when seen down the electron microscope. The pressure of the proton reservoir on the outside of the membrane forces protons through the drive shaft to rotate the head; for each three protons that pass through the drive shaft, the head cranks around by 120°, so three cranks complete a turn. There are three binding sites on the head, and these are where the ATP is assembled. Each time the head rotates, the tensions exerted force chemical bonds to form or break. The first site binds ADP; the next crank of the head attaches the phosphate onto the ADP to form ATP; and the third releases the ATP. In humans, a complete turn of the head requires 9 protons and releases 3 molecules of ATP. Just to complicate matters, in other species, the ATPase often requires different numbers of protons to rotate the head.
The ATPase is freely reversible. Under some circumstances it
can go into reverse, whereupon it splits ATP, and uses the energy released to pump protons up the drive shaft, back across the membrane against the pressure of the reservoir. In fact the very name ATPase (rather than ATP synthase) signifies this action, which was discovered first. This bizarre trait hides a deep secret of life, and we’ll return to it in a moment.
The deeper meaning of respiration
In a broad sense, respiration generates energy using proton pumps. The energy released by redox reactions is used to pump protons across a membrane. The proton difference across the membrane corresponds to an electric charge of about 150 mV. This is the proton-motive force, which drives the ATPase motor to generate ATP, the universal energy currency of life.
Something very similar happens in photosynthesis. In this case, the sun’s energy is used to pump protons across the chloroplast membrane in an analogous fashion to respiration. Bacteria, too, function in the same way as mitochondria, by generating a proton-motive force across their outer cell membrane. For anyone who is not a microbiologist, there is no field of biology more confusing than the astonishing versatility with which bacteria generate energy. They seem to be able to glean energy from virtually anything, from methane, to sulphur, to concrete. This extraordinary diversity is related at a deeper level. In each case, the principle is exactly the same: the electrons pass down a redox chain to a terminal electron acceptor (which may be and others). In each case the energy derived from the redox reactions is used to pump protons across a membrane.
Such a deep unity is noteworthy not just for its universality, but perhaps even more because it is such a peculiar and roundabout way of generating energy. As Leslie Orgel put it, ‘Few would have laid money on cells generating energy with proton pumps.’ Yet proton pumping is the secret of photosynthesis, and all forms of respiration. In all of them, the energy released by redox reactions is used to pump protons across a membrane, to generate a proton-motive force. It seems that pumping protons across a membrane is as much a signature of life on earth as DNA. It is fundamental.
In fact the proton-motive force has a much broader significance than just generating ATP, as Mitchell realized. It acts as a kind of force field, enveloping bacteria with an impalpable source of power. Proton power is involved in several fundamental aspects of life, most notably the active transport of molecules in and out of the cell across the external membrane. Bacteria have dozens of membrane transporters, many of which use the proton-motive force to pump nutrients into the cell, or waste products out. Instead of using ATP to power active transport, bacteria use protons: they hive off a little energy from the proton gradient to power active transport. For example, the sugar lactose is transported into the cell against a concentration gradient by coupling its transport to the proton gradient: the membrane pump binds one lactose molecule and one proton, so the energetic cost of importing lactose is met by the dissipation of the proton gradient, not by ATP. Similarly, to maintain low sodium levels inside the cell, the removal of one sodium ion is paid for by the import of one proton, again dissipating the proton gradient without consuming ATP.
Sometimes the proton gradient is dissipated for its own sake, to produce heat. In these circumstances, respiration is said to be uncoupled, for electron flow and proton pumping continue as normal, but without ATP production. Instead, the protons pass back through pores in the membrane, thereby dissipating the energy bound up in the proton gradient as heat. This can be useful in itself, as a means of producing heat, as we shall see in Part 4, but it also helps to maintain electron flow during times of low demand, when ‘stagnant’ electrons are prone to escape from the respiratory chain to react with oxygen, producing destructive oxygen free radicals. Think of this like a hydroelectric dam on a river. At times of low demand there is a risk of flooding, which can be lowered by having an over-flow channel. Similarly, in the respiratory chain, a through-flow of electrons can be maintained by uncoupling electron flow from ATP synthesis. Instead of flowing through the main hydroelectric dam gates (the ATPase), some protons are diverted through the overflow channels (the membrane pores). This through-flow helps to prevent any problems that may arise from having an overflowing reservoir of electrons, ready to form free radicals; and there are important health consequences, as we shall see in later chapters.
Besides active transport, the proton force can be put to other forms of work. For example, bacterial locomotion also depends on the proton-motive force as shown by the American microbiologist Franklin Harold and his colleagues in the 1970s. Many bacteria move around by rotating a rigid corkscrew-like flagella attached to the cell surface. They can achieve speeds of up to several hundred cell-lengths per second by this process. The protein that rotates the flagellum is a tiny rotary motor, not dissimilar to the ATPase itself, which is powered by the proton current through a drive shaft.
In short, bacteria are basically proton-powered. Even though ATP is said to be the universal energy currency, it isn’t used for all aspects of the cell. Both bacterial homeostasis (the active transport of molecules in and out of the cell) and locomotion (flagellar propulsion) depend on proton power rather than ATP. Taken together, these vital uses of the proton gradient explain why the respiratory chain pumps more protons than are required for ATP synthesis alone, and why it is hard to specify the number of ATP molecules that are formed from the passage of one electron—the proton gradient is fundamental to many aspects of life besides ATP formation, all of which tap off a little.
The importance of the proton gradient also explains the odd propensity of the ATPase to go into reverse, pumping protons at the cost of burning up ATP. On the face of it, such a reversal of the ATPase might seem to be a liability, because it swiftly drains the cell of its ATP reserves. This only begins to make sense when we appreciate that the proton gradient is more important than ATP. Bacteria need a fully charged proton-motive force to survive, just as much as a galactic cruiser in Star Wars needs its protective force field fully operational before attacking the Empire’s star fleet. The proton-motive force is usually charged up by respiration. However, if respiration fails, then bacteria generate ATP by fermentation. Now everything goes into reverse. The ATPase immediately breaks down the freshly made ATP and uses the energy released to pump protons across the membrane, maintaining the charge—which amounts to an emergency repair of the force field. All other ATP-dependent tasks, even those as essential as DNA replication and reproduction, must wait. In these circumstances, it might be said that the main purpose of fermentation is to maintain the proton-motive force. It is more important for a cell to maintain its proton charge than it is to have an ATP pool available for other critical tasks such as reproduction.
To me, all this hints at the deep antiquity of proton pumping. It is the first and foremost need of the bacterial cell, its life-support machine. It is a deeply unifying mechanism, common to all three domains of life, and central to all forms of respiration, to photosynthesis, and to other aspects of bacterial life, including homeostasis and locomotion. It is in short a fundamental property of life. And in line with this idea, there are good reasons to think that the origin of life itself was tied to the natural energy of proton gradients.
6
The Origin of Life
How life began on Earth is one of the most exhilarating fields of science today—a wild west of ideas, theories, speculations, and even data. It is too large a subject to embark on in detail here, so I will limit myself to a few observations on the importance of chemiosmotics. But for perspective let me paint a quick picture of the problem.
The evolution of life depends in very large measure on the power of natural selection—and this in turn depends on the inheritance of characteristics that can be subjected to natural selection. Today we inherit genes made of DNA; but DNA is a complicated molecule and can’t have just ‘popped’ into existence. Moreover, DNA is chemically inert, as we noted in the Introduction. Recall that DNA does little more than code for proteins, and even this is achieved by way of a more active
intermediary, RNA, which in various forms physically translates the DNA code into the sequence of amino acids in a protein. In general, proteins are the active ingredients that make life possible—they alone have the versatility of structure and function needed to fulfil the multifarious requirements of even the simplest forms of life. Individual proteins are honed to the requirements of their particular tasks by natural selection. First among these tasks, proteins are needed to replicate DNA and to form RNA from the DNA template, for without heredity natural selection is not possible; and for all their glories proteins are not repetitive enough in structure to form a good heritable code. The origin of the genetic code is therefore a chicken and egg problem. Proteins need DNA to evolve, but DNA needs proteins to evolve. How did it all get started?
The answer agreed by most of the field today is that the intermediary, RNA, used to be central. RNA is simpler than DNA, and can even be put together in a test tube by chemists, so we can bring ourselves to believe that it may once have formed spontaneously on the early Earth or in space. Plenty of organic molecules, including some of the building blocks of RNA, have been found on comets. RNA can replicate itself in a similar manner to DNA, and so forms a replicating unit that natural selection can act upon. It can also code for proteins directly, as indeed it does today, and so provides a link between template and function. Unlike DNA, RNA is not chemically inert—it folds into complex shapes and is able to catalyse some chemical reactions in the same way as enzymes (RNA catalysts are called ribozymes). Thus, researchers into the origin of life point to a primordial ‘RNA world’, in which natural selection acts upon independently self-replicating RNA molecules, which slowly accrue complexity, until being displaced by the more robust and efficient combination of DNA and proteins. If this whistlestop tour whets your appetite for more, I can recommend Life Evolving by Christian de Duve as a good place to start.