Everything that living things do can be understood in terms of the jiggling and wiggling of atoms …
RICHARD FEYNMAN1
Hamlet: How long will a man lie i’ the earth ere he rot?
Gravedigger: Faith, if he be not rotten before he die—as we have many pocky corses now-a-days, that will scarce hold the laying in,—he will last you some eight year or nine year: a tanner will last you nine year.
Hamlet: Why he more than another?
Gravedigger: Why, sir, his hide is so tanned with his trade that he will keep out water a great while; and your water is a sore decayer of your whoreson dead body.
WILLIAM SHAKESPEARE, Hamlet, ACT V, SCENE I, “A CHURCHYARD”
SIXTY-EIGHT MILLION years ago, in the period we now call the late Cretaceous, a young Tyrannosaurus rex was making its way through a sparsely wooded river valley cut into a semitropical forest. At around eighteen years old, the animal had not yet reached maturity, but it stood nearly five meters tall. With every lumbering step it accelerated many tons of dinosaur meat forward with a momentum sufficient to flatten trees or any smaller creatures unfortunate enough to get in its way. That its body could retain its integrity while being subject to these flesh-sundering forces was due to the fact that every bone, sinew and muscle was held in place by tough but elastic fibers of a protein known as collagen. This protein acts as a kind of glue that bonds flesh, and it is an essential component of all animal bodies, including our own. Like all biomolecules, it is made and unmade by the most remarkable machines in the known universe. Our focus in this chapter is on how these biological nanomachines*1 work; and from there we will explore the recent discovery that the gears and levers of these engines of life dip into the quantum world to keep us and every other living organism alive.
But first, back to that ancient valley. On this particular day, the dinosaur’s bulk, built by millions of nanomachines, would be its undoing, because those limbs that had been so effective at chasing down and dismembering its prey would prove to be of little use in extricating it from the sticky mud of the soft riverbed into which it stumbled. After many hours of fruitless struggling, the Tyrannosaur’s huge jaws filled with murky water and the dying animal sank into the mud. Under most circumstances the animal’s flesh would have suffered the same rapid decay as Hamlet’s gravedigger’s “corses,” but this individual dinosaur sank so fast that its entire body was soon entombed in thick, flesh-preserving mud and sand. Over the years and centuries, finely grained minerals permeated cavities and pores in its bones and flesh, replacing the animal’s tissues with stone: the dinosaur corpse became a dinosaur fossil. Up on the surface, the rivers continued to wander over the landscape, depositing successive layers of sand, mud and silt, until the fossil lay beneath tens of meters of sandstone and shale.
About forty million years later the climate warmed, the rivers dried up and the rock layers covering the long-dead bones eroded in hot desert winds. Another twenty-eight million years passed before members of another biped species, Homo sapiens, walked into the river valley; but these upright primates mostly shunned this dry and hostile country. When, in more modern times, European settlers arrived they named this inhospitable area the Badlands of Montana and called the dry river valley Hell Creek. In 2000, a team of palaeontologists led by the most famous fossil hunter of them all, Jack Horner, was camping there. One of that group, Bob Harmon, was having his lunch when he noticed a large bone jutting out of the rock just above him.
Over the course of three years, nearly half of the entire skeleton of the animal was carefully excavated out of the surrounding stone—a task that involved the Army Corps of Engineers, a helicopter and a lot of graduate students—and transported to the Museum of the Rockies in Bozeman, Montana, where it was designated specimen MOR-1125. The dinosaur’s femur had to be cut in two before it could be winched onto a helicopter, and in the process a chunk of fossilized bone was broken off. Jack Horner gave several of the fragments to his palaeontologist colleague, Dr. Mary Schweitzer from North Carolina State University, whom he knew to be interested in the chemical make-up of fossils.
When Schweitzer opened the box, she got a surprise. The first fragment she looked at seemed to have very unusual-looking tissue on the inner (marrow-cavity) side of the bone. She placed the bone in an acid bath, which would dissolve its outer stony minerals to reveal its deeper structures. However, on this occasion she accidentally left the fossil in the bath for too long, and by the time she returned all of its minerals had dissolved away. Schweitzer expected the entire fossil to have disintegrated, but she and her colleagues were astonished to discover that a pliable fibrous substance remained which, under the microscope, looked just like the kind of soft tissue that you would find in modern bones. And, just as in modern bones, this tissue appeared to be packed full of blood vessels, blood cells and those long chains of collagen fibers, the biological glue that had kept the lumbering live animal in one piece.
Fossils that preserve the structure of soft tissue are rare but far from unknown. The Burgess Shale fossils found high up in the Canadian Rockies of British Columbia between 1910 and 1925 preserve astonishingly detailed impressions of the flesh of animals that swam in the Cambrian seas nearly six hundred million years ago, as does the famous feathered archaeopteryx from the Solnhofen quarry in Germany, which lived some hundred and fifty million years ago. But conventional soft-tissue fossils preserve only the impression of biological tissue, not its substance; yet the pliable material that remained in Mary Schweitzer’s acid bath appeared to be the dinosaur’s soft tissue itself. When, in 2007, Schweitzer published her finding in the journal Science,2 her paper was initially met with surprise and a considerable degree of skepticism. But, although the survival of biomolecules for millions of years is indeed astonishing, it is what happened next in this story that is the focus of our interest. To prove that the fibrous structures were indeed made of collagen, Schweitzer first demonstrated that proteins that stick to modern collagen also stuck to the fibers in her ancient bone. As a final test, she mixed the dinosaur tissue with an enzyme called collagenase, one of the many biomolecular machines that make and unmake collagen fibers in animal bodies. Within minutes, collagen chains that had held fast for sixty-eight million years were broken by the enzyme.
Enzymes are the engines of life. Those that are probably most familiar to us have somewhat mundane everyday uses, such as the proteases added to “biological” detergents that help to remove stains, the pectinase added to fruit to make wines or the rennet added to milk to help it to coagulate and become cheese. We may also appreciate the role that the various enzymes in our stomachs and intestines play in digesting our food. But these are fairly trivial examples of the action of nature’s nanomachines. All life depends or depended on enzymes, from those first microbes that oozed out of the primordial soup, to the dinosaurs that stomped through the Jurassic forests, to every organism alive today. Every cell in your body is filled with hundreds or even thousands of these molecular machines that help to keep that continual process of assembly and recycling of biomolecules, the process that we call life, moving.
Here, “help” is the key word that defines what enzymes do: their job is to speed up (catalyze) all sorts of biochemical reactions that would otherwise proceed far too slowly. Thus, protease enzymes added to detergents speed up the digestion of proteins in stains, pectin enzymes speed up the digestion of polysaccharides in fruit, and rennet enzymes speed up the coagulation of milk. Similarly, enzymes in our cells speed up metabolism: the process by which trillions of biomolecules inside our cells are continually transformed into trillions of other biomolecules to keep us alive.
The collagenase enzyme that Mary Schweitzer added to her dinosaur bones is just one of these biomachines whose regular job in animal bodies is to disintegrate collagen fibers. The rate of speed-up provided by enzymes can be roughly estimated by comparing the time taken to digest collagen fibers in their absence (clearly, more than sixty-eight million years) and in the presence of the right enzyme (about thirty minutes): a trillion-fold difference.
In this chapter we’ll be exploring how it is that enzymes such as collagenase manage to achieve these astronomical chemical accelerations. One of the surprises of recent years is the discovery that quantum mechanics plays a key role in the action of at least some enzymes; and, since they are central to life, they are our first port of call on the voyage through quantum biology.
The exploitation of enzymes pre-dated their discovery and characterization by many millennia. Several thousand years ago our ancestors were transforming grain or grape juice into beer or wine by the addition of yeast—essentially a microbial bag of enzymes.*2 They also understood that extracts from the stomach lining of calves (rennet) accelerated the transformation of milk into cheese. For many centuries it was believed that these transforming properties were performed by vital forces associated with living organisms, endowing them with the vitality and speed of change that distinguished the living (the “quick” in the biblical reference in this section heading) from the dead.
In 1752, inspired by the mechanistic philosophy of René Descartes, the French scientist René Antoine Ferchault de Réaumur set out to investigate one of these supposed vital activities, digestion, with an ingenious experiment. It was generally believed at the time that animals digested their food by a mechanical process brought about by pounding and churning within their digestive organs. This theory seemed especially pertinent to birds, whose gizzards contained small stones that were thought to macerate their food—a mechanical action consistent with René Descartes’s view (outlined in the previous chapter) that animals were mere machines. But de Réaumur was puzzled by how birds of prey, whose gizzards lacked digestive stones, also managed to digest their food. So he fed his pet falcon small pieces of meat enclosed in tiny metal capsules punctured by small holes. When he recovered the capsules he discovered that the meat was completely digested, despite the fact that, protected within the metal, it could not have been subject to any mechanical action. Descartes’s cogs, levers and grinders were clearly insufficient to account for at least one of life’s vital forces.
A century after de Réaumur’s work, another Frenchman, the chemist and founder of microbiology Louis Pasteur, studied another biological transformation hitherto attributed to “vital forces”: the conversion of grape juice into wine. He showed that the transforming principle of fermentation appeared to be intrinsically associated with living yeast cells that were present in the “ferments” used in the brewing industry, or in the leaven used to make bread. The term “enzyme” (Greek: “in yeast”) was then coined by the German physiologist Wilhelm Friedrich Kühne in 1877 to describe the agents of these vital activities, such as those performed by living yeast cells, or indeed any transformations promoted by substances extracted from living tissue.
But what are enzymes and how do they quicken life’s transformations? Let’s return to the enzyme that opened our story in this chapter, collagenase.
Collagen is the most abundant protein in animals (including humans). It acts as a kind of molecular thread woven into and in between our tissues, holding flesh together. Like all proteins, it is composed of basic chemical building blocks: strings of amino acids that come in about twenty varieties, of which some (for example, glycine, glutamine, lysine, cysteine, tyrosine) may be familiar to you as nutritional supplements that can be bought in health-food stores. Each amino-acid molecule is made of between ten and fifty or so atoms of carbon, nitrogen, oxygen, hydrogen and occasionally sulphur, held together by chemical bonds in their own uniquely characteristic three-dimensional shape.
Several hundred of these twisted amino-acid molecular shapes are then themselves strung together to form a protein, rather like oddly shaped beads on a string. Each bead is linked to the next via a peptide bond, which connects a carbon atom in one amino acid to a nitrogen atom in the next. Peptide bonds are very strong; after all, those that held the T. rex collagen fibers together had survived for sixty-eight million years.
Collagen is an especially strong protein, which is crucial to its role as the internal webbing that maintains the shape and structure of our tissues. The proteins are twisted together in triple strands, which are in turn bonded into thick ropes, or fibers. These fibers are threaded through our tissues to sew our cells together; they are also present in tendons, which attach our muscles to our bones, and in ligaments, which bind bone to bone. This dense network of fibers is called the extracellular matrix and it basically holds us together.
Anyone who isn’t a vegetarian is already familiar with the extracellular matrix as the stringy gristle that you might encounter within an indigestible sausage or in one of the cheaper cuts of meat. Cooks will also be aware of the insolubility of this sinewy material, which fails to tenderize even after hours of boiling a stew. But however unwelcome the extracellular matrix may be on a dinner plate, its presence in the bodies of the diners is absolutely vital. Without collagen, our bones would fall apart, our muscles would drop off our bones and our internal organs would become a kind of jelly.
But the collagen fibers present in your bones, muscles or dinner are not indestructible. Boiling them in strong acids or alkalis will eventually break the peptide bonds between the amino-acid beads and transform these tough fibers into soluble gelatin, the jelly-like substance that is used to make marshmallows and jelly (Jell-o in the United States). Film fans might remember the Stay Puft Marshmallow Man in Ghostbusters as the giant lumbering mass of wobbly white flesh that terrorized New York. But Marshmallow Man was easily defeated by being liquidized into molten marshmallow cream. Peptide bonds between the amino-acid beads of collagen fibers are the difference between Marshmallow Man and Tyrannosaurus rex. Tough collagen fibers make real animals tough.
There is a problem, however, when you scaffold an animal body with tough, long-lasting materials such as collagen. Consider what happens when you cut or bruise yourself or even break an arm or a leg: tissues are destroyed and the supporting extracellular matrix, that internal stringy mesh, is likely to be damaged or broken. If a house is damaged by a storm or an earthquake, repair has to be preceded by stripping out the broken framework. Similarly, animal bodies use the enzyme collagenase to cut away damaged parts of the extracellular matrix so that the tissue can be repaired—by another set of enzymes.
Even more crucially, the extracellular matrix has to be constantly remodeled as an animal grows: the internal scaffold that sustained an infant will not serve to support the much larger adult. This problem is particularly acute—and its solution therefore particularly instructive—in amphibians, whose adult form is very different from the juvenile. The most familiar example is amphibian metamorphosis: the transformation from a spherical egg to a wriggling tadpole, which later matures into a hopping frog. Fossils of these short-bodied, tail-less amphibians with their unmistakable powerful rear limbs are found in Jurassic rocks dating back to the middle of the Mesozoic Era two hundred million years ago, known as the Age of Reptiles. But they can also be found in rocks dating from the Cretaceous period. So it seems likely that frogs swam through that same Montana river where the dinosaur that became MOR 1255 met its end. But, unlike dinosaurs, frogs managed to survive the great Cretaceous extinction and remain common in our own ponds, rivers and swamps, allowing generations of schoolchildren, and scientists, to study how bodies are formed and re-formed.
The transformation of a tadpole into a frog involves a considerable amount of dismantling and reshaping of, for example, the animal’s tail, which is gradually reabsorbed into the body and its flesh recycled to form the frog’s new limbs. All of this requires the collagen-based extracellular matrix that supported the animal’s tail structure to be rapidly dismantled before being reassembled in its newly forming limbs. But, remember those sixty-eight million years under the Montana rocks: collagen fibers are not easily broken. Frog metamorphosis would take a very long time if it relied on the chemical breakdown of collagen solely by inorganic processes. Clearly an animal can’t boil its tough sinews in hot acid, and therefore needs a much milder means of dismantling its collagen fibers.
This is where the enzyme collagenase comes in.
But how does it—and all its fellow enzymes—work? The vitalist belief that enzyme activity was mediated by some kind of mysterious living force persisted until the late nineteenth century. At that point, one of Kühne’s colleagues, the chemist Eduard Buchner, demonstrated that nonliving extracts from yeast cells could stimulate precisely the same chemical transformations brought on by the live cells. Buchner went on to make the revolutionary proposal that the vital force was nothing more than a form of chemical catalysis.
Catalysts are substances that accelerate ordinary chemical reactions and were already familiar to chemists in the nineteenth century. Indeed, many of the chemical processes that drove the industrial revolution depended crucially on catalysts. For example, sulphuric acid was an essential chemical that spurred both the industrial and agricultural revolutions, used in iron and steel manufacture, in the textile industry and for the manufacture of phosphate fertilizer. It is produced by a chemical reaction that starts off with sulphur dioxide (SO2) and oxygen (the reactants), both of which react with water to form the product: sulphuric acid (H2SO4). However, the reaction is very slow and was therefore initially difficult to commercialize. But in 1831 Peregrine Phillips, a vinegar manufacturer from Bristol, England, discovered a way to speed it up by passing the sulphur dioxide and oxygen over hot platinum, which acted as a catalyst. Catalysts differ from the reactants (the initial substances participating in the reaction) because they help to speed up the reaction without taking part in it or being changed by it. Buchner’s claim was therefore that enzymes were no different in principle from the kind of inorganic catalyst discovered by Phillips.
Decades of subsequent biochemical research have largely confirmed Buchner’s insight. Rennet, produced in calves’ stomachs, was the first enzyme to be purified. The ancient Egyptians stored milk in bags made from the lining of calves’ stomachs, and it is they who are usually credited with the discovery that this unlikely material accelerated the conversion of milk into the better-preserved cheese. This practice continued until the end of the nineteenth century. By then, calves’ stomachs themselves were being dried and sold as “rennets” in apothecaries’ shops. In 1874, the Danish chemist Christian Hansen was being interviewed for a job at an apothecary’s when he overheard an order arriving for a dozen rennets and, on inquiring what they were, came up with the idea of using his chemical skills to provide a less unsavory source of rennet. He returned to his laboratory, where he developed a method for converting the foul-smelling liquid obtained from rehydrating calves’ stomachs into a dry powder, and made his fortune by commercializing the product, which was sold the world over as Dr. Hansen’s Rennet Extract.
Rennet is actually a mixture of several different enzymes, the most active of which for the purposes of cheese-making is called chymosin, itself one of a huge family of enzymes called proteases that accelerate the cleavage of proteins. Its action in cheese-making is to cause milk to coagulate so it can be separated into curds and whey; but its natural role in a young calf’s body is to curdle the milk it ingests so that it remains longer in the digestive tract, giving more time for it to be absorbed. Collagenase is another protease, but methods for its purification weren’t developed until fifty years later when Jerome Gross, a clinical scientist at Harvard Medical School in Boston in the 1950s, was intrigued by the question of how tadpoles absorb their tails to become frogs.
Gross was interested in the role of collagen fibers as an example of molecular self-assembly, which he considered to “hold a major secret of life.”3 He decided to work on the rather massive tail of the bullfrog tadpole, which can be several inches long. Gross correctly guessed that the process of tail reabsorption must involve a lot of assembly and disassembly of the animal’s collagen fibers. To detect collagenase activity he developed a simple test in which a Petri dish was filled with a layer of milky-looking collagen gel, packed full of those tough, durable collagen fibers. When he placed fragments of tissue from tadpole tails on the gel’s surface, he noticed a zone surrounding the tissue where those tough fibers were being degraded and turned into soluble gelatin. He then went on to purify the collagen-digesting substance, the enzyme collagenase.
Collagenase is present in the tissue of frogs and other animals, including the dinosaur that left its bones in Hell Creek. The enzyme performed the same function sixty-eight million years ago that it performs today, breaking down collagen fibers; but the enzyme was inactivated when the animal died and fell into the swamp, so its collagen fibers remained intact until Mary Schweitzer added some fresh collagenase to the bone fragments.
Collagenase is just one of millions of enzymes on which all animals, microbes and plants depend to perform nearly all the vital activities of life. Other enzymes make the collagen fibers of the extracellular matrix; yet others make biomolecules such as proteins, DNA, fats and carbohydrates, and a whole different set of enzymes degrade and recycle these biomolecules. Enzymes are responsible for digestion, respiration, photosynthesis and metabolism. They are responsible for making all of us; and they keep us alive. They are the engines of life.
But are enzymes just biological catalysts, providing the same kind of chemistry that is used to make sulphuric acid and scores of other industrial chemicals? A few decades ago, most biologists would have agreed with Buchner’s view that the chemistry of life is no different from the kinds of processes that take place inside a chemical plant, or even a child’s chemistry set. But in the last couple of decades that view has radically changed as a number of key experiments have provided remarkable new insights into the way enzymes work. It seems that life’s catalysts are able to reach down into a deeper level of reality than plain old classical chemistry and make use of some neat quantum trickery.
But to understand why quantum mechanics is needed to account for life’s vitality, we must first investigate how the far more mundane industrial catalysts work.
Catalysts operate by a variety of different mechanisms, but most can be understood through an idea called transition state theory (TST)4 that provides a simple explanation of how catalysts speed up reactions. To understand TST, it is probably useful to first turn the problem around and consider why catalysts are needed to accelerate reactions. The answer is that most common chemicals in our environment are rather stable and unreactive. They neither spontaneously break down nor readily react with other chemicals; after all, if they did either of these things, they would not be common now.
Figure 3.1: Reactant molecules, represented by gray dots, can be converted into product molecules, represented by black dots, but first they have to climb over an energy hill. Cool molecules seldom possess sufficient energy to make the ascent, but hot molecules can easily hike over the summit.
The reason why common chemicals are stable is that their bonds are not often broken by the inevitable molecular turbulence that always exists within matter. We can visualize this as the reactant molecules needing to negotiate a landscape, climbing over the top of a hill that stands between them and conversion into products (figure 3.1). The energy needed to ascend the “hillside” is mostly provided by heat, which speeds the motion of atoms and molecules, causing them to move or vibrate faster. This molecular bumping and jostling can break the chemical bonds that hold the atoms together within molecules and even allow them to form new bonds. But the atoms of more stable molecules—those that are common in our environment—are held together by bonds strong enough to resist the surrounding molecular turbulence. So the chemicals we find around us are common because their molecules are, by and large, stable,*3 despite the energetic molecular jostling of their environment.
Even stable molecules can, however, be ripped apart if they are provided with sufficient energy. One possible source of that energy is more heat, which speeds up molecular motion. Heating up a chemical will eventually break its bonds. This is why we cook so much of our food: the heat speeds up the chemical reactions responsible for transforming the raw ingredients—the reactants—into tastier products.
A convenient way to visualize how heat accelerates chemical reactions is to imagine the reactant molecules as the grains of sand in the left-hand chamber of an hourglass lying on its side (figure 3.2a). If left alone all the sand grains will remain where they are until the end of time since they do not possess sufficient energy to reach the neck of the hourglass and pass across to the right chamber, which represents the final products of the reaction. The reactant molecules in a chemical process can be provided with more energy by heating them up, thereby causing them to move and vibrate faster, and providing some of them with sufficient energy to be converted into products. We can envisage this as simply giving the hourglass a good shake so that some of the sand grains will be thrown into the right-hand chamber and change from reactants to products (figure 3.2b).
But another way of converting reactants to products is to lower the energy barrier they need to climb over. This is what catalysts do. They perform the equivalent of making the neck of the hourglass wider so that sand in the left-hand chamber can flow into the right-hand chamber with only a minimal amount of thermal agitation (figure 3.2c). The reaction is thereby greatly accelerated by the catalyst’s ability to change the shape of the energy landscape in such a way as to allow substrates*4 to become products much faster than they can do in the absence of a catalyst.
Figure 3.2: Changing the energy landscape. (a) Molecules can pass from the reactant (R) to the product (P) state, but they must first possess sufficient energy to ascend to the transition state (the neck of the hourglass). (b) Turning the hourglass puts the reactant (substrate) into a higher-energy state than the product, allowing it to flow through easily. (c) Enzymes work by stabilizing the transition state, effectively lowering its energy (the neck of the hourglass), allowing substrates to flow through more easily into the product state.
We can illustrate how this works at a molecular level by first considering the very slow reaction responsible for breaking down a collagen molecule in the absence of the collagenase*5 enzyme (figure 3.3). As we have already explained, collagen is a string of amino acids, each one attached to the next by a peptide bond (shown as a heavy line in the figure) between a carbon and a nitrogen atom. The peptide bond is just one of several types of bond that hold atoms together within molecules. It consists essentially of a pair of electrons that are shared between the nitrogen and carbon atoms. These shared negatively charged electrons attract the positively charged atomic nuclei of the atoms on either side of the bond, thereby acting as a kind of electronic glue that holds the atoms together in the peptide bond.*6
Peptide bonds are very stable because breaking them, by forcing the shared electrons to separate, requires a high “activation energy”: the bond has to climb a very tall energy hill before it reaches the neck of the reaction hourglass. In practice, the bond doesn’t usually break of its own accord and needs a helping hand from one of the surrounding water molecules in a process known as hydrolysis. For this to take place, the water molecule must first wander close enough to the peptide bond to donate one of its electrons to the bond’s carbon atom, forming a new weak bond that tethers the water molecule in place, represented by dotted lines in figure 3.3. This intermediate stage is called a transition state (hence transition state theory) and is the unstable peak of the energy hill that needs to be climbed if the bond is to be broken, represented by the neck of the hourglass. Note from the figure that this donated electron from water has traveled all the way down to the oxygen atom adjacent to the peptide bond, which having acquired an extra electron is now negatively charged. The water molecule that donated the electron meanwhile has been left with an overall positive charge in the transition state.
Figure 3.3: Proteins such as collagen (a) consist of chains of amino acids made up from atoms of carbon (C), nitrogen (N), oxygen (O) and hydrogen (H) linked by peptide bonds. One of these bonds is represented by the thick bold line in the figure. The peptide bond can be hydrolyzed by a water molecule (H2O), which breaks the peptide bond (c), but it must first pass through an unstable transition state, which consists of at least two different structures that mutually interconvert (b).
Here is where the process gets slightly trickier to grasp. Think of this water molecule (H2O) as positively charged not because it has lost an electron, but because it now contains a bare hydrogen nucleus, a proton, represented by the + sign in the figure. This positively charged proton is no longer held firmly in place within the water molecule and becomes delocalized in the quantum mechanical sense we discussed in the last chapter. Although it spends most of its time still associated with its water molecule (the left-hand structure in figure 3.3b), some of the time it can be found farther away, closer to the nitrogen atom (the right-hand structure in figure 3.3b) at the other end of the peptide bond. In this position, the roving proton can tug one of the peptide bond electrons out of its position, thereby breaking the bond.
But this will not usually happen. The reason is that transition states, such as the one illustrated in figure 3.3b, are very short-lived; they are so unstable that the slightest “nudge” can dislodge them. For example, the negatively charged electron that was donated by the water molecule is easily reclaimed so that the initial reactants are re-formed (shown by the thick arrow in the figure). This is a far more likely scenario than the forward reaction in which the bond gets broken. So peptide bonds usually don’t break. In fact, in neutral solutions, which are neither acidic nor alkaline, the time taken for half the peptide bonds in a protein to break, known as the half-life of the reaction, is more than five hundred years.
All this, of course, is what happens without enzymes: we have yet to describe how the enzyme comes in to help the hydrolysis process. According to transition state theory, catalysts speed up chemical processes, such as the breaking of the peptide bond, by making the transition state more stable, thereby increasing the chances of the final products forming. There are various ways this can happen. For example, a positively charged metal atom near the bond can neutralize the negatively charged oxygen atom in the transition state to stabilize it (so that it is no longer in such a hurry to give back the electron donated by the water molecule). By stabilizing transition states, catalysts are lending a helping hand by performing the equivalent of widening the neck of the hourglass.
We now need to consider whether transition state theory, viewed through our hourglass analogy, can also account for the way enzymes accelerate all those other reactions necessary for life.
The collagenase enzyme that Mary Schweitzer used to shatter those ancient Tyrannosaurus collagen fibers is the same enzyme that Jerome Gross detected in frogs. You will remember that this enzyme is needed to dismantle the tadpole’s extracellular matrix so that its tissues, cells and biomolecules can be reassembled into an adult frog. It performed the same function in the dinosaur, and continues to perform that function in our bodies: dismantling collagen fibers to allow growth and re-formation of tissue during development and after injury. To see this enzymatic process in action, we will borrow an idea from a science-transforming lecture delivered by Richard Feynman to an audience at the California Institute of Technology in 1959 entitled “There’s Plenty of Room at the Bottom.” The lecture is generally acknowledged as having been the intellectual foundation of the field of nanotechnology: engineering on the scale of atoms and molecules. Feynman’s ideas are also said to have inspired the 1966 film Fantastic Voyage, in which a submarine and its crew were shrunk small enough to be injected into a scientist’s body to find and repair a potentially fatal blood clot in his brain. To investigate how it works we will take a trip in an imaginary nanosubmarine. Our destination will be the tail of a tadpole.
First we must find our tadpole. A visit to the local pond reveals a clutch of frogspawn, and we carefully remove a handful of the jelly-like black-dotted spheres and transfer them to a glass water tank. It isn’t long before we observe wriggling within the spawn and, within days, tiny tadpoles emerging from their eggs. After making a quick note of their principal features under a magnifying glass—a relatively large head with snout above a small mouth, lateral eyes, and feathery gills in front of a long powerful tail fin—we supply the tadpoles with sufficient food (algae) and return daily for observations. For several weeks we notice little change in the form of the animal but are impressed by its rapid increase in length and girth. By about eight weeks we notice that the animal’s gills have retracted into its body, revealing front limbs. Another two weeks and rear legs emerge from the base of its sturdy tail. At this stage we must make more frequent observations since the rate of change in the metamorphosing animal appears to be accelerating. The tadpole’s gills and gill pouches completely disappear and its eyes migrate higher up its head. Alongside these dramatic changes to the tadpole’s front end, its tail starts to shrink. This is the cue we have been waiting for: so we board our nanosubmarine and launch it into the glass tank to investigate one of nature’s most remarkable transformations.
As our craft shrinks we can see more detail of the frog’s metamorphosis, including dramatic changes to the tadpole’s skin, which has become thicker, tougher and embedded with mucus-secreting glands that will keep it moist and supple when it leaves the pond and walks onto land. We dive into one of these glands, which leads us through the animal’s skin. After safely passing through several cell barriers, we arrive within its circulatory system. Cruising through the animal’s veins and arteries, we can witness from the inside the many changes taking place within its body. From their sac-like beginnings, its lungs form, expand and fill with air. The tadpole’s long spiral gut, which was suitable for digesting algae, is straightened into one typical of a predator. Its translucent cartilaginous skeleton, including the notochord (a primitive form of backbone that runs the length of its body), becomes dense and opaque as cartilage is replaced by bone. Continuing our mission, we follow the developing spine down into the tail of the tadpole, which is just beginning the process of being absorbed into the growing body of the frog. At this scale we can see thick striated muscle fibers packed into its length.
Another round of shrinkage allows us to see that each muscle fiber is composed of long columns of cylindrical cells whose periodic contractions are the source of the tadpole’s locomotion. Surrounding these muscle cylinders is a dense netting of stringy ropes: the extracellular matrix that is the target of our investigation. The matrix itself appears to be in a state of flux as individual ropes are unraveling to release trapped muscle cells that break free to join a growing mass cell migration out of the disappearing tadpole tail and into the frog’s body.
Shrinking down further, we home in on one of those unraveling ropes of the disintegrating extracellular matrix. As its girth expands we see that, like a rope, it is woven from thousands of individual protein cords, each of which is itself a bundle of collagen fibers. Each fiber is made of three collagen protein strings—those amino-acid beads on a string that we met earlier when discussing the dinosaur bone—but wound around one another to make a tough helical thread, a bit like DNA but triple-stranded rather than double-stranded. And here at last we spot the target of our expedition: a collagenase enzyme molecule. It shows up as a clam-like structure clamped onto one of the collagen fibers, and slides down the fiber, unzipping the triple helix strands before simply clipping apart the peptide bonds connecting the amino-acid beads. The chain that might otherwise remain intact for millions of years is broken in an instant. We will now zoom in even farther to see exactly how this clipping action works.
Our next bout of shrinkage takes us down to the molecular scale of just a few nanometers (millionths of a millimeter). It is difficult to grasp just how minute this scale actually is, so to give you a better idea, consider the size of the letter “o” on this page: if you were to shrink down from your normal size to the nanometer scale, then to you that “o” would appear to be roughly the size of the whole of the United States of America. At this scale we can see that the interior of the cell is densely packed with water molecules, metal ions*7 and a vast and diverse variety of biomolecules that include lots of those oddly shaped amino acids. This busy and crowded molecular pond is in a state of constant agitation and turbulence, with the molecules spinning and vibrating and bouncing off one another in that billiard-ball-like molecular motion that we met in the last chapter.
And there, among all this randomly turbulent molecular activity, are those clam-like enzymes sliding along the collagen fibers, moving in a very different way. At this scale we can zoom in on a single enzyme as it clips its way along the collagen protein chain. At first sight, the overall form of the enzyme molecule looks rather lumpy and amorphous, giving the false impression that it is a rather disorganized assembly of parts. But collagenase, like all enzymes, has a precise structure, with every atom occupying a specific location within the molecule. And, in contrast to the random molecular jostling of the surrounding molecules, the enzyme is performing an elegant and precise molecular dance as it wraps itself around the collagen fiber, unwinding the fiber’s helical turns and precisely snipping the peptide bonds that link the amino acids in the chain before unwrapping itself and moving along to clip the next peptide bond in the chain. These are not shrunken-down versions of manmade machines whose operations are, at a molecular level, driven by the chaotic billiard-ball-like motion of trillions of randomly moving particles. These nanomachines of nature are performing, at a molecular level, a carefully choreographed dance whose actions have been precision engineered by millions of years of natural selection to manipulate the motion of the fundamental particles of matter.
To get a closer look at the cutting action, we descend into the enzyme’s jaw-like cleft that holds the substrates in place: the collagen protein chain and a single water molecule. This is the active site of the enzyme—its business end that is speeding up the breaking of peptide bonds by bending the neck of the energy hourglass. The choreographed action taking place within this molecular steering center is very different from all the random jostling going on outside and around the enzyme, and it plays a disproportionately important role in the life of the entire frog.
The enzyme’s active site is illustrated in figure 3.4. By comparing this diagram with figure 3.3, you can see that the enzyme is restraining the peptide bond in the unstable transition state that has to be reached before the bond can be broken. The substrates are tethered by weak chemical bonds, indicated by dotted lines in the figure, which are essentially electrons that are shared between the substrate and the enzyme. This tethering holds the substrates in a precise configuration ready for the chopping action of the enzyme’s molecular jaws.
As the jaws of the enzyme close, they do something far subtler than simply “biting down” on the bond: they provide the means through which catalysis can take place. We notice a big positively charged atom hanging directly beneath the target peptide bond being swung into position. This is a positively charged zinc atom. If we consider the active site of the enzyme to be its jaws, then the zinc atom is one of its two incisors. The positively charged atom plucks an electron out of the oxygen atom from the substrates to stabilize the transition state and thereby deform the energy landscape: the hourglass has just had its neck widened.
Figure 3.4: Breakage of the peptide bond (in bold) of collagen at the active site of collagenase. The transition state of the substrate is shown by the dashed lines. The sphere to the left of center at the bottom is the positively charged zinc ion; the top carboxyl group (COO) is from a glutamate amino acid at the enzyme active site. Note that molecular distances are not drawn to scale.
The rest of the job is carried out by the enzyme’s second molecular incisor. This is one of the enzyme’s own amino acids, called glutamate, which has swung into position to hang its negatively charged oxygen atom over the target peptide bond. Its role is first to pluck a positively charged proton out of the tethered water molecule. It then spits this proton into the nitrogen atom at one end of the target peptide bond, giving it a positive charge which draws electrons out of the peptide bond. You may remember that electrons provide the glue of chemical bonds; so drawing the electron out is like pulling the glue out of a bonded joint, causing it to weaken and break.5 A few more electron rearrangements and the products of the reaction, the broken peptide chains, are expelled from the enzyme’s molecular jaws. A reaction that might otherwise take upward of sixty-eight million years has been completed in nanoseconds.
But where does quantum mechanics come into the picture? To appreciate why we need quantum mechanics to explain enzyme catalysis, we will pause for a moment to consider again those insights provided by the quantum mechanics pioneers. We have already mentioned the special role played by those few particles at the active site of the enzyme whose choreographed motions are in stark contrast to the random molecular jostling going on elsewhere in the molecular environment. Here, highly structured biomolecules interact in very specific ways with other highly structured biomolecules. This can be seen as either Jordan’s dictatorial amplification or Erwin Schrödinger’s “order from order” that goes all the way down from the developing frog through its organized tissues and cells down to the fibers that hold those tissues and cells together and the choreographed motion of fundamental particles within the active site of collagenase that remodels those fibers and thereby affects the development of the entire frog. Whether we choose Jordan’s model or Schrödinger’s, what is going on here is clearly very different from the chaotic molecular motion that pushes trains up hillsides.
But does this molecular order allow a different set of rules to come into play in life, as Schrödinger claimed? To discover the answer to this question we need to know a little more about that different set of rules that operates at the scale of the very small.
Does such choreographed molecular motion necessarily involve quantum mechanics? We have discovered that the ability of collagenase to accelerate the breakage of peptide bonds involves several of the catalytic mechanisms that chemists routinely use to accelerate chemical reactions, without recourse to quantum mechanics. For example, the zinc metal atom at the active site of the enzyme appears to be playing a similar role to the hot platinum metal that Peregrine Phillips used in the nineteenth century to accelerate the manufacture of sulphuric acid. These inorganic catalysts rely on random molecular motions, rather than choreographed actions, to bring their catalytic groups close to their substrates and thereby accelerate their chemical reactions. Is enzyme catalysis just a collection of several straightforward classical catalytic mechanisms packed into active sites, thereby providing the vital spark that ignites life?
Up until recently, nearly all enzymologists would have said yes; standard transition state theory, with its description of the different processes that help extend the life of the intermediate transition state, was considered to be the best explanation of how enzymes work. But after all the known contributing factors were taken into account, some doubts emerged. For example, the different possible mechanisms that can speed up the peptide cleavage reaction discussed earlier in this chapter are each well understood and give rise, individually, to rate enhancement factors of up to about a hundredfold. But even if you multiply all these factors together, the most that can be achieved is about a million-fold enhancement in reaction rate. This is a puny number compared to the kinds of rate enhancement that enzymes are known to deliver: there seems to be an embarrassingly large gap between theory and reality.
Another puzzle is how enzyme activity is affected by various kinds of change to the structure of the enzymes themselves. For example, like all enzymes, collagenase consists essentially of a protein chassis on a string that supports the jaws and teeth of the enzyme within its active site. We would expect that changing the amino acids that form its jaws and teeth would have a big impact on an enzyme’s efficiency, and indeed it does. What is more surprising is the discovery that changing amino acids within the enzyme that are far from the active site can also have dramatic effects on its efficiency. Why these supposedly innocuous modifications to enzyme structure make such a dramatic difference remains something of a mystery within standard transition state theory; but it turns out that they make sense if quantum mechanics is brought into the picture. We will return to this discovery in the last chapter of the book.
Yet another problem is that transition state theory has so far failed to deliver artificial enzymes that work as well as the real ones. You may remember Richard Feynman’s famous dictum, “What I cannot create, I do not understand.” This is relevant to enzymes because, despite knowing so much about enzyme mechanisms, no one has so far managed to design an enzyme from scratch that can produce anything like the rate enhancements delivered by natural enzymes.6 According to Feynman’s criterion, we do not yet understand how enzymes work.
But take another look at figure 3.4 and ask the question: What is the enzyme doing? The answer is pretty obvious: enzymes manipulate individual atoms, protons and electrons, within and between molecules. Up to now in this chapter we have considered these particles as behaving pretty much as though they were tiny lumps of electric charge being pushed and pulled from one place to another in ball-and-stick-like molecules. But as we saw in our explorations of the last chapter, electrons, protons and even whole atoms are very different from such classical balls because they adhere to the rules of quantum mechanics, including the weird ones that depend on coherence but are normally filtered out at the macroscopic level of billiard balls by that process of decoherence. Billiard balls are not, after all, good models for fundamental particles; so, to understand the real action that goes on inside the active sites of enzymes, we must leave our classical preconceptions behind and enter the weird world of quantum mechanics where objects can be doing two or a hundred things at once, can possess spooky connections and can pass through apparently impenetrable barriers. These are feats that no billiard ball has ever accomplished.
As we have discovered, one of the key activities of enzymes is to move electrons around within substrate molecules, as for example when collagenase pushes and pulls electrons within the peptide molecule. But as well as being pushed around within molecules, electrons can also be transferred from one molecule to another.
A very common type of electron transfer reaction in chemistry takes place during a process called oxidation. This is what happens when we burn carbon-based fuels, such as coal, in air. The essence of oxidation is movement of electrons from a donor to an acceptor molecule. In the case of burning a lump of coal, high-energy electrons from carbon atoms move to form lower-energy bonds within oxygen atoms, giving rise to carbon dioxide. The surplus energy is released as the heat of a coal fire. We harness this thermal energy to heat our homes, cook our food and turn water into the steam that drives an engine or powers a turbine to generate electricity. But the burning of coal and internal combustion engines are fairly crude and inefficient devices for utilizing electron energy. Nature long ago discovered a far more efficient means of capturing this energy, through the process of respiration.
We tend to think of respiration as the process of breathing: taking the oxygen we need into our lungs and expelling carbon dioxide as a waste product. But breathing is in fact a combination of just the first (the delivery of oxygen) and last (the expulsion of carbon dioxide) steps of a far more complex and orderly molecular process that goes on within all our cells. It takes place inside complex organelles*8 called mitochondria, which look a bit like bacterial cells trapped inside our own larger animal cells, since they too have internal structures such as membranes and even their own DNA. In fact, mitochondria almost certainly evolved from a symbiotic bacterium that made a home inside the ancestor of animal and plant cells hundreds of millions of years ago and then lost the ability to live independently. But their ancestry as an independently living bacterial cell probably explains why they are capable of executing such an extraordinarily intricate process as respiration. In fact, in terms of chemical complexity, respiration is probably second only to photosynthesis, which we will meet in the next chapter.
To home in on the role that quantum mechanics plays here, we will need to simplify how respiration works. And even when simplified, it still involves a remarkable sequence of processes that beautifully convey the wonder of these biological nanomachines. It starts off with the burning of a carbon-based fuel, in this case the nutrients we get from our food. For example, carbohydrates are broken down in our gut to yield sugars, such as glucose, that are loaded into the bloodstream and then delivered to cells hungry for energy. The oxygen needed to burn this sugar fuel is delivered by the blood from the lungs to the same cells. Just as with the burning of coal, electrons in the outer orbits of carbon atoms within a molecule are transferred to a molecule called NADH. But instead of being used immediately to bond to the oxygen atoms, the electrons are passed from one enzyme to another along a respiratory chain of enzymes inside our cells, rather like the baton being passed from one runner to another in a relay race. At each transfer step the electron is dropped into a lower-energy state and the difference in energy is used to power enzymes that pump protons out of the mitochondria. The resulting proton gradient from the outside to the inside of the mitochondria is then used to drive the rotation of another enzyme, called ATPase, which makes a biomolecule called ATP. ATP is very important in all living cells as it acts as a kind of energy battery that can easily be transported around the cell to power lots of energy-hungry activities, such as moving or building bodies.
The function of the electron-driven proton-pumping enzymes is a bit like that of hydroelectric pumps that store excess energy by pumping water up a hillside. The stored energy can then be released by letting the water flow down the hillside to rotate a turbine engine that generates electrical power. Similarly, respiratory enzymes pump protons out of the mitochondria. When the protons flow back inside, they power the rotations of the turbine-like ATPase enzyme. These rotations drive another set of choreographed molecular motions that bolt a high-energy chemical phosphate group onto a molecule within the enzyme to make ATP.
Extending the analogy of this energy-capturing process as a relay race, we can imagine the baton being replaced by a bottle of water (representing the electron energy), with each runner (enzyme) taking a sip of water and then passing on the bottle, before finally the remainder of the water is poured into a bucket called oxygen. This capturing of the electron energy in small chunks makes the whole process much more efficient than simply pouring it directly into oxygen, as very little of it is lost as waste heat.
So the key events of respiration actually have very little to do with the process of breathing, but consist instead of an orderly transfer of electrons through a relay of respiratory enzymes inside our cells. Each electron transfer event, between one enzyme and the next in the relay, takes place across a gap of several tens of angstroms—a distance of many atoms—much farther than was thought to be possible for conventional electron-hopping. The puzzle of respiration is how these enzymes are able to shift the electrons so quickly and efficiently across such big molecular gaps.
This question was first asked as far back as the early 1940s by the Austro-Hungarian–American biochemist Albert Szent-Györgyi, who won the Nobel Prize in medicine in 1937 for his part in the discovery of vitamin C. In 1941, Szent-Györgyi delivered a lecture entitled “Towards a New Biochemistry” in which he proposed that the way electrons flow easily through biomolecules is similar to how they move in semiconductor materials such as the silicon crystals used in electronics. Unfortunately, it was realized just a few years later that proteins are in fact rather poor conductors of electricity, so electrons would not easily flow through the enzymes in the way that Szent-Györgyi envisaged.
Major advances in chemistry were made during the 1950s, in particular by the Canadian chemist Rudolph Marcus, who developed a powerful theory that is today named after him (Marcus theory) and which explains the rate at which electrons can move or jump between different atoms or molecules. He too eventually received a Nobel Prize, in chemistry, in 1992 for his work.
But half a century ago, the issue of how respiratory enzymes in particular were able to encourage such rapid transfer of electrons across relatively large molecular distances remained a puzzle. One suggestion was that proteins might rotate in sequence like clockwork machines, bringing distant molecules close together so that the electrons could easily hop across. An important prediction of these models was that this mechanism would slow down dramatically at low temperatures, when there is less thermal energy to drive the clockwork motion. Yet in 1966, one of the very first real breakthroughs in quantum biology came from experiments carried out at the University of Pennsylvania by two American chemists, Don DeVault and Britton Chance, who showed that, contrary to all expectations, the rate of electron-hopping in respiratory enzymes did not drop at low temperatures.7
Don DeVault was born in Michigan in 1915 but moved west with his family during the Depression. He studied at Caltech and Berkeley in California and received a PhD in chemistry in 1940. He was a committed human rights activist and spent time in prison during the Second World War for his stance as a conscientious objector. In 1958, he resigned his post as professor of chemistry at the University of California to move to Georgia so that he could be directly involved in the struggle for racial equality and integration in the South. His strength of conviction, dedication to the cause and adherence to peaceful protest exposed him to the risk of physical attack during marches with black activists. He even had his jaw broken on one occasion when his racially mixed group of protesters was attacked by a mob. But this didn’t deter him.
In 1963, DeVault went to work at the University of Pennsylvania with Britton Chance, a man just two years his senior who had already established a worldwide reputation as one of the leading scientists in his field. Chance had obtained not one but two PhDs, the first in physical chemistry and the other in biology. So his “field” of expertise was very wide and his research interests diverse. He had spent much of his career working on the structure and function of enzymes—while taking time out to win a gold medal in sailing for the United States in the 1952 Olympics.
Britton Chance had been intrigued by a mechanism by which light can promote the transfer of electrons from the respiratory enzyme cytochrome to oxygen. Together with Mitsuo Nishimura, Chance found that this transfer takes place in the bacterium Chromatium vinosum even when its cells are cooled to a chilly liquid nitrogen temperature of −190°C.*9 But how this process varied with changing temperature, which might provide clues to the molecular mechanism involved, was still unknown. What was required, Chance realized, was to initiate the reaction very rapidly with a very brief, but intense, flash of light. This is where Don DeVault’s expertise came in. He had spent some years working as an electrical consultant for a small company developing a laser that could provide just such short light pulses.
Together, DeVault and Chance designed an experiment in which a ruby laser delivered a brief flash of bright red light for just 30 nanoseconds (30 billionths of a second) to bacterial cells packed full of respiratory enzymes. They found that as they reduced the temperature the rate of electron transfer fell until, at about 100 K (or −173°C), the electron transfer reaction time was about a thousand times slower than it was at room temperature. This was to be expected if the process of electron transfer was driven primarily by the amount of thermal energy involved. However, something odd happened when DeVault and Chance reduced the temperature below 100 K. Instead of dropping to lower values, the rate of electron transfer seemed to have reached a plateau, remaining constant despite further reduction in temperature, right down to 35 degrees above absolute zero (−238°C). This indicated that the electron transfer mechanism cannot be due solely to the “classical” electron-hopping described earlier. The answer, it seems, lies in the quantum world, specifically in the weird process of quantum tunneling that we met in chapter 1.
You may remember from chapter 1 that quantum tunneling is the peculiar quantum process that allows particles to pass through impenetrable barriers as easily as sound passes through walls. It was first discovered in 1926 by the German physicist Friedrich Hund and was soon after used successfully to explain the concept of radioactive decay by George Gamow, Ronald Gurney and Edward Condon, all using the then new mathematics of quantum mechanics. Quantum tunneling became a staple feature of nuclear physics, but it was later appreciated as a phenomenon that applied more widely in material science and chemistry. As we have already seen, it is essential for life on earth as it allows pairs of positively charged hydrogen nuclei in the interior of the sun to fuse together in the first step of converting hydrogen to helium, thereby releasing the sun’s vast energy. However, until recently, it was not thought to be involved in any living processes.
One way of thinking about quantum tunneling is as a means by which particles can get from one side of a barrier to the other in a way that common sense tells us should be impossible. By “barrier” we mean here a physically impassable region of space (without sufficient energy)—think of force fields used in science fiction stories. This region could consist of a narrow insulating material separating two sides of electric conductors or even empty space, such as the gap between two enzymes in a respiratory chain. It can also be the kind of energy hill that we described earlier, which limits the rate of chemical reactions (figure 3.1). Consider the example of a ball being kicked up a small hill. In order for it to reach the top and roll down the other side it has to be given a firm enough kick. As it climbs the slope it will gradually slow down, and without sufficient energy (a hard enough kick) it will simply stop and roll back again the way it came. According to classical Newtonian mechanics, the only way a ball can get across the barrier is for it to possess sufficient energy to be lifted over the energy hill. But if that ball were an electron, say, and the hill a repulsive energy barrier, then there would be a small probability that the electron would flow through the barrier as a wave, essentially making an alternative and more efficient passage through. This is quantum tunneling (figure 3.5).
An important feature of quantum mechanics is that the lighter the particle, the easier it is for it to tunnel. It is not surprising, therefore, that once this process was understood to be a ubiquitous feature of the subatomic world it was the tunneling of electrons that was found to be most common as they are very light elementary particles. The field emission of electrons from metals was explained as a tunneling effect in the late 1920s. Quantum tunneling also explained how radioactive decay takes place: when certain atomic nuclei such as those of uranium occasionally spit out a particle. This became the first successful application of quantum mechanics to the problems of nuclear physics. In chemistry, quantum tunneling of electrons, protons (hydrogen nuclei) and even heavier atoms is today well understood.
A crucial feature of quantum tunneling is that, like many other quantum phenomena, it depends on the spread-out wave-like nature of matter particles. But for a body made up of very many particles to tunnel it has to maintain the wave aspects of all its constituents marching in step, with peaks and troughs of waves coinciding, something we refer to as the system being coherent, or simply “in tune.” Decoherence describes the process whereby all the many quantum waves very rapidly get out of step with one another and wash away any overall coherent behavior, thus destroying the body’s ability to quantum tunnel. For a particle to quantum tunnel, it must remain wavy in order to seep through the barrier. This is why big objects, such as footballs, do not quantum tunnel: they are made up of trillions of atoms that cannot behave in a coordinated coherent wave-like fashion.
By quantum standards, living cells are also big objects, so at first glance it would seem unlikely that quantum tunneling would be found inside hot, wet living cells whose atoms and molecules would mostly be moving incoherently. But, as we have discovered, the interior of an enzyme is different: its particles are engaged in a choreographed dance rather than a chaotic rave. So let us explore how this choreography can make a difference to life.
It took several years for the unexpected temperature profile of DeVault and Chance’s 1966 experiment to be fully explained. Another American scientist whose work spanned many disciplines, ranging from molecular biology to physics to computer science, is John Hopfield. Best known for his work on developing neural networks in computing, Hopfield was nevertheless very interested in the physical processes involved in biology. In 1974 he published a paper entitled “Electron transfer between biological molecules by thermally activated tunneling,”8 in which he developed a theoretical model to explain the DeVault and Chance result. Hopfield pointed out that at high temperature the vibrational energy of the molecules would be sufficient to allow the electrons to hop over the top of a barrier without tunneling. As the temperature is reduced, there shouldn’t be enough vibrational energy for the enzymatic reaction to take place. But DeVault and Chance had found that the reaction did proceed at low temperatures. Hopfield therefore suggested that at these lower temperatures the electron is raised to a state sitting halfway up the energy slope, where the distance it needs to traverse is shorter than it is at the bottom of the slope, enhancing its chances of quantum tunneling through the barrier. And he was right: the tunneling-mediated transfer of electrons takes place even at very low temperatures, just as DeVault and Chance found.
Few scientists now doubt that electrons travel along respiratory chains via quantum tunneling. This places the most important energy-harnessing reactions in animal and (nonphotosynthetic) microbial cells (we will be dealing with the photosynthetic sort in the next chapter) firmly within the sphere of quantum biology. But electrons are very light, even by the standards of the quantum world, and their behavior is inevitably very “wave-like.” They should not therefore be regarded as moving and bouncing about like tiny classical particles, despite the fact that they are still treated this way in many standard biochemistry texts that continue to use the “solar system” model of the atom. A much more appropriate representation of the electrons in an atom is as a spread-out, wavy cloud of “electronness” surrounding the tiny nucleus, the “cloud of probability” that we discussed in chapter 1. It is perhaps not so surprising, therefore, that electron waves can pass through energy barriers rather like sound waves passing through walls, as we described in that first chapter, even in biological systems.
But what about bigger particles, such as protons or even whole atoms? Can these also tunnel in biological systems? At first glance you would think the answer would be no. Even a single proton is two thousand times as heavy as an electron, and quantum tunneling is known to be exquisitely sensitive to how massive the tunneling particle is: small particles tunnel readily whereas heavy particles are far more resistant to tunneling unless the distances to be covered are very short. But recent remarkable experiments indicate that even these relatively massive particles are able to quantum tunnel in enzymatic reactions.
You may remember that, as well as promoting electron transfer, one of the key activities of the enzyme collagenase (figure 3.4) is moving protons to promote breaking of the collagen chain. As already mentioned, this kind of reaction is one of the most common particle manipulation tricks performed by enzymes. About one-third of all enzyme reactions involve moving a hydrogen atom from one place to another. Note here that “hydrogen atom” can mean several things: it could be a neutral atom of hydrogen (H) consisting of an electron around its nucleus (a proton); it could be a positively charged hydrogen ion (H+), which is just a bare nucleus—a proton without its electron; or it could even be a negatively charged hydride ion, which is a hydrogen atom with an extra electron (H-).
As any self-respecting chemist or biochemist will quickly tell you, moving hydrogen atoms (well, protons) around within and between molecules does not necessarily imply any quantum effect; or at least, none that requires us to appeal explicitly to the weirder processes of the quantum world, such as tunneling. Indeed, for most chemical reactions occurring at the kind of temperatures at which life operates, protons are thought to move mostly by non-quantum thermal hopping from one molecule to another. But proton tunneling is involved in a few chemical reactions that can be identified by their relative indifference to temperature, just as DeVault and Chance had demonstrated for electron tunneling.
Life operates at high temperatures (by the standards of the quantum world). So, for most of the history of biochemistry, scientists assumed that enzymatic transfer of protons was mediated entirely by the (non-quantum) mechanism of hopping over the energy barrier.*10 But this view changed in 1989 when Judith Klinman and her colleagues at Berkeley provided the first direct evidence for proton tunneling in enzyme reactions.9 Klinman is a biochemist who has long argued for the importance of proton tunneling in the molecular machinery of life. Indeed, she has gone so far as to claim that it is one of the most important and prevalent mechanisms in the whole of biology. Her breakthrough came from a study of a particular enzyme in yeast called alcohol dehydrogenase (ADH), whose job it is to transfer a proton from an alcohol molecule to another small molecule called NAD+ to form NADH (nicotinamide adenine dinucleotide, a molecule we have met already as the cell’s principal electron carrier). The team was able to confirm the presence of proton tunneling by using an ingenious technique called the kinetic isotope effect. This idea is well known in chemistry and deserves a careful explanation here, for it helps provide one of the main pieces of evidence for quantum biology and will crop up a few times more in this book.
Have you ever cycled up a steep hill only to find yourself being overtaken by people on foot? On level ground, you have no problem cycling effortlessly past any number of pedestrians, even runners, so why is cycling so much less efficient on hills?
Imagine that instead of cycling you got off the saddle and walked the bicycle either along the flat ground or up the hill. Now, the issue becomes obvious. On the hill, you have to push the bicycle as well as yourself up the incline. The weight of the bicycle, which was pretty irrelevant to its horizontal motion along a flat road, is now working against you when you try to get up the hill: you have to raise its weight many meters against the gravitational pull of the earth. This is why racing-bike manufacturers make a big deal of how light their bikes are. Obviously, the weight of an object can make a big difference to the ease of moving it; but our bicycle example illustrates that this difference depends on what kind of motion we’re talking about.
Now, imagine that you wanted to discover whether the terrain between two towns, let’s call them town A and town B, was flat or hilly, but were unable to travel between the towns yourself. A possible strategy emerges when you discover that a postal service exists between the towns manned by postmen who ride either a light or a heavy bicycle. To discover whether the intervening terrain is flat or hilly you need only post a set of identical packages between the towns, sending half via postmen riding the light bicycles and the rest by postmen on the heavier ones. If you discover that all the packages take about the same time to be delivered then you can conclude that the terrain between the two towns is probably quite flat; but if all the packages that arrived on heavy bicycles took much longer, you will conclude that the terrain between A and B is probably hilly. Your cycling postmen thereby act as probes of the unknown terrain.
Atoms of each chemical element come, like bicycles, in groups of different weights. Let’s take hydrogen as our example since it is both the simplest atom and the one of most interest to us here. An element is determined by the number of protons it has in its nucleus (along with the corresponding equal number of electrons surrounding the nucleus). So, hydrogen has one proton in its nucleus, helium has two, lithium has three, and so on. But the nuclei of atoms also contain another type of particle: the neutron, which we met in chapter 1 when discussing the fusion of hydrogen nuclei inside the sun. Adding neutrons to the nucleus makes the atom heavier and therefore changes its physical attributes. Atoms of a particular element that have different numbers of neutrons are called isotopes. The normal isotope of hydrogen is the lightest one, consisting of just the single proton and electron. This is the most abundant form of hydrogen. But there are two rarer, heavier isotopes of hydrogen: deuterium (D), which has a neutron in addition to the proton in its nucleus, and tritium (T), which contains two neutrons.
Since the chemical properties of elements are determined mostly by the number of electrons their atoms contain, different isotopes of the same element, with different numbers of neutrons in their nuclei, will have very similar, but not identical, chemistries. The kinetic isotope effect involves measuring how sensitive a chemical reaction is to the changing of atoms from light to heavy isotopes, and is defined as the ratio of reaction rates observed with heavy and light isotopes. For example, if water is involved in a reaction then the hydrogen atoms in the H2O molecules can be replaced with their heavier cousins, deuterium or tritium, to make D2O or T2O. Just like our cycling postmen, the reaction may or may not be sensitive to the changing weight of the atoms, depending on the route that the reactants take to be converted into products.
There are several mechanisms responsible for significant kinetic isotope effects, and one of them is quantum tunneling, which, like cycling, is extremely sensitive to the mass of the particle that is trying to tunnel. Increasing the mass makes the particle’s behavior less wave-like and hence less likely to be able to seep through an energy barrier. So doubling the mass of the atom, for example changing from normal hydrogen to deuterium, causes its probability of quantum tunneling to plummet.
Finding a big kinetic isotope effect may therefore be evidence that the reaction mechanism—the route between reactants and products—involves quantum tunneling. However, it would not be conclusive since the effect might be attributable to some classical (non-quantum-driven) chemistry. But if quantum tunneling is involved, then the reaction should also show a peculiar response to temperature: its rate should plateau out at low temperatures, just as DeVault and Chance had demonstrated for electron tunneling. This is precisely what Klinman and her team discovered for the ADH enzyme; and the result provided strong evidence that quantum tunneling was involved in the reaction mechanism.
Klinman’s group has gone on to amass substantial evidence that proton tunneling occurs commonly in many enzymatic reactions at the kinds of temperature at which life operates. Several other groups, such as that of Nigel Scrutton at the University of Manchester, have performed similar experiments with other enzymes and demonstrated kinetic isotope effects that point strongly toward quantum tunneling.10 Yet how enzymes maintain quantum coherence to promote tunneling remains a very controversial topic. It has been known for some time that enzymes are not static, but are constantly vibrating during their reactions. For example, the jaws of the collagenase enzyme open and close every time they break a collagen bond. It was thought that these motions were either incidental to the reaction mechanism or were involved in capturing the substrates and bringing all the reactive atoms into the correct alignment. However, quantum biology researchers now claim that these vibrations are so-called “driving motions” whose primary function is to bring atoms and molecules into close enough proximity to allow their particles (electrons and protons) to quantum tunnel.11 We will be returning to this topic, one of the most exciting and fast-moving fields of quantum biology, in the last chapter of the book.
Enzymes have made and unmade every single biomolecule inside every living cell that lives or has ever lived. Enzymes are as close as anything to the vital factors of life. So the discovery that some, and possibly all, enzymes work by promoting the dematerialization of particles from one point in space and their instantaneous materialization in another provides us with a novel insight into the mystery of life. And while there remain many unresolved issues related to enzymes that need to be better understood, such as the role of protein motions, there is no doubt that quantum tunneling plays a role in the way they work.
Even so, we should address a criticism made by many scientists who accept the findings of Klinman, Scrutton and others, but nevertheless claim that quantum effects have as relevant a role in biology as they have in the workings of a steam train: they are always there but are largely irrelevant to understanding how either system works. Their argument is often positioned within a debate about whether or not enzymes evolved to take advantage of quantum phenomena such as tunneling. The critics argue that the appearance of quantum phenomena in biological processes is inevitable given the atomic dimensions of most biochemical reactions. To a certain extent, they are right. Quantum tunneling is not magic; it has been taking place in the universe since its birth. It is certainly not a trick that was somehow “invented” by life. Yet we would argue that its appearance in enzyme activity is far from inevitable, given those hot, wet and busy conditions inside living cells.
Remember that living cells are extraordinarily crowded places, crammed with complex molecules in a state of constant agitation and turbulence, similar to that billiard-ball-like molecular motion we explored in the last chapter that is responsible for driving steam trains up hillsides. If you remember, it is this kind of random motion that scatters and disrupts the delicate quantum coherence and makes our everyday world appear “normal” to us. Quantum coherence would not be expected to survive within this molecular turbulence, so the discovery that quantum effects, such as tunneling, manage to persist in the sea of molecular agitation that is a living cell is very surprising. After all, it was only a decade or so ago that most scientists dismissed the idea that tunneling and other delicate quantum phenomena could be taking place in biology. The fact that they have been found in these habitats suggests that life takes special measures to capture advantages provided by the quantum world to make its cells work. But what measures? How does life keep that enemy of quantum behavior, decoherence, at bay? This is one of the biggest mysteries of quantum biology, but one that is slowly being unraveled, as we will discover in the final chapter.
But before we move on, we must return to where we left our nanosubmarine: at the active site of the collagenase enzyme inside the disappearing tail of a tadpole. We quickly exit the active site as the jaws reopen, allowing the broken collagen chain (and us!) to break free, and leave the clam-like enzyme ready to clip the next peptide bond in the chain. We then take a short cruise through the rest of the tadpole’s body to witness the orderly activities of a few of the other enzymes that are equally vital for life. Following the migrating cells out of the shrinking tail and into the developing rear limbs, we witness new collagen fibers being laid down, like new railroad tracks, to support the building of the adult frog’s body, often from cells migrating out of the disappearing tail. These new fibers are being built by enzymes that capture the amino-acid subunits released by collagenase and bolt them together again into new collagen fibers. Although we do not have the time to dive into these enzymes, within their active sites are the same kind of choreographed motions that we witnessed inside the collagenase enzyme, but now performing the reverse reaction. Elsewhere, all the biomolecules of life—fats, DNA, amino acids, proteins, sugars—are being made and unmade by different enzymes. Also, every action the growing frog performs is similarly mediated by enzymes. For example, when the animal spots a fly, the nerve signals that carry the message from its eyes to its brain are mediated by a group of neurotransmitter enzymes crammed into nerve cells. As it flings out its tongue, the muscular contractions that draw in the fly are driven by another enzyme, called myosin, crammed into muscle cells, which cause those cells to contract. When the fly enters the frog’s stomach, a whole battery of enzymes is released to speed its digestion and release its nutrients so they can be absorbed. Yet more enzymes transform those nutrients into frog tissue, or capture their energy via respiratory enzymes within its mitochondrial cell organelles.
Every vital activity of frogs and other living organisms, every process that keeps them—and us—alive, is accelerated by enzymes, the engines of life, whose extraordinary catalytic power is provided by their ability to choreograph the motions of fundamental particles and thereby dip into the quantum world to harness its strange laws.
But tunneling isn’t the only potential advantage provided to life by quantum mechanics. In the next chapter we will discover that the most important chemical reaction in the biosphere involves another trick of the quantum world.
*1 “Nano” refers to structures on the scale of one nanometer, or one-billionth of a meter.
*2 Yeasts are single-celled fungi.
*3 There are, of course, very important exceptions: principally chemicals such as oxygen that, although reactive, are continuously replenished by processes occurring on our planet, most notably those of living organisms such as the plants that pour oxygen into our atmosphere.
*4 The initial chemical in a reaction is called the reactant; however, when the reaction is helped along by a catalyst such as an enzyme then this initial chemical is referred to as the substrate.
*5 Most enzymes’ names start with that of the initial molecule, the “substrate,” that is consumed in the reaction, and end in -ase; so collagenase is an enzyme that acts on collagen.
*6 This type of bond is known as a covalent bond.
*7 An ion is an atom or molecule that carries an electrical charge as a result of having missing electrons (positive ion) or additional electrons (negative ion).
*8 As you may remember from chapter 2, organelles are the “organs” of cells: internal structures that perform particular functions, such as respiration.
*9 Most scientists use the Kelvin (K) as their unit of temperature. A 1 K change in temperature corresponds to a 1˚C change. However, the Kelvin scale starts at what is called absolute zero, which is equivalent to −273˚C. So, for example, human body temperature is 310 K.
*10 You might wonder why quantum tunneling is therefore necessary to explain the fusion processes inside the sun. But there, even the incredibly high temperatures and pressures are not enough to overcome the electrical repulsion that prevents the fusion of the two positively charged protons, and so quantum mechanics is needed to offer a helping hand.
