The discovery of ATP, glycolysis, and the Krebs cycle during the first half of the 20th century went a long way in answering the question of how energy from food molecules, such as glucose, is harnessed by the cell. But a huge question remained –namely, how is the bulk of the energy of food molecules converted to ATP?
Chemical energy is contained in electrons. Since electrons can move between different molecules, that energy can travel as well. By harnessing high-energy electrons from each glucose molecule, glycolysis generates ATP from a precursor molecule called ADP (Figure 1). Similarly, the Krebs cycle and other energy pathways also generate ATP, which serves as a kind of energy currency for the cell.
Figuring out the ATP generation process took decades and involved many researchers, but it was off to a good start by the 1930s. At that time, Sir Hans Adolf Krebs was beginning his research. Using newly available instruments, such as the manometer (developed by Krebs’ mentor, Otto Warburg, one of the giants of biochemistry of that era, see Figure 2), biochemists were able to hone in on specific quantities of ATP that are made in different biochemical reactions. They found that glycolysis (the splitting of a glucose molecule into two molecules of pyruvate) generates two or three molecules of ATP for each molecule of glucose that is consumed. The Krebs cycle also generates two ATP molecules for each glucose molecule that is broken down. Additionally, one other reaction – the breakdown of pyruvate to produce acetate that goes into the Krebs cycle – generates two ATP molecules for each glucose molecule that is consumed.
Adding up the ATP molecules generated for each glucose molecule during glycolysis (2-3 ATP), the Krebs cycle (2 ATP), and the conversion of pyruvate to acetate (2 ATP) yields 6-7 ATP molecules per glucose molecule. However, using the Warburg manometer in slightly different experiments (mostly involving liver and muscle tissue), Krebs and his colleagues realized that more than 6-7 ATP molecules are actually generated from each molecule of glucose, a great deal more. Their measurements told them that each glucose molecule actually generates well over 30 ATP molecules, provided that oxygen is available to the cell.
To mid-20th century biochemists, the discrepancy between 30 molecules of ATP generated by the cell and just 6-7 ATP molecules generated by known reactions could mean only one thing. Clearly, the remainder of the ATP must be generated indirectly from other chemical products generated during glycolysis, the Krebs cycle, and the conversion of pyruvate to acetate.
These “other chemical products” are nicotinamide adenine dinucleotide (NADH) and FADH2. For each glucose molecule consumed, Krebs worked out that his cycle generated six molecules of NADH and two molecules of FADH2 (Figure 3). Additionally, it was known that NADH also was produced during glycolysis and during the conversion of pyruvate to acetate. Scientists also observed that NADH and FADH2 are produced during the breakdown of fats (a process called beta oxidation). Just like ATP, both NADH and FADH2 seemed to be all over the cell and connected with energy reactions. This was the state of knowledge on cellular energy during the late 1940s, when Krebs was tweaking the details of his famous cycle.
But as for the specific role of NADH and FADH2 – how the cell could use them to obtain energy – that was a mystery. It was clear that they were carrying the bulk of the energy extracted from glucose, fats, and other body fuels, but it was not clear how that energy was harnessed to produce ATP.
Accounting for the rest of the ATP
At the midpoint of the 20th century, Krebs and other biochemists knew that NADH and FADH2 disappear after their production and transform into slightly different molecules. Using very straightforward chemistry techniques, they saw that both NADH and FADH2 undergo a chemical process called oxidation. In the oxidation reaction, NADH is converted, or oxidized, to a compound called NAD+ and FADH2 is converted (oxidized) to a compound called FADH+. In becoming NAD+ and FADH+, NADH and FADH2 each give up one hydrogen atom and two electrons.
Thus, biochemists began describing NADH and FADH2 as carriers of a sort: carriers of electrons. In their oxidized forms, NAD+ and FADH+, the electron carriers contain less energy than they do in what’s called their reduced forms, NADH and FADH2. The reduced forms of the molecules are more energetic than the oxidized forms, because the electrons physically hold the energy. By liberating a pair of electrons, NADH and FADH2 return to their oxidized forms. That much was clear to Krebs, but this raised the question of what happens to the electrons. They don’t just disappear into nothingness. Clearly, their energy must be used to generate all the rest of the ATP that is not made directly during glycolysis and the other reactions.
Figuring out exactly how this worked required a new researcher, and his name was Peter Mitchell. In the 1960s, Mitchell introduced an idea that he called “chemiosmosis.” It explains how the cell harnesses energy from electrons, and it depends on another phenomenon called electron transport, which had to be discovered first, by other researchers.
Born in England, September 29, 1920, Mitchell showed an interest in science throughout childhood and entered Cambridge University in 1939 at age 19. There, he was influenced especially by two instructors: biochemist Ernest Baldwin and nerve physiology instructor Edgar Adrian. As a child and throughout most of his undergraduate career, Mitchell often did not perform well on tests, but he improved enough for admission to graduate school where he pursued biochemistry.
As a graduate student, Mitchell conceived of numerous, novel experiments but often failed to complete them. Generally, it was his assistant, Jennifer Moyle, who completed the needed laboratory work. On top of that, his first PhD thesis idea was rejected and he was directed to spend an additional three years researching penicillin, a topic that didn’t excite him very much. Mitchell lacked the patience for the nitty-gritty work that goes along with experimental science. But he put in the needed laboratory work to earn his PhD and later to show the world that his chemiosmosis idea was correct.
The fate of the high-energy electrons
Tracking the pathways of NADH and FADH2 over the next few years led to the discovery that these two compounds were actually electron carriers. Like ATP, they were a kind of energy currency, but the currency of NADH and FADH2 is less versatile compared with that of ATP. Imagine ATP as a universal energy currency for the cell; it can be used in many different ways, and thus each molecule of ATP can be likened to a dollar bill. In contrast NADH and FADH2 can be likened to a store-specific gift card. They carry energy value, but that value can be used only for a special purpose. That purpose is the generation of ATP, and the way that ATP is generated begins with the high-energy electrons that NADH and FADH2 acquired during glycolysis, the Krebs cycle, and other pathways.
Once scientists realized that energy could be carried through the cell by transferring electrons between different molecules, researchers began contemplating how this could happen. Harnessing the energy from the high-energy electrons of NADH and FADH2 occurs in two interconnected processes: electron transport and oxidative phosphorylation.
In the 1930s, two Soviet researchers, Vladimir Aleksandrovitch Belitser and Elena Tsybakova, identified the movement of electrons through a series of special enzymes. This transfer of electrons from one enzyme molecule to another was called “electron transport.” In 1953, Dutch researcher Edward Charles Slater identified the various enzymes of the chain and began researching how they operate. The enzymes are embedded in the inner of two membranes that surround each mitochondrion, the powerhouse organelle of eukaryotic cells. They are also in the membranes of certain microorganisms. Within the membrane, the enzymes are lined up, forming a chain, known as the electron transport chain (ETC) (Figure 4). Like NADH and FADH2, each electron carrier enzyme of the ETC is capable of accepting electrons from other molecules, holding those electrons temporarily, and then releasing them to a different electron carrier.
Arriving at the membrane of each mitochondrion, both NADH and FADH2 easily unload their high-energy electrons to the ETC. In addition to being lined up, the electron carriers of the mitochondrial ETC are arranged into four groups known as complexes. Today, we know that NADH “drops off” its electrons at complex I, while FADH2 drops off its electrons at complex II. This is because the electrons donated by NADH actually have more energy than the electrons donated by FADH2. NADH is like a high-energy package; whereas FADH2 is like a lower energy package.
ATP and oxygen
By the mid 20th century, biochemists had an idea that electrons give off their energy gradually while moving through the ETC. The generation of ATP from ADP is called “phosphorylation,” which refers to the addition of a group of atoms known chemically as a phosphate (PO43-) group. Biochemists observed that electrons move through the ETC as ADP is phosphorylated into ATP. Since it was observed to happen in the presence of oxygen, researchers began using the term “oxidative phosphorylation” to describe the generation of ATP connected to electron transport. This contrasts with ATP production that occurs directly in biochemical reactions, such as glycolysis and the Krebs cycle, which is called “substrate level phosphorylation.”
In certain types of human cells, glycolysis (but not the breakdown of fats) can proceed in the absence of oxygen. This means that substrate level phosphorylation of glycolysis can occur in the absence of oxygen. This is called anaerobic glycolysis and when it happens, small amounts of ATP are produced along with pyruvate. This keeps cells alive and working, but there are consequences. Without oxygen powering the ETC to draw the high-energy electrons from NADH, the cell needs a different way to oxidize NADH back to NAD+. That’s because the supply of NAD+ is limited. If NAD+ runs out because it has all been converted to NADH, glycolysis will stop.
To solve the problem, when oxygen supplies are low, the cell converts pyruvate (made during glycolysis) into lactic acid. In being converted to lactic acid, pyruvate receives electrons from NADH. Thus, NADH is oxidized, converted back to NAD+, which then is available to glycolysis. But it’s only a temporary solution. During anaerobic glycolysis in muscle cells, lactic acid builds up, causing pain and cramps. That’s why you feel pain if you start exercising too quickly. But as you exercise more, oxidative phosphorylation kicks in gradually as mitochondria start working harder. NADH from glycolysis moves into mitochondria and delivers electrons to the ETC. Large amounts of ATP are then generated. By giving up electrons, NADH is converted back to NAD+, which then is available to glycolysis, so the production of lactic acid stops.
The big question in the mid 20th century, though, was how does oxidative phosphorylation work? How is energy of the electrons that are delivered to the ETC harnessed for the production of ATP? That’s the question that Peter Mitchell set out to answer.
Transferring the energy
By the early 1960s, a chemist named Robert Joseph Paton Williams proposed a new idea: The energy from electrons delivered to the ETC is converted to ATP using protons (hydrogen atoms without their electrons) as intermediates. To explain how the proton idea might operate, exploring the possible ways protons act as intermediates for ATP production, Williams proposed a very complex chemical mechanism. At the same time as Williams developed these ideas, however, Mitchell also independently proposed that protons couple electron transport with ATP production, but through an entirely different mechanism. Mitchell came up with something simpler called the “proton motive force.”
Imagine blowing up a balloon - forcing air into the balloon stores up energy. And, once forced into the balloon, air will flow out through any hole with great force. Similarly, Mitchell imagined energy being stored, not with air, but with protons forced into the space between the two membranes of a mitochondrion, using energy obtained during electron transport. If there is an opening in the membrane, then the protons will stream out, like air from a balloon, with force. In chemistry, this is known as a proton gradient, but Mitchell used the term proton motive force, because he imagined cells using it for power.
Mitchell used the term chemiosmosis” to describe the overall mechanism of ATP generation that he imagined taking place within mitochondria, and within microorganisms that thrive in oxygen. He hypothesized his proton motive force being harnessed by enzymes to convert ADP to ATP, and also to power other cell processes. For instance, in chloroplasts (organelles that use sunlight to make food in cells of plants and certain other eukaryotes) and photosynthetic bacteria, he imagined the proton gradient transferring sunlight energy to energize various molecules. He also hypothesized proton gradients being used to transform chemical energy into mechanical energy. Many bacteria and other microorganisms move around with a tail like structure called a flagellum, and Mitchell imagined the tiny protons causing a flagellum to move.
Mitchell: An unconventional thinker
When Mitchell proposed chemiosmosis in 1961, his colleagues thought the idea was crazy. Mocking the idea of the proton motive force, which Mitchell abbreviated PMF, his colleagues joked that PMF stood for the “Peter Mitchell Force.” This was mostly because Mitchell lacked evidence to support the idea at the time, but also because he looked and acted rather unorthodox.
Holding fast to chemiosmosis and ignoring those who mocked him, Mitchell did the needed lab work and also watched carefully for discoveries by others that could be relevant to his idea. During the 1960s and 1970s, such relevant discoveries revolved around how the ETC could transfer energy from electrons into a gradient of protons. While the physics is beyond the scope of this module, the take-home message is this: As electrons move along the chain, giving up their energy gradually, special enzymes take that energy and literally pump protons into the intermembrane space (see Figure 4). Recall that the ETC enzymes are lined up, embedded in the membrane. As electrons move along the ETC in a conveyor belt fashion, protons are pumped in a direction perpendicular to the movement of the electrons.
The other research relevant for Mitchell revolved around an enzyme called ATP synthase. Located in the inner mitochondrial membrane, this enzyme proved to be a key component since it acts as a doorway for protons which want to move out from between the two membranes (Figure 6).
Thinking of the balloon analogy from earlier, imagine if there was a wind turbine capturing mechanical energy from the air moving out of the balloon. Like a mini-turbine, the enzyme ATP synthase harnesses the power of the protons streaming out from between the membranes and uses the energy to generate ATP. Vindicated due to the growing understanding of ATP synthase and other discoveries related to membranes, Mitchell was awarded the Nobel Prize in Chemistry in 1978.
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