Reactive oxygen species (ROS) are not produced only under unusual or harmful conditions. They are generated naturally as part of the everyday processes that keep us alive. Three of the main sources are mitochondria, white blood cells and the process of producing and excreting uric acid.
Cellular respiration: mitochondria, where electrons go astray
From nutrients to the electron transport chain
Something as seemingly trivial as losing electrons helps us produce the energy we need to stay alive. And the place where this remarkable process takes place is the mitochondrion. To see how our bodies produce ATP (adenosine triphosphate), the molecule that supplies energy for our cells, let's briefly follow the process. After we take in nutrients from food, they are carefully broken down, step by step, until they reach the form of pyruvate through a process called glycolysis. The pyruvate is then whisked from the cytoplasm into the mitochondrial matrix, where it is converted into acetyl-CoA, which in turn enters a repeating cycle called the TCA cycle (tricarboxylic acid cycle). The matrix is the innermost space of a mitochondrion, enclosed by its inner membrane. The TCA cycle operating here captures energy from nutrients in molecules called NADH and FADH₂. The energy carried by these molecules is then used in the process that ultimately produces ATP. This takes place through the electron transport chain (ETC), located in the inner mitochondrial membrane. The system consists of four protein complexes and two mobile electron carriers. Here, NADH and FADH₂ give up the electrons they have been carrying, and those electrons are passed along through numerous redox centres within the complexes. Eventually, they reach oxygen molecules, which accept the electrons and combine with hydrogen to form water.
Why electrons take the long way round
But if the electrons are destined for oxygen anyway, why not simply hand them over directly? Why make them travel through such a complicated series of steps? There is a reason. As electrons are transferred, energy is released. Quite a considerable amount of it, in fact. What would happen if all that energy were released at once, in an amount our bodies could not handle? That would be quite frightening. So instead of sending the electrons straight to their destination, the system takes them along a winding route, releasing the energy little by little, in amounts our bodies can manage.
How the electron transport chain produces ATP
But is the energy released as electrons are transferred the very energy our bodies use to carry out their activities? Not quite. That energy is actually used to pump hydrogen ions, or protons, from the mitochondrial matrix into the space between the inner and outer membranes. Mitochondria have two membranes: an inner one and an outer one. The space between them reminds me of the moat surrounding a medieval castle. As mentioned earlier, the electron transport chain lies in the inner membrane, next to the matrix. When NADH and FADH₂ give up their electrons, they also release hydrogen, and the energy generated through electron transfer is used to pump these protons into the space between the two membranes — our castle moat, so to speak. As more and more protons accumulate in this space, their concentration becomes much higher than inside the mitochondrial matrix. This marked difference in concentration, known as a chemical gradient, allows the tightly packed protons to rush back into the mitochondrion. The force generated by this movement is used to produce ATP, the energy currency of our bodies. It works rather like water rushing down to turn a waterwheel, or water falling from a dam and driving a turbine. To turn ADP, which has two phosphate groups (di means two), into ATP, which has three (tri means three), another inorganic phosphate (P) must be attached. The energy needed to form this bond comes from the difference in proton concentration.
Cellular respiration is, of course, a complex process that deserves an article of its own. But why have I spent so much time explaining it here, even if only in a very simplified way? Because this essential, everyday biological process also produces a considerable amount of reactive oxygen species. Their formation is, in a sense, an unavoidable part of the process. The electrons are carefully carried along towards oxygen, but inevitably some stray from their route, go missing or leak out along the way. Instead of reaching the oxygen waiting at the final destination, they end up somewhere they were never meant to go. Think of it as a delivery gone wrong. And it happens more often than we might expect. When these stray electrons react with oxygen, they produce superoxide.
We'll explore radicals in more detail in the articles to come. For now, the point to remember is that mitochondria provide an environment where superoxide radicals and even hydrogen peroxide can readily form. Electrons can escape during their journey through the electron transport chain, while protons and oxygen molecules are present in abundance. The human body is estimated to contain around 30 trillion cells on average [1]. And although the number varies depending on the tissue, a single cell is said to contain roughly 1,000–2,500 mitochondria [2]. ATP production through cellular respiration accounts for about 90% of the body's total ATP production. And because ATP cannot be stored in any substantial amount once it has been made, it must be produced continuously. So just how much ATP do our mitochondria produce in a single day? An average adult is said to produce and use around 100–150 moles of ATP daily, equivalent to approximately 6 × 10²⁵–9 × 10²⁵ molecules. Expressed in terms of energy, this amounts to roughly 730–1,090 kcal per day.[3] An astonishing amount of activity, to say the least. It makes me think, once again, that each of us is a universe in our own right. What matters here is that these figures give us some sense of the enormous scale of ATP production, and therefore the scale on which electrons can inevitably stray or escape along the way. Reactive oxygen species may be produced far more frequently, and on a much greater scale, than we might imagine.
Respiratory burst: the weapons white blood cells use against invaders
When microorganisms such as bacteria, fungi and viruses invade our bodies, macrophages and neutrophils, two types of white blood cells, are among the first to respond and arrive at the scene. They form part of our immune system's first line of defence. After engulfing pathogens, these cells produce reactive oxygen species and use them as weapons to destroy and dispose of the intruders. Phagocytosis, the process of engulfing pathogens, is one of the most basic yet important ways our bodies defend themselves against foreign invaders. But simply swallowing a pathogen is not enough. The captured intruder must also be destroyed. To accomplish this, an enzyme complex called NADPH oxidase is activated, producing a large amount of reactive oxygen species in a short burst. This is known as the respiratory burst. Armed with their powerful oxidising ability, these reactive oxygen species attack and damage various parts of pathogens, including their proteins and cell membranes, helping to kill them. We'll take a closer look at this fascinating process in the next article.
Purine metabolism: reactive oxygen species produced during uric acid formation
Another source of reactive oxygen species is the process in which purines, components of nucleic acids, are broken down into uric acid. An enzyme involved in this process can transfer electrons to oxygen, producing reactive oxygen species along the way. When we hear the words uric acid, gout is probably the first thing that comes to mind. Gout is a painful condition that can cause severe swelling and excruciating pain, particularly in the joint of the big toe. When the concentration of uric acid in the blood becomes too high, monosodium urate crystals can form and accumulate in the joints. The immune system detects these crystals and triggers a powerful inflammatory response, resulting in a gout attack.
Where does uric acid come from?
So where exactly does this troublesome uric acid come from? We often associate gout with the food we eat, but what we mainly consume through food is not uric acid itself. Rather, it is purines, the raw materials from which uric acid is made. Some of the nucleic acids and purine compounds in our food are converted into uric acid as they are digested and metabolised. But our own bodies are constantly producing uric acid as well. Purines are part of the nucleotides that make up DNA and RNA, as well as other nucleotides we use every day, such as ATP and GTP. As these substances undergo metabolism within our cells, their purine components can be broken down. For example, after an mRNA molecule has served its purpose following transcription, it is eventually broken down. Some of its components are recycled, but purine components that are not reused can pass through several metabolic steps and ultimately be converted into uric acid. So whether purines come from the food we eat or are produced within our own bodies, uric acid is formed as they are broken down.
To understand where uric acid comes from, then, we should take a closer look at purines themselves. And to understand purines, we first need to look at the structure of nucleotides. I've explained this in greater detail in my series on nucleic acids, so you may wish to refer to that as well.
To put it simply, nucleic acids are DNA and RNA, the molecules that carry the genetic information of living organisms. Nucleic acids are made up of basic units called nucleotides. Each nucleotide consists of three components: a five-carbon sugar (pentose), a nitrogenous base and a phosphate group. A long chain of these connected nucleotides forms a nucleic acid. Think of the beautiful double-helix structure of DNA that we are all familiar with. Its outer backbone is made of sugar and phosphate, while the nitrogenous bases are positioned on the inside. These bases, belonging to the purine and pyrimidine families, pair with one another and carry the actual genetic information. RNA, although generally single-stranded, is likewise made up of a long chain of nucleotides. The nitrogenous bases found in nucleic acids fall into two families: purines and pyrimidines. A purine has a structure consisting of a six-membered ring joined to a five-membered ring, whereas a pyrimidine has a single six-membered ring. The purine bases are adenine and guanine, while the pyrimidine bases are cytosine, thymine (in DNA) and uracil (in RNA). These bases, which serve as units of genetic information, are constantly broken down and recycled during the normal metabolism of nucleic acids within our cells. Nucleic acids are also broken down when cells are damaged or die, and purines that are not recycled eventually enter the metabolic pathway that produces uric acid.
Uric acid is the final product of purine base metabolism in humans. Unlike most other animals, humans lack uricase, the enzyme that breaks down uric acid one step further. As a result, purines are converted into uric acid, which is then excreted mainly through the kidneys in urine, although some is also eliminated through the intestines. The concentration of uric acid in the blood therefore depends not only on how much is produced but also on how efficiently it is removed from the body. This is something worth bearing in mind when thinking about gout, as mentioned earlier. There is another interesting point I'd like to bring up here: uric acid itself acts as an important antioxidant in the blood. As we continue exploring uric acid, this may begin to seem rather ironic. I've often wondered why humans lost the ability to break down uric acid further, while most other animals still have it. Could we perhaps have evolved this way because of the antioxidant benefits of uric acid? It's a question that has always intrigued me.
The pathways by which the two purine bases, adenine and guanine, are broken down into uric acid are slightly different. Although they belong to the same purine family, the chemical groups attached to their purine rings are not identical. Adenine has an amino group (–NH₂) attached to carbon 6, whereas guanine has an amino group at carbon 2 and an oxygen-containing carbonyl group (=O) at carbon 6. Adenine passes through several metabolic steps before being converted into hypoxanthine, which is then oxidised into xanthine by an enzyme called xanthine oxidoreductase (XOR), and subsequently converted into uric acid. Guanine, on the other hand, skips the hypoxanthine stage and is converted directly into xanthine, before being broken down into uric acid and eventually excreted from the body. But what exactly are hypoxanthine and xanthine? They are not substances with entirely different molecular frameworks from adenine and guanine. They retain the same basic purine ring, but the chemical groups attached to it have changed. To look a little more closely, hypoxanthine has one oxygen atom at position 6 of the purine ring. Xanthine has oxygen atoms at positions 2 and 6, while uric acid has an additional oxygen atom at position 8. Guanine already has an oxygen atom at position 6, so when the amino group at position 2 is removed and replaced with an oxygen-containing group, it becomes xanthine directly. Looking at the sequence hypoxanthine (C₅H₄N₄O) → xanthine (C₅H₄N₄O₂) → uric acid (C₅H₄N₄O₃), we can see that the basic purine framework remains intact while one oxygen atom is added at each stage. Both the conversion of hypoxanthine into xanthine and that of xanthine into uric acid are oxidation reactions catalysed by XOR.
So why have I spent so much time tracing the family tree of purines and explaining how they are metabolised? Because the breakdown of purines into uric acid is not simply a matter of disposing of waste. It can also be an important point at which reactive oxygen species are produced. Now let's take a closer look at how the reactions that oxidise purines can give rise to reactive oxygen species.
Xanthine oxidoreductase (XOR)
XOR oxidises its substrates, accepts the electrons released in the process, carries them through the enzyme and eventually passes them on to another molecule. It is quite a large enzyme, equipped with several cofactors that help it do its job. A purine substrate binds to the active site of XOR. The enzyme then catalyses the conversion of hypoxanthine into xanthine by incorporating an oxygen atom derived from water (H₂O). During this reaction, hypoxanthine loses two electrons.
Hypoxanthine + H₂O → Xanthine + 2H⁺ + 2e⁻
Since hypoxanthine has lost electrons, it has been oxidised. XOR, on the other hand, has accepted those electrons and has therefore been reduced. Whenever electrons are lost somewhere, something else must receive them. XOR passes the electrons it has gained through the enzyme until they eventually reach a cofactor called FAD. FAD then transfers these electrons to another nearby molecule, usually NAD⁺. (NAD⁺ + 2e⁻ + H⁺ → NADH) Now, this is where things become particularly interesting. Scientists initially believed that xanthine dehydrogenase (XDH) and xanthine oxidase (XO) were two separate enzymes. But when scientists examined their amino acid sequences, they discovered that the two were actually different forms of the same protein.[4]
The two faces of XOR: XDH and XO
So why does XOR have two faces? Both forms perform essentially the same task of oxidising hypoxanthine, accepting electrons and passing them on to another molecule. But there is a crucial difference in where those electrons are ultimately delivered. Under normal physiological conditions, XOR generally exists in its XDH form. However, under stressful conditions such as low oxygen levels, ischaemia, inflammation or tissue damage, changes in the protein structure around FAD, or even the cleavage of part of the protein, can alter its three-dimensional arrangement and convert XDH into XO. The problem is that these structural changes make it difficult for NAD⁺, the molecule that normally accepts the electrons, to reach FAD. The smaller oxygen molecule (O₂), however, can still gain access relatively easily. Under ordinary conditions, therefore, XOR in its XDH form transfers most of its electrons to NAD⁺, producing relatively little ROS. But when XOR takes the XO form, it can become a problem because the electrons are instead transferred to oxygen molecules. Why is that? In an earlier article, we looked closely at how oxygen can gain electrons one at a time to become superoxide and eventually be converted into hydrogen peroxide. That very process can unfold here. As XO produces reactive oxygen species such as superoxide (O₂•⁻) and hydrogen peroxide (H₂O₂), the consequences for the cell can change dramatically. The same substance is being oxidised, but where the electrons end up makes all the difference. In other words,
O₂ + e⁻ → O₂•⁻
O₂ + 2e⁻ + 2H⁺ → H₂O₂
An oxygen molecule can gain one electron and become superoxide, or it can accept two electrons and two protons to form hydrogen peroxide. The two equations above show how oxygen can become a reactive oxygen species by accepting electrons. So the very process of producing and excreting uric acid can, under certain conditions, become a powerful source of oxidative stress. Indeed, XOR is known to contribute to tissue damage by producing reactive oxygen species in situations such as ischaemia–reperfusion injury, organ transplantation and inflammatory diseases.[4]
We have now looked at three of the main ways reactive oxygen species are produced. Good or bad, these processes are simply part of the normal workings of life. What I think we should remember is that reactive oxygen species do not necessarily arise through some extraordinary process. They are produced quite naturally in the course of our bodies' everyday activities.
[References]
[1] Sender R, Fuchs S, Milo R. Revised Estimates for the Number of Human and Bacteria Cells in the Body. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991899/
[2] Pizzorno J. Mitochondria - Fundamental to Life and Health. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684129/
[3] Physiology, Adenosine Triphosphate https://www.ncbi.nlm.nih.gov/books/NBK553175/
[4] Xanthine Oxidoreductase-Derived Reactive Species: Physiological and Pathological Effects https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707389/
[Image sources]
Image 1. Melissa Hardy. Electron Transport Chains in Respiration and Photosynthesis. College Biology I. CC BY-NC 4.0.
Image 2. Mango Slices (2016). Phagocytosis. Wikimedia Commons. CC BY-SA 4.0.
