The constant movement of electrons: redox reactions are the processes of life
Oxidation and reduction, collectively known as redox reactions, involve the movement of electrons between molecules and form the very foundation of life's processes. If we look closely enough, right down to the atomic level, life itself could be seen as an endless series of processes driven by the movement of electrons. After all, it is through these processes that our bodies produce energy, our cells carry out their functions, and signals are transmitted. This is why I find myself agreeing so wholeheartedly with the Hungarian Nobel laureate Albert Szent-Györgyi, who once said, "Life is nothing but an electron looking for a place to rest."
Yet the production of reactive oxygen species (ROS) is an inevitable by-product of these processes. As we saw in the previous articles, ROS are generated during ATP production and the breakdown of biological molecules such as nucleic acids. Immune cells even produce ROS deliberately to destroy invading pathogens. When excessive amounts of ROS accumulate inside cells for various reasons, they can cause oxidative stress and damage biological molecules. Fortunately, however, our cells have mechanisms for detecting and responding to changes in ROS levels. When ROS levels rise, cells can strengthen their antioxidant defences by regulating the expression of genes needed to cope with and adapt to the change. In other words, our bodies have their own systems for maintaining homeostasis by keeping oxidation and antioxidant defences in balance.
But here is something interesting: our bodies do not treat reactive oxygen species merely as substances that need to be eliminated. Apart from attacking pathogens or being removed by antioxidant enzymes, what other roles might these ROS play inside our cells?
Maintaining cellular homeostasis
For a long time, ROS were regarded as toxic by-products of redox reactions, known for damaging biological macromolecules such as DNA, RNA, proteins and lipids, potentially causing cell death or even malignant changes. They have also been blamed for various diseases arising from tissue damage, as well as ageing itself, and have therefore been treated as something to guard against. However, as research has progressed, it has become increasingly clear that ROS are not simply harmful substances that cause damage to our bodies. Scientists have discovered that ROS can regulate physiological signalling pathways depending on their concentration. Hydrogen peroxide (H₂O₂), one of the most representative ROS, provides a good example. Under the tight control of cellular signalling and antioxidant systems, H₂O₂ can be maintained within an appropriate physiological range, allowing it to help regulate cell proliferation, differentiation and death. But if H₂O₂ accumulates beyond the cell's ability to control it, it can damage biological molecules such as proteins, DNA and lipids. When its concentration exceeds this range, various antioxidant systems come into action, attempting to neutralise the excess oxidants and protect the cell. Yet ROS do more than simply trigger these defensive responses. Even under normal conditions, they act as important signalling molecules that regulate cellular functions. Their involvement in inflammatory signalling, an essential part of our immune system, is also well established.
Oxidative eustress
In fact, cells have mechanisms that allow them to regulate the production of H₂O₂, one of the most important ROS, and restrict where it is generated to particular regions or compartments within the cell. For example, when H₂O₂ is produced at a specific location, proteins nearby may be affected. This is because H₂O₂ can oxidise particular amino acid residues in proteins, changing their activity and thereby activating or inhibiting signalling pathways. But what stops the H₂O₂ produced in one location from spreading throughout the cell and affecting other proteins as well? Fortunately, enzymes capable of breaking down H₂O₂ are found throughout the cell, helping to remove it before it can travel too far. As a result, H₂O₂ can act mainly where it is produced and in the surrounding area, allowing cells to transmit signals relatively selectively. This control also reduces the likelihood of ROS affecting other parts of the cell unnecessarily or causing damage.
Through enzymes such as catalase and various other antioxidant defence systems, cells maintain a balance between oxidation and antioxidant activity, helping to preserve cellular homeostasis. The concept of oxidative eustress was introduced to describe the beneficial role of H₂O₂ as a signalling molecule within these regulatory systems, contributing to normal physiological activity. It stands in contrast to oxidative distress, which refers to the harmful state that arises when oxidation becomes excessive.
So how do cells use ROS such as hydrogen peroxide to regulate specific functions? When ROS alter the redox state of a protein, its activity may change, and that change can influence gene expression through signalling pathways. This principle can be seen not only in antioxidant defence but also in cell survival, adaptation to environmental changes, immunity and inflammation. Let's look at how hydrogen peroxide and redox signals influence the activity of four transcription factors.
Four transcription factors regulated by hydrogen peroxide and redox signalling
1. The Keap1–Nrf2 activation pathway
The interaction between Keap1 and Nrf2 (nuclear factor erythroid 2-related factor 2) acts as a thiol-based redox switch that responds to various forms of oxidative stress and plays a crucial role in maintaining redox homeostasis. Nrf2 is a transcription factor that regulates the expression of antioxidant and cell-protective genes, while Keap1 can be thought of as a sensor protein that detects the cell's redox state and regulates Nrf2 activity.
Even under normal conditions, Nrf2 is continuously produced. However, Keap1 captures Nrf2 and connects it to an enzyme complex containing a protein called CUL3. Through the action of this complex, a small protein called ubiquitin is attached to Nrf2, a process known as ubiquitination. Here, ubiquitin acts as a label marking the protein for destruction. Nrf2 carrying this label is rapidly broken down by the proteasome, something like a protein shredder inside the cell. As a result, Nrf2 activity remains low under normal conditions. But why would our bodies keep producing Nrf2 if it is only going to be destroyed? So that they can respond quickly when needed. By continuously producing and breaking down Nrf2, cells can rapidly increase its activity simply by inhibiting its degradation when their redox state changes.
So what happens when the concentration of hydrogen peroxide (H₂O₂) rises? Sulphur atoms in the cysteine residues of Keap1 can become oxidised, leading to various oxidative modifications, including the formation of disulphide bonds (discussed below). These changes inhibit Keap1's ability to promote the degradation of Nrf2. As a result, newly produced Nrf2 is no longer broken down as rapidly and can move into the cell nucleus. As degradation is suppressed, more Nrf2 accumulates inside the cell, and consequently more of it enters the nucleus.
Once inside the nucleus, Nrf2 works with other proteins to bind to antioxidant response elements (AREs), specific DNA sequences found in the regulatory regions of its target genes. It acts as a master transcription factor, regulating the expression of hundreds of genes involved in cellular protection and metabolism. Antioxidant defence also involves what we might call the three main ROS-fighting enzymes: superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). Nrf2 plays a particularly important role in regulating glutathione synthesis and regeneration, as well as the expression of various detoxifying enzymes. An increase in hydrogen peroxide, then, is not simply a sign that the cell is beginning to suffer damage. It can also serve as a signal that allows the cell to detect changes in its redox state and adjust its defence systems accordingly.
☞ Disulphide bonding: A disulphide bond forms when the sulphur atoms of two cysteine residues lose hydrogen through oxidation and form a covalent bond. Such bonds help stabilise the folded structures that give proteins their more complex three-dimensional forms and functions, including tertiary and quaternary structures. Disulphide bonds are particularly important for protein structure. Of the 20 standard amino acids, only methionine and cysteine contain sulphur. In methionine, however, the sulphur atom lies within the side chain rather than at its end, so it cannot form this type of bond with another sulphur atom. Disulphide bonds therefore form between cysteine residues, which contain a thiol group (–SH). When two cysteine residues are oxidised and lose hydrogen, their sulphur atoms form a strong covalent bond with the structure R–S–S–R′. This bond can connect two cysteine residues within the same polypeptide chain or link two separate polypeptide chains. Also known as a disulphide bridge or S–S bond, it is a relatively stable covalent bond that helps maintain the spatial arrangement of a folded protein. When formed in appropriate positions, disulphide bonds can make proteins more resistant to external influences. As their formation through oxidation suggests, disulphide bonds can also arise within or between proteins involved in antioxidant defence pathways under oxidative stress, contributing to the regulation of cell signalling.
The right-hand side of the illustration above shows how disulphide bonds form between cysteine (Cys) residues as a polypeptide chain folds, helping to stabilise its three-dimensional structure. Disulphide bonds play an important role in the formation of numerous biological proteins, including enzymes and hormones. According to one study, cysteine accounts for only about 2.26% of the amino acids found in proteins from humans, cattle and mice, yet approximately 92% of the human proteins analysed contained at least one cysteine residue. Considering that cysteine makes up such a small proportion of amino acids but is present in most proteins, we can begin to appreciate just how important it is in our bodies.[1]
2. FOXO (forkhead box class O) transcription factors
ROS signalling is not limited to antioxidant defence. It also plays a part in how cells survive, repair damage and regulate metabolism. FOXO is another transcription factor that illustrates these functions.
When FOXO proteins are phosphorylated through the AKT signalling pathway, they generally remain in the cytoplasm, where their activity is suppressed. However, they can respond to changes in the cellular redox state by moving into the nucleus and becoming active. In particular, researchers have identified a mechanism in which FOXO4 forms a disulphide bond with a protein called TNPO1 in response to ROS, allowing it to move into the nucleus. TNPO refers to a transport system that helps proteins enter the nucleus, and TNPO1 is the transport protein that assists FOXO4 in this process.
Once inside the nucleus, FOXO promotes the expression of antioxidant genes. One example is the gene encoding Mn-SOD, an enzyme that uses manganese as a cofactor to convert superoxide anion radicals into hydrogen peroxide in mitochondria. Like the Nrf2 transcription factor we examined earlier, FOXO also regulates a wide range of physiological functions beyond antioxidant defence. Its target genes are involved in cell cycle control, the regulation of programmed cell death (apoptosis), DNA repair and glucose metabolism, among other processes. This shows how changes in ROS such as hydrogen peroxide can influence not only antioxidant responses but also the mechanisms that govern cell survival and metabolic activity.
3. Hypoxia-inducible factor (HIF)
Now imagine a situation in which oxygen becomes scarce. How do cells detect this change in their environment and adjust their functions accordingly? Interestingly, ROS produced by mitochondria can also play a part in this process of adaptation.
HIF is a transcription factor that activates and increases the expression of various genes to help cells respond to low oxygen levels, a condition known as hypoxia. Our bodies produce ATP through the electron transport chain (ETC) in the inner mitochondrial membrane, while also maintaining redox balance. If the ETC fails to function properly or becomes inhibited, H₂O₂ production may increase. Under hypoxic conditions, the flow of electrons through the ETC can also change, potentially increasing ROS production, particularly at complex III. The superoxide produced in this way can be converted into hydrogen peroxide (H₂O₂), which may then act as a signal involved in the cell's adaptation to low oxygen levels.
So how is HIF activated? Under normal oxygen conditions, HIF-α is modified by an enzyme that uses oxygen and is subsequently broken down. When oxygen becomes scarce, however, this degradation process is suppressed, allowing HIF-α to accumulate. ROS produced by mitochondria can also influence the stabilisation of HIF-α. Under hypoxic conditions, therefore, ROS can act as signalling molecules involved in how cells detect oxygen availability and adapt to its decline. Once stabilised, HIF-α moves into the nucleus, where it joins HIF-β to form a transcriptional complex. This complex binds to specific DNA sequences called hypoxia response elements (HREs), located in the regulatory regions of target genes, and increases the expression of various genes that help cells cope with and adapt to low oxygen levels. These include genes involved in blood vessel formation, such as vascular endothelial growth factor (VEGF); factors that stimulate red blood cell production, such as erythropoietin (EPO); and enzymes involved in glycolysis, which helps cells produce energy when oxygen is limited. By increasing glycolytic metabolism and promoting the formation of blood vessels and red blood cells, these responses help improve the supply of oxygen to the body's tissues.
Changes in ROS levels during hypoxia, then, are not necessarily harmful to cells. They can also contribute to how cells detect and adapt to an environment where oxygen is scarce. It is important to remember, however, that the main reason HIF becomes activated is the suppression of HIF-α degradation when oxygen levels fall. ROS should be understood as additional signals that help regulate this process.
4. The NF-κB pathway
Immunity and inflammation are two other areas in which ROS act as signalling molecules. In our earlier discussion of the respiratory burst, we saw how ROS attack invading pathogens. Here, let's look at how ROS participate in the signalling pathways that regulate immune and inflammatory responses.
NF-κB (nuclear factor kappa B) is a transcription factor found in almost every type of cell and plays an important role in immune responses, inflammation, cell survival and proliferation. It is one of those essential transcription factors that seems to crop up everywhere. Under resting physiological conditions, NF-κB remains inactive in the cytoplasm, where it is tightly controlled by an inhibitory protein called IκB (inhibitor of NF-κB). NF-κB carries a nuclear localisation signal (NLS) that allows it to enter the nucleus and regulate gene expression. However, IκB masks this signal, keeping NF-κB in the cytoplasm. When inflammatory signals such as cytokines increase, or certain signalling pathways involving ROS become activated, an enzyme called IκB kinase (IKK) is activated and phosphorylates IκB. The phosphorylated IκB is then tagged with ubiquitin, marking it for degradation by the proteasome. Once IκB has been removed, the nuclear localisation signal of NF-κB is exposed. Nuclear transport proteins can now recognise this signal and help NF-κB move into the nucleus at last. Once there, NF-κB binds to specific DNA sequences known as κB sites and regulates gene expression.
What makes H₂O₂ particularly interesting as a signalling molecule in this pathway is that it can either stimulate or suppress NF-κB activation, depending on the circumstances. In the cytoplasm, H₂O₂ can influence upstream signalling pathways that regulate the phosphorylation and degradation of IκB, thereby helping to activate NF-κB. For example, by oxidising cysteine residues in certain phosphatases and inhibiting their activity, H₂O₂ can strengthen the phosphorylation signals required for NF-κB activation. Conversely, oxidative modification of a specific cysteine residue (Cys179) in IKKβ, a component of the IKK complex, can inhibit IKKβ activity. This reduces the phosphorylation and degradation of IκB and may consequently suppress NF-κB activation. NF-κB itself is also subject to redox regulation. Even after NF-κB has entered the nucleus, oxidative reactions involving H₂O₂ can modify a cysteine residue (Cys62) in its p50 subunit. This can interfere with NF-κB binding to DNA and thereby reduce its transcriptional activity. Whether H₂O₂ promotes or inhibits NF-κB activation therefore depends on several factors, including the particular conditions inside the cell, where H₂O₂ acts, which proteins it targets, its concentration and how long the exposure lasts.
What I find particularly interesting here is that the very same hydrogen peroxide can increase or suppress NF-κB activity, depending on the circumstances. Think of a valve controlling the flow of water. Depending on which protein is inhibited, the flow of a signal may become stronger or be cut off altogether. The NF-κB pathway shows us that ROS do not simply act as switches that turn functions on. Even within the same signalling pathway, they can produce very different outcomes depending on which protein they oxidise and where that oxidation takes place.
We have now looked at several ways in which H₂O₂, one of the best-known ROS, acts as a signalling molecule inside cells. Superoxide radicals have relatively greater difficulty crossing biological membranes than H₂O₂, while hydroxyl radicals are so reactive that they are poorly suited to acting as selective signalling molecules. Hydrogen peroxide, being more stable and more capable of crossing membranes, is therefore generally considered the most important ROS involved in cell signalling. There are, of course, reports that superoxide radicals can activate the MAPK/ERK pathway and act as growth signals in various cells, and that singlet oxygen can also participate in signalling pathways and promote programmed cell death. However, ROS are highly reactive and often short-lived, and their concentrations can change rapidly depending on where and when they are produced inside the cell. This makes them difficult to measure accurately in living cells. Determining exactly which ROS was produced, where it originated and which protein it acted upon is even more challenging. Further research is therefore needed to understand the physiological signalling roles of different ROS in greater detail.
In the previous articles, we explored where and how reactive oxygen species are produced. This time, we have looked at how these same ROS are put to work as signalling molecules inside cells. Their roles are more varied than we might expect, from strengthening antioxidant defences and regulating cell survival and metabolism to helping cells adapt to low oxygen levels and controlling immune and inflammatory responses. What I find especially fascinating is that even the same ROS can produce different outcomes depending on which protein it acts upon and which signalling pathway is involved. Our bodies, then, do not simply treat reactive oxygen species as unwanted by-products to be eliminated. They make use of them in many different ways to sustain life and respond to changes in the environment.
[References]
[1] Relationship Between the Occurrence of Cysteine in Proteins and the Complexity of Organisms
Redox signaling at the crossroads of human health and disease
An overview of mechanisms of redox signaling
Signaling Functions of Reactive Oxygen Species
Redox Signaling, Vascular Function, and Hypertension
Reactive oxygen species or reactive sulfur species: why we should consider the latter
The molecular mechanisms regulating the KEAP1-NRF2 pathway
Redox-dependent control of FOXO/DAF-16 by transportin-1
Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing
Crosstalk of reactive oxygen species and NF-κB signaling
[Image sources]
Image 1. Keap1–Nrf2 signalling pathway. CC BY 4.0.
