12.3: The Chemistry and Biochemistry of Dioxygen

Search Fundamentals of Biochemistry

Learning Goals 

 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Dioxygen Chemistry: Electronic Structure and Reactivity

• Explain why ground-state dioxygen is a triplet diradical using molecular orbital theory, and describe how this electronic structure accounts for both the kinetic sluggishness of its reactions with organic molecules and the requirement for spin-paired electron transfer.
• Describe the stepwise one-electron reduction products of dioxygen — superoxide, peroxide, and hydroxyl radical — and explain why each constitutes a reactive oxygen species (ROS) capable of damaging biological macromolecules.
• Compare the reactivity of triplet and singlet dioxygen toward organic substrates, and explain how singlet oxygen is generated biologically and photochemically.

Biological Defenses Against Oxidative Damage

• Explain the catalytic mechanisms of superoxide dismutase and catalase, including the roles of metal cofactors and protein structural features (such as the electrostatic guidance of superoxide to the SOD active site and the hydrophobic channel selectivity of catalase).
• Describe how glutathione, ferritin, and transferrin function collectively to limit oxidative damage, distinguishing between direct ROS scavenging and iron sequestration as protective strategies.
• Explain the Mst1/2–Keap1–Nrf2 signaling axis by which macrophages sense ROS and upregulate antioxidant gene expression.

Oxidative Damage to Biomolecules and Disease

• Identify the major oxidative modifications of proteins (carbonylation, disulfide formation, methionine sulfoxidation), lipids (lipid peroxidation), and DNA (8-oxoguanine formation), and connect each to specific disease processes, including atherosclerosis, neurodegeneration, and cancer.
• Explain how oxidized LDL is recognized by macrophage scavenger receptors and how this initiates atherosclerotic plaque formation, using the structural basis of scavenger receptor–ligand interactions to support the argument.

Evolutionary and Physiological Context of Oxygen

• Describe the Great Oxidation Event and the second oxygenation event, explaining how the rise of atmospheric dioxygen drove both mass extinctions and the evolution of aerobic metabolism, eukaryotes, and multicellular animals.

The History of Oxygen

Oxygen may be considered one of the most important elements in chemistry. Not counting hydrocarbons, there is a greater diversity of molecules with oxygen than with carbon. Given its role in the molecular world, very little time is spent on the chemistry of oxygen in undergraduate chemistry classes. Why is oxygen so special?

Oxygen reacts with atoms of all elements except the Noble gases to form molecules. Water is one of the most important molecules in biology. It :

• provides a perfect solvent for biomolecules
• moderates the Earth's climate
• is the source of almost all the dioxygen in the air

From a chemical point of view, water is a(n):

• nucleophile and electrophile
• acid and base
• oxidizing agent and reducing agent
• a protic solvent that can form H-bonds

The formation of the Earth and the development of life:

The gaseous and dusty environment from which the Earth was formed contained metals and water, which, as you remember from introductory chemistry, can react to form hydrogen gas. H₂ reacts with nonmetals (under various conditions of temperature and pressure) to form H₂S, HCl, CH₄, and NH₃, contributing to the reducing nature of the early atmosphere. This kept the transition metals in their lowest oxidation states. Many metals, including the coinage metals (Cu, Ag, and Au) and the platinum group (Ru, Rh, Pd, Pt), were stable in elemental form.

Then, around 2.7-2.8 billion years ago, photosynthetic organisms (blue/green algae- also called cyanobacteria) developed that could oxidize water to form dioxygen. Oxygen was generally unavailable for redox chemistry before then, as photosynthesis, the process that would evolve to oxidize water to produce dioxygen, was unavailable. Remember that oxidizing water to dioxygen, which is a strong oxidizing agent, requires a stronger oxidizing agent than dioxygen and lots of energy. Fossilized remains of cyanobacteria are found in stromatolites. Using knowledge of how atmospheric oxygen can alter the chemistry of different sulfur isotopes of SO₂, it has been shown that O₂ did not exceed 1 ppm earlier than 2.4 billion years ago, although there might have been isolated pockets with higher concentrations. After that, it rose, presumably due to cyanobacteria. Before this, bacteria oxidized a similar molecule, H₂S, to form elemental sulfur. It could do this by photosynthetically reducing CO₂ with H₂S. Volcanic gases like H₂ might have kept oxygen levels from rising between 2.7 billion and 2.4 billion years ago, when its build-up began. Hydrogen in the form of H₂ and methane probably decreased around 2.4 billion years ago as methane, with its hydrogen atoms, escaped to the upper atmosphere and space. Methane levels would also decrease due to its easy reaction with dioxygen in the presence of UV light, forming CO₂. This would paradoxically lead to cooling of the Earth and pronounced glaciation, as a more potent greenhouse gas, methane, was replaced by a less potent one, carbon dioxide.

Over the next billion years, dioxygen rose to perhaps 0.2-2% (compared to present levels of 20%). Why? Because the early atmosphere was reducing, the added oxygen combined with a large "sink" of reduced metals (such as elemental Cu and Fe) or nonmetals (such as C and ammonia), preventing a large buildup. Only after these reduced substances were "titrated" did dioxygen build up to present levels. In addition, the oxygen might have increased weathering (by oxidation) of sulfur deposits, which can lead to sulfides entering the ocean, which could precipitate ocean iron ions necessary for cyanobacterial chemistry. This would constrain cyanobacterial growth until atmospheric dioxygen levels increased enough to convert sulfides to sulfates. This first increase in atmospheric oxygen is often called the Great Oxidation Event, as it correlated and presumably caused one of the greatest mass extinctions (of anaerobic organisms) of all time.

Around 2.3 billion years ago, redox chemistry changed as trace amounts of dioxygen accumulated in the atmosphere, although isotope evidence suggests that little dioxygen was present in water. Around 1.8-1.5 billion years ago, Earth's atmosphere became somewhat oxygenated, coinciding with the emergence of eukaryotic organisms. Until then, life was restricted to the oceans because there was no ozone layer to absorb dangerous UV radiation. The buildup of dioxygen in the air must have led to another extinction of anaerobic organisms, since, as we shall see, oxygen metabolism products are very toxic. Some evolved to use dioxygen. Ozone developed, and life could migrate from the sea to the land. It wasn't until around 600 million years ago that animals arose, however. Was this event associated with developing a fully oxygenated (20%) atmosphere? Recent evidence shows that substantial oxygen wasn't available in the deep sea until about 600 million years ago, suggesting that. Based on an analysis of iron compounds in Newfoundland waters, oxygen levels were very low in the sea 580 million years ago, during the Gaskiers glaciation. Immediately after that, it rose to levels consistent with atmospheric dioxygen levels of 15%, which is necessary for large animals. Similar trends in carbon and sulfur isotopes in marine rocks in Oman also suggest large increases in oxygen at the end of the Gaskiers glaciation period. What caused this second great oxygenation event? One possibility is that organic matter was sequestered from reactions with atmospheric dioxygen, as clays bound organic molecules in the ocean, and lichens and zooplankton facilitated weathering and the production of insoluble organic material in the oceans.

Dioxygen is critically important to higher organisms, so understanding its chemistry is essential. This chapter will show that dioxygen is a ground-state diradical with low solubility in an aqueous solution. It reacts kinetically slowly in the oxidation reaction and forms toxic byproducts upon reduction. Life forms evolved ways to deal with these problems, including increasing their solubility (with dioxygen-binding and transport proteins) and using enzymes (that could activate it kinetically and detoxify oxygen by-products). Dioxygen is toxic to many cells. Obligate aerobes die in an oxygen environment as this excellent oxidizing agent oxidizes many of their cellular components. Several strains of bacteria swim away from high dioxygen levels. A graph of log survival vs. log pO2 is linear, with a negative slope across various organisms, including mice, fish, rats, rabbits, and insects. Pure oxygen can cause chest soreness, coughing, and a sore throat. Premature infants placed in pure oxygen environments often developed blindness due to retrolental fibroplasia (a buildup of fibrous tissue behind the lens). The trade-off for this toxicity is clear. Energy is derived from organic molecules through oxidation. Before dioxygen became available to power aerobic catabolism of reduced molecules like fatty acids and less reduced sugars, such molecules were only partially oxidized. The glycolytic pathway, found in most organisms, oxidizes glucose (6 Cs) to two pyruvate molecules (3 Cs). With the availability of dioxygen, pathways evolved (Krebs Cycle, mitochondrial electron transport/oxidative phosphorylation) that allowed pyruvate to be fully oxidized to carbon dioxide, releasing much more energy.

The Properties of Dioxygen

It is important to understand the properties of dioxygen since oxidation reactions involving O₂ power not only our bodies but our entire civilization. We will concentrate on biological reactions, but even these show the same characteristics as non-biological ones.

• Oxidation of organic molecules by oxygen is thermodynamically favored but kinetically slow.
• Pure oxygen environments are toxic to cells and organisms.

First, we will try to understand these properties of oxygen, and then we will see how organisms overcome these problems to use dioxygen.

We can understand both properties by examining the molecular orbitals of oxygen and its reduction products, as shown in the diagrams below. Ground-state oxygen is a diradical, which explains its paramagnetic behavior. The two unpaired electrons have a spin state of 1/2, giving a total resultant spin S of 1, making ground-state oxygen a triplet (2S+1) = 3. Organic molecules typically undergo 2-electron oxidation steps. Consider the stepwise oxidation of methane below. The oxidation number of C in methane is -4, -2 in methanol, 0 in formaldehyde, +2 in formic acid, and finally, +4 in carbon dioxide, indicating two electron losses in each step. These states are shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\):

The two electrons lost by the organic substrate are added to oxygen. However, since the two lost electrons are spin-paired, a spin flip must occur to allow the electrons to enter the unfilled oxygen orbitals. Alternatively, energy can be put into ground-state dioxygen to produce excited-state singlet oxygen (S=0, 2S+1 = 1). The large activation energy required (about 25 kcal, or 105 kJ/mol) to flip the electron spin accounts for the sluggish kinetics of dioxygen reactions with organic reactants.

A traditional Lewis structure for ground-state dioxygen cannot be easily written, since the electrons are added in pairs and dioxygen is a diradical. There are 6 electrons in the sigma molecular orbitals from second shell electrons (two each in σ2s, σ2s*, and σ2p,) and 6 electrons in the pi molecular orbitals from second shell electrons (two each in two different π2p orbitals, and one electron each in two different π2p*), so the net number of electrons in bonding orbitals is 4, giving a bond order (or number of 2). In contrast, it is easy to write the Lewis structure of the singlet excited-state oxygen, since all electrons are paired, with two net bonds (1 sigma, 1 pi) connecting the oxygen atoms. Figure \(\PageIndex{2}\) shows the molecular orbital diagram for ground and excited state dioxygen.

Figure \(\PageIndex{2}\): Molecular orbital diagram for ground and excited state dioxygen

This Lewis structure will be used to represent singlet, excited oxygen, which should react more quickly with organic molecules. The excited state singlet on the right is unstable and decays to the middle singlet state. The middle state is approximately 94.3 kJ/mol higher in energy than the ground state triplet (on the left). In quantum mechanical parlance, the transition from the ground state triplet to the singlet state is forbidden for several reasons, making it unlikely that absorption of a photon will induce the transition.

The Reduction of Dioxygen

When oxygen oxidizes organic molecules, it is reduced. By adding electrons one at a time to the molecular orbitals of ground-state dioxygen, we produce the step-wise reduction products of oxygen. On the addition of one electron, superoxide is formed. A second electron produces peroxide. Two more produce two separated oxides since no bonds connect the atoms (the number of electrons in antibonding and bonding orbitals is identical). Each of these species can react with protons to produce species such as HO₂, H₂O₂ (hydrogen peroxide), and H₂O. The first two reactive reduction products of dioxygen make it potentially toxic. Figure \(\PageIndex{3}\) shows the MO diagrams for the reduction products of dioxygen.

Figure \(\PageIndex{3}\): MO diagrams for the reduction products of dioxygen

How are the potential problems in oxygen chemistry dealt with biologically?

Kinetic sluggishness: Enzymes that utilize dioxygen must activate it somehow, decreasing the activation energy. Enzymes that use dioxygen are typically metalloenzymes and often heme-containing proteins. Since metals such as Fe²⁺ and Cu²⁺ are themselves free radicals (i.e., they have unpaired electrons), they react readily with ground-state oxygen, which itself is a radical. The molecular orbitals of the metal and oxygen combine to produce new orbitals, which for oxygen are more singlet-like. Likewise, dioxygen reacts more readily with organic molecules, which can form reasonably stable free radicals, such as flavin adenine dinucleotide (FAD), as we shall see later.

Dioxygen toxicity: Since toxicity arises from oxygen reduction products, enzymes that use oxygen have evolved to bind oxygen and its reduction products tightly (through metal-oxygen bonds), preventing their release into the cells, where they can cause damage. In addition, enzymes that detoxify free dioxygen reduction products are widely found in nature. For example:

• superoxide dismutase catalyzes the dismutation (self-redox) of 2 superoxides into dioxygen and hydrogen peroxide;
• catalase converts hydrogen peroxide into water and oxygen;
• peroxidase catalyzes the reaction of hydrogen peroxide with an alcohol to form water and an aldehyde;
• peroxiredoxins react with peroxides and thioredoxin (a small electron donor) to form water and oxidized thioredoxin.

Finally, free radical scavengers such as vitamins A, C, E, and selenium can react with reactive free radicals to produce more stable free radical derivatives of the vitamins and Se. More on this later.

The Reactions of Dioxygen and Its Reduction Products

Triplet O₂ - Ground State

Here are some reactions for the ground state (triplet O₂):

a. Metal ions - Metal ions are radicals themselves, so they can easily react with dioxygen (think about rust). Here is one example

\[\ce{Fe^{2+} + O2 <=> [ Fe^{2+}-O2 <=> Fe^{3+}-O2^{-.}] <=> Fe^{3+} + \underbrace{O2^{-.}}_{superoxide}} \nonumber\]

b. Autoxidation of organic molecules to produce peroxides - These are multistep reactions that have initiation, propagation, and termination steps.

RH → R^. (Initiation)
R^. + O₂ → ROO^. (Propagation)
ROO^. + RH → R^. + ROOH (Propagation)
R^. + R^. → R-R (Termination)
ROO^. + ROO^. → ROOR + O₂ (Termination)
ROO^. + R^. → ROOR (Termination

Figure \(\PageIndex{4}\) summarizes the triplet ground state dioxygen reactions.

Figure \(\PageIndex{4}\): Reactions of triplet ground state dioxygen

The initiation step above occurs mostly at C atoms, which can produce the most stable free radicals (allylic, benzylic position, and 3^o > 2^o >> 1⁰ carbons).

Single O₂ - Excited State

Figure \(\PageIndex{5}\) shows some reactions for singlet dioxygen in which dioxygen is shown with a double bond and two lone pairs on each oxygen.

Figure \(\PageIndex{5}\): Some reactions for singlet dioxygen

Alkenes react with oxygen to form hydroperoxides, potentially through an epoxide intermediate. Dienes react with oxygen in a Diels-Alder pericyclic reaction to form endoperoxides. A molecular orbital perspective (that you may remember from chemistry classes) on this cycloaddition reaction is shown in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): Pericyclic cycloaddition reactions using molecular orbitals

Singlet oxygen can be made from triplet oxygen by photoexcitation. Alternatively, it can be generated by collision with an excited molecule, which relaxes to the ground state after a radiationless energy transfer to triplet oxygen, forming reactive singlet oxygen. This latter process accounts for the photobleaching of colored clothes, as conjugated dye molecules absorb UV and Vis light and relax to the ground state by transferring energy to triplet oxygen, which then forms singlet oxygen. That can more readily react with the dye's conjugated double bonds. These processes are summarized in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): Photosensitization and the reaction of dyes with singlet state dioxygen

Superoxide

Common reactions of superoxide are shown below.

1. Dismutation: This reaction involves a specified reactant undergoing oxidation, followed by another molecule of the same reactant undergoing reduction. \[\ce{O2^{-.} + O2^{-.} + 2H^{+} <=> H2O2 + O2} \tag{slow}\] \[\ce{HO2^{.} + O2^{-.} + H^{+} <=> H2O2 + O2} \tag{fast}\]
2. Acid/Base: \[\ce{HO2^{.} <=> O2^{-.} + H^{+}} \tag{pKa = 4.8}\]
3. With metal ions: \[\underbrace{\ce{Fe^{3+}}}_{\text{as in heme}} + \ce{O2^{-.}} → \ce{O2} + \ce{Fe^{2+}} \nonumber\]

The enzyme superoxide dismutase catalyzes the dismutation reaction. The common eukaryotic cytosolic form contains Cu²⁺ and Zn²⁺ ions coordinated by histidine side chains. The reaction proceeds in two steps or half-reactions. The first is an electron's removal (oxidation) from superoxide (O₂^-.) through its reduction by Cu²⁺. This reaction forms Cu¹⁺ and nontoxic O₂.

\[\ce{Cu^{2+}-SOD + O2^{−.} → Cu^{+}-SOD + O2} \nonumber\]

(reduction of Cu; oxidation of superoxide)

The second is the addition (reduction) of an electron from a second superoxide to Cu¹⁺ to reform the catalytic Cu²⁺ and, in the process, form the reactive peroxide O₂^2- (unfortunately), which when protonated forms \(\ce{H2O2}\).

\[\ce{Cu^{+}-SOD + O2^{−.} + 2H^{+} → Cu^{2+}-SOD + H2O2} \nonumber\]

(oxidation of copper; reduction of superoxide)

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the electrostatic potential surface of the superoxidase dismutase dimer showing Cu and Zn ions (2SOD).

 

Figure \(\PageIndex{8}\): Electrostatic potential surface of the superoxidase dismutase dimer showing Cu and Zn ions (2SOD) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...zozKPVKr4MzES9

Two dimers are shown with a C2 axis of rotation separating them. Red indicates a negative surface potential, and blue indicates a positive one. Catalytic Cu²⁺ and Zn²⁺ ions are in each subunit and are shown in orange (Cu) and gray (Zn) spheres and labeled (disregard the label not centered on the orange and gray spheres). Note that the Cu²⁺ and Zn²⁺ ions are in the center of a large blue surface (positive potential), which helps "sweep up" any negatively charged superoxide O₂^- nearby. Rotate the complex around the C2 axis, and you will see another large positive blue patch on the backside. These two positive electrostatic surfaces facilitate electrostatic attraction and binding of the dangerous superoxide anion from the larger 3D region surrounding the enzyme.

Zn²⁺ in SOD is not redox active. What is its role? Both metals appear to increase the thermostability of the individual monomer and the dimer. In the presence of metal ions (a holo form of the enzyme), the dimer dissociates into monomers at a higher urea concentration than the apo-dimer. Ion binding reduces the flexibility of groups found in the dimer interface. The protein has a disulfide bond between Cys 57 and Cys 146. This bond stabilizes a metal-ion-binding loop that contributes to the dimer's binding interface. The enzyme operates at diffusion-controlled rates (k_cat/K_M is between 10⁸ and 10⁹ M^-1s^-1), partly due to the attraction of any negatively charged superoxide by the electrostatic field around the dimer.

There are two other superoxide dismutases: one that uses either Mn²⁺ or Fe²⁺ (bacterial, mitochondrial, chloroplasts, protists) and another that uses Ni²⁺(some prokaryotes).

Peroxide

In contrast to dioxygen, which contains multiple bonds between the O atoms, peroxide has only one bond. It is quite weak and requires only 38 kcal/mol (160 kJ/mol) to break it. Remember, bonds can be broken heterolytically (both electrons in a bond go to one of the atoms) or homolytically (one electron goes to each atom).

Figure \(\PageIndex{9}\) shows typical reactions of peroxides.

Figure \(\PageIndex{9}\): Reactions of peroxides

The reaction with Fe²⁺, the Fenton Reaction, is similar to that of triplet O₂ with Fe²⁺. In this reaction, the homolytic cleavage of the O-O bond occurs, generating OH^- and the hydroxy free radical, OH^., which will react with any molecule it encounters. Thermal or photochemical homolytic cleavage of peroxide also forms free radicals that react like the hydroxy-free radical.

The enzyme catalase facilitates the decomposition of the reactive H₂O₂ to water and dioxygen. As such, it offers protection similar to that against superoxide offered by superoxide dismutase. This is the next reaction catalyzed by the enzyme catalase:

The human enzyme is a homotetramer, with each monomer having a heme at the active site. The tetramer also binds NADP⁺ (2 per tetramer), but its function is unclear. Figure \(\PageIndex{10}\) shows a potential mechanism for catalase.

Figure \(\PageIndex{10}\): Human catalase reaction mechanism (adapted from Putnam et al. J. Mol. Biol. 296, 2000,https://doi.org/10.1006/jmbi.1999.3458)

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of human erythrocyte catalase with bound ligand (CN^-) and NADPH (1DGG)

Figure \(\PageIndex{11}\): human catalase with bound ligand (CN^-) and NADPH (1DGG). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...TD7yJsfrNcwSJ7

The cyanide ligands in this structure bind to the heme instead of peroxide. The reaction in mammalian catalases appears to involve a tyrosine radical.

H₂O₂ is very similar in structure to H₂O. How does the enzyme differentiate between them? Both approach the heme through a long 25 Å water channel, with a hydrophobic constriction partway along, leading to the active-site heme. Four waters in the crystal structure are positioned in the constricted opening and in a widened opening just at the heme. The amino acid side chains forming the constriction are Val 74, Val 116, Pro 129, Phe 153, Phe 154, and Trp 186. These allow only small molecules to enter. Two of the four waters form hydrogen bonds to side chains (one to His 75 and Asn 148, and another to Gln 168 and Asp 12. The others don't form hydrogen bonds, are more dynamic, and are more likely to leave the channel. H₂O₂ is bigger and can form bridging hydrogen bonds to side chains that mobile waters can't. They probably leave the opening. H₂O₂ is more polar and has a higher dipole moment (2.26 Debye) than water (1.86 Debye), which implies that hydrogen bonds would be differentially stabilized in this hydrophobic site compared to the less polar water.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of human erythrocyte catalase (monomer) showing the hydrophobic constriction leading to the active site (1DGF)

Figure \(\PageIndex{12}\): Human erythrocyte catalase (monomer) showing hydrophobic constriction leading to the active site (1DGF). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...e48pAa9X4zhcM7

Hydroxyl Free Radical

We won't specifically discuss the reaction of the hydroxyl free radical (^.OH^-) since it will react with anything nearby to produce another free radical. General reactions of the radical are shown in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\): Chemical reactions of the hydroxyl radical. Fabrice Collin. Int. J. Mol. Sci. 2019, 20, 2407; doi:10.3390/ijms20102407. Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

In summary, we reap the benefits of using dioxygen as an oxidizing agent, as it allows aerobic, and hence complete, oxidation of carbohydrates and lipids to CO₂ and H₂O. Yet we pay the price for using O₂ as an oxidizing agent, as its partial reduction products, the superoxide radical, peroxides, and hydroxy free radical, collectively known as reactive oxygen species (ROS), can react with and damage proteins, nucleic acids, and lipids, as we will see below. Figure \(\PageIndex{13}\) summarizes their reactions.

Figure (\PageIndex{14}\): Basis of Reactive Oxygen Species (ROS) generation. Fabrice Collin, ibid.

We have presented the role of superoxide dismutase and catalase in removing ROS in cells. In addition to these elegantly designed proteins, another simpler biomolecule, the tripeptide glutathione (γGlu-Cys-Gly), can reduce ROS in cells and prevent oxidative damage. Glutathione serves as a major antioxidant in cells. It exists in reduced (GSH) and oxidized (GSSG) states, with the ratio depending on cellular oxidative stress. It can react with and detoxify peroxides and alkyl free radicals by the following net reactions:

2 GSH + R₂O₂ → GSSG + 2 ROH

2GSH + 2R^. → GSSG + 2RH

Protection from Fe²⁺ - Ferritin and Transferrin

The Fenton reaction highlights the potential problem of free Fe²⁺ ions in a chemical state that readily supports this reaction and ROS generation. Hence, much of the Fe²⁺ in the body is sequestered in Fe²⁺ binding cofactors like heme and FeS clusters. It is transported in the blood by the protein transferrin and stored in cells, such as erythrocytes, in ferritin. The iron ions in transferrin and ferritin are in the +3 oxidation state (Fe³⁺). This strongly positive cation is quite insoluble in the presence of anions like hydroxide, phosphate, and carbonate. The K_sp value for the hydroxide salt of Fe³⁺ is 3.8x10^-38. Let's detour for a bit and look at the structures of ferritin and transferrin and how they work, starting with ferritin.

Ferritin

The biologically functional form of ferritin is a 24-mer. The structure encapsulates a large volume that can hold many Fe ions (up to 4500) in the central cavity. The ions are stored in the more insoluble form, Fe³⁺, in complexes of oxide and hydroxide. Mammalian ferritin contains both heavy (H) and light (L) chains, so they are hetero 24-mers.

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of ferritin, the intracellular Fe storage protein (1fha).

Figure \(\PageIndex{15}\): Ferritin, the intracellular Fe storage protein (1fha). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...MQXAbPAwNnVya8

The ferritin chains are shown in purple. This structure shows only 24 Fe ions (orange spheres with yellow halos).

Two questions arise. How does Fe²⁺ get into the internal volume of ferritin, and how does it convert to Fe³⁺? There must be a channel through which Fe²⁺ can diffuse. Its conversion to Fe³⁺ requires catalysis by a Fe cluster in the heavy chain (H) subunits. Figure \(\PageIndex{16}\)s shows a generic diagram outlining the processes of uptake and conversion to Fe³⁺ salts inside the central cavity of ferritin.

Figure \(\PageIndex{16}\): (A) Schematic outline of phases involved in the uptake, oxidation, and storage of Fe(II). Hagen et al. Metallomics, 9,(2017). https://doi.org/10.1039/c7mt00124j. Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by/3.0/),

Heart and brain ferritin are enriched in the heavy (H) chain. These two organs require safety from toxic Fe²⁺ ions. Ferritins in organs like the liver and spleen, which store lots of iron, are enriched in the light (L) chain. The two chains are about 50% homologous, but the H chain harbors a dinuclear ferroxidase iron site that catalyzes the Fe²⁺ to Fe³⁺ conversion. Once inside, the L chain surface provides a nucleation site for the deposition of Fe³⁺ into a ferrihydrite "precipitate" ((Fe³⁺)₂O₃•0.5H₂O). The general reaction is:

2Fe²⁺ + O₂ → [Fe³⁺-O-O-Fe³⁺] → [Fe³⁺-O(H)-Fe³⁺]

Figure \(\PageIndex{17}\) shows a possible generic mechanism for the oxidation of the Fe cluster from Fe²⁺ to Fe³⁺.

Figure \(\PageIndex{17}\): Possible mechanism for ferroxidase activity of ferritin heavy chain. (adapted from Hagen et al. Metallomics, 9 (2017). https://doi.org/10.1039/c7mt00124j)

It acts as a ferroxidase, suggesting that dioxygen serves as a ligand to oxidize the two Fe²⁺ ions in the cluster to Fe³⁺.

Figure \(\PageIndex{18}\)s shows a closeup of the interactions of the di-Fe cluster and two other Fe ions bound in the human H chain with water ligands.

Figure \(\PageIndex{18}\): Interactions of the diFe cluster and two other Fe ions bound in the site with water ligands (image made with ChimeraX)

The dinuclear Fe core is shown in the central area of the figure.

Figure \(\PageIndex{19}\) shows an interactive iCn3D model of human heavy-chain ferritin monomer with bound Fe (4zjk)

Figure \(\PageIndex{19}\): Human heavy-chain ferritin monomer with bound Fe (4zjk). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?Ux6ED11dT5dyBb2XA

The crystal structure of the full ferritin structure shows multiple binding sites and a channel to the oxidase site.

Now, let's look at how the light chain L might nucleate the formation of the iron precipitates. Crystal structures show how mineralization likely occurs at a specific site on the light chains that protrude from the inside surface of ferritin. Figure \(\PageIndex{20}\) shows a closeup of the interactions of a di-Fe cluster and two other Fe ions bound in the human L chain with water and peroxide ligands. This site probably represents the nucleation and mineralization site.

Figure \(\PageIndex{20}\): Closeup of the interactions of a di-Fe cluster and two other Fe ions bound in the human L chain with water and peroxide ligands at the probable mineralization site

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of human light chain ferritin with a possible nuclear site for mineralization (5LG8). The structure was made after 60 minutes of mineralization.

Figure \(\PageIndex{21}\): Human light chain ferritin with a possible nuclear site for mineralization (5LG8). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...LKo7WJtoHeLBq9

The structure suggests the presence of a μ(3)-oxo)Tris[( μ (2)-peroxo)] triiron(III) cluster assembled at subsite on the L chains containing the carboxylate ligands Glu60, Glu61, and Glu64 side chains. A Glu57, which is along the incoming path of Fe ions, is involved in Fe delivery and coordination. Figure \(\PageIndex{22}\) shows the electrostatic surface around the nucleation site.

Figure \(\PageIndex{22}\): Electrostatic surface around the nucleation site in light chain ferritin. Here is a link to an interactive iCn3D model: https://structure.ncbi.nlm.nih.gov/i...9LKs4NFkurnhX8

Note that the Fe ions are embedded in a site of negative electrostatic potential arising partly from the localization of the glutamic acid side chains in the site. this

Why doesn't the heavy chain of ferritin perform the same nucleation function in preparation for the crystallization of ferrihydrite? A comparison of Figures 18 and 20 shows that the H chain (which has the ferroxidase activity) has a His 65 ligand instead of a glutamic acid (position 60) as one of the coordinating ligands. This gives Glu 61 greater flexibility, which must inhibit nucleation and mineralization.

Figure \(\PageIndex{23}\) shows two top views (left and center image) and one side view of three contiguous subunits of human heavy-chain ferritin monomer with bound Fe (4zjk). The gray/black sphere is actually a Ca²⁺ ion (which is larger than Fe²⁺) from the crystal structure. This three-monomer cluster would be replicated 8 times to form the full ferritin shell. There is a threefold C3 axis passing through the central calcium atom in the figure, where all the monomers meet. Fe²⁺ must move through these central ions into the internal cavity.

Figure \(\PageIndex{23}\): Opening in ferritin formed by 3 contiguous subunits of human heavy-chain ferritin monomer (4zjk)

Transferrin

Iron ions are moved in the circulation bound to the iron-binding protein transferrin. It binds to a transferrin receptor and can be endocytosed into the cell, where the Fe ions are transferred and stored in ferritin. The transferrin receptor can also bind and internalize circulating ferritin (see pdb 6GSR).

Figure \(\PageIndex{24}\) shows an interactive iCn3D model of transferrin (2N and 2C-lobes) binding to the ectodomain of the transferrin receptor (1SUV).

Figure \(\PageIndex{24}\): Transferrin (2N and 2C-lobes) binding to the ectodomain of the transferrin receptor (1SUV). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...cBhTyxoSDQgH47

The transferrin receptor is shown in gray (2 monomers) bound to the N-lobes (cyan) and C-lobes (magenta) of transferrin. Each transfer lobe has a bound CO₃^2- (spacefill with CPK colors) and a Fe³⁺ ion (orange).

Oxidative Modification of Proteins

Many amino acid side chains can be oxidized in cells, as shown in Table \(\PageIndex{1}\).

Table \(\PageIndex{1}\): Amino acid side chain oxidation products. Berlett and Stadtman. JBC, 272, 20313-20316 (1997). DOI:https://doi.org/10.1074/jbc.272.33.20313. Creative Commons Attribution (CC BY 4.0)

Amino acids	Oxidation products
Cysteine	Disulfides, cysteic acid
Methionine	Methionine sulfoxide, methionine sulfone
Tryptophan	2-, 4-, 5-, 6-, and 7-Hydroxytryptophan, nitrotryptophan, kynurenine, 3-hydroxykynurinine, formylkynurinine
Phenylalanine	2,3-Dihydroxyphenylalanine, 2-, 3-, and 4-hydroxyphenylalanine
Tyrosine	3,4-Dihydroxyphenylalanine, tyrosine-tyrosine cross-linkages, Tyr-O-Tyr, cross-linked nitrotyrosine
Histidine	2-Oxohistidine, asparagine, aspartic acid
Arginine	Glutamic semialdehyde
Lysine	α-Aminoadipic semialdehyde
Proline	2-Pyrrolidone, 4- and 5-hydroxyproline pyroglutamic acid, glutamic semialdehyde
Threonine	2-Amino-3-ketobutyric acid
Glutamyl	Oxalic acid, pyruvic acid

We will focus on one here: the oxidation of the ε-amino group of lysine by H₂O₂ to form α-aminoadipic semialdehyde, where the amine is replaced with an aldehyde. This reaction is called the Fenton reaction.  It is actually a carbonylation reaction. Figure \(\PageIndex{25}\) shows a hypothetical mechanism for the oxidation of lysine side chains.

Figure \(\PageIndex{25}\):

The next result is the oxidation of the lysine ε-amino group to an aldehyde. Oxidized levels of proteins (as evidenced by increased carbonylation levels) increase dramatically with age (especially after age 40 ). The reactions seem to be catalyzed by metals and may proceed by the generation of hydroxy free radicals. Diseases associated with premature aging (Werner's Syndrome, another link to Werner's Syndrome, Progeria) show very high levels of oxidized proteins at an early age. Fibroblasts from 10-year-old children with progeria have levels of oxidized proteins usually not seen until age 70. Beta-amyloid protein deposits (found in Alzheimer's and Down's Syndrome) cause neurotoxicity and death, partly by increasing superoxide production by endothelial cells, causing vasoconstriction/dilation and, ultimately, disease progression. Beta-amyloid aggregates appear to increase H₂O₂ levels in a process facilitated by Fe²⁺ and Cu⁺. Free radical scavengers (antioxidants) may help to prevent this damage.

Carbonylation of proteins appears to be irreversible and nonrepairable. Increased carbonylation leads to misfolding and protein aggregation that protein chaperones cannot reverse. The graphs in Figure \(\PageIndex{26}\) (the summation of many experiments) show the correlation (negative) of increasing carbonylation of proteins (red line), a measurement of oxidative damage, and the resulting decrease in protein function (green).

Figure \(\PageIndex{26}\): Susceptibility of proteoforms to oxidative modifications determines the onset of their oxidation over time and leads to their functional decline. Krisko and Radman, (2019) Open Biol.9. http://doi.org/10.1098/rsob.180249. Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/.

Red dots in the top representations of protein show carbonylation. Variants of the same protein (proteoforms) that have more intrinsically disordered regions (m1) are more susceptible to carbonylation compared to more ordered variants (m3). Cancer starts to increase around 40 years of age, and the levels of carbonylation correlate with increased cancer rates and may, in part, cause it.

Lou Gehrig's Disease (Amyotrophic Lateral Sclerosis) is a disease of progressive motor neuron degeneration, which affects 1/100,000 people and is 10-15% familial. Among familial cases, about 25% harbor a mutation in superoxide dismutase 1, a copper-zinc enzyme. About 2-3% of ALS patients carry 1 of 60 dominant mutations in this enzyme. Mutations often decrease protein stability, reducing Zn²⁺ affinity by 5-50 fold. The A4V mutation (valine at amino acid 4 substituted for Ala) has the weakest Zn affinity and causes rapid disease progression. Without Zn²⁺, the apoprotein somehow seems to induce cell death in neurons. This superoxide dismutase also expresses a second activity. It also acts as a peroxidase, catalyzing the reaction of ROH + H₂O₂ to form an RHO (an aldehyde) and water. In some cases, the enzyme retains normal superoxide activity but altered peroxidase activity.

Figure \(\PageIndex{27}\)s shows the extent of carbonylation of wild type (WT) and two mutant forms of α-synuclein after exposure (in vitro) to increasing doses of γ-radiation. α-synuclein forms aggregates (Lewy bodies) in Parkinson's Disease.

Figure \(\PageIndex{27}\): Wild-type version of α-synuclein is characterized by the highest oxidation resistance. The plot displays the differential resistance to γ radiation of the human α-synuclein protein isomorph. Krisko and Radman, ibid.

The mutants, especially the A53T one, show significantly greater extents of oxidative damage. This particular mutation is associated with early-onset Parkinson's Disease (around 30).

Is your hair going white? Wood et al. have shown that millimolar concentrations of hydrogen peroxide accumulate in hairs that have grayed or whitened. This was associated with a decrease in catalase and increases in Met oxidation (to Met-sulfoxide) in proteins, also associated with a decrease in the repair enzyme Met-sulfoxide reductase, Met 374 in the active site of tyrosinase, an enzyme required for the production of melanin in hair follicles, is also damaged, leading to lack of melanin, a pigment necessary for hair coloration and "senile hair graying".

ROS and Protein Folding

As discussed earlier, the cytoplasm has sufficient concentrations of "β-mercaptoethanol"-like molecules (used to reduce disulfide bonds in proteins in vitro), such as glutathione (γ-Glu-Cys-Gly) and reduced thioredoxin (with an active site Cys), to prevent disulfide bond formation in cytoplasmic proteins. Disulfide bonds in proteins are typically found in extracellular proteins, which keep multisubunit proteins together as they become diluted in the extracellular milieu. These proteins destined for secretion are cotranslationally inserted into the endoplasmic reticulum (see below), which presents an oxidizing environment to the folding protein and where sugars are covalently attached to the folding protein and disulfide bonds are formed (see Chapter 3D: Glycoproteins - Biosynthesis and Function). Protein enzymes involved in disulfide bond formation contain free Cys residues, which form mixed disulfides with their target substrate proteins. The enzymes (thiol-disulfide oxidoreductases, protein disulfide isomerases) have a Cys-XY-Cys motif and can promote disulfide bond formation or their reduction to free sulfhydryls. They are especially redox-sensitive since their Cys side chains must cycle between free and disulfide forms.

Reactive oxygen species (ROS) can significantly affect redox chemistry and, if present in excess, can place the cell under "oxidative" stress. ROS can indiscriminately oxidize lipids, nucleic acids, and proteins, but more specifically, they may also oxidize proteins involved in creating and maintaining the normal disulfide bond formation in proteins. As ROS concentration increases, the concentration of cytoplasmic proteins with incorrect disulfides should increase. Using a two dimension PAGE system (first dimension run under nonreducing and the second reducing conditions) of neural cell proteins derived from cells exposed to normal and differing oxidative conditions (hydrogen peroxide or decreased intracellular glutathione levels, Cumming et al showed that oxidizing stress increased the levels of disulfide bonds in redox-sensitive enzymes and, unexpectedly, among other cytoplasmic proteins involved in many aspects of life, affecting the activity of many cellular processes, suggesting that disulfide bond formation may have not only a structural but regulatory role.

Oxidative Modification of Lipids:

Figure 4 shows the most likely position in organic molecules that can form stable free radicals (allylic, benzylic position, and 3^o > 2^o >> 1⁰ carbons), which are likely targets for reaction with ROS. Hence, unsaturated fatty acids are extra reactive at the methylene C that separates the double bonds, as shown in Figure \(\PageIndex{28}\).

Figure \(\PageIndex{28}\): Reactions of fatty acids with ROS

Lipid and protein oxidation - cardiovascular disease

The initial stages of cardiovascular disease appear to involve the formation of fatty streaks beneath arterial walls. Macrophages are immune cells that have receptors that recognize oxidized lipoproteins in the blood, which they take up. The cells then further differentiate into fat-containing foam cells, forming the streaks. Oxidation of fatty acids in lipoproteins could produce lipid peroxides and, along with the Fenton reaction, lead to the oxidation of apoproteins in LDL. Cortical neurons from fetal Down Syndrome patients show 3-4 times higher levels of intracellular reactive oxygen species and increased lipid peroxidation levels than control neurons. This damage is prevented by treating cultured neurons with free radical scavengers or catalase. Figure \(\PageIndex{29}\) shows key events in atherosclerotic plaque initiation.

Figure \(\PageIndex{29}\): Key events in atherosclerotic plaque initiation. Blood LDL can accumulate within the arterial wall, in the sub-endothelial intima. This accumulated LDL can be chemically modified or oxidized: this new lipid particle species (e.g., oxLDL) promotes chronic inflammation, which promotes the trans-endothelial migration of immune cell types and foam cell development. Abbreviations: endothelial cell, EC; intercellular adhesion molecule-1, ICAM-1; interferon-γ, IFNγ; low-density lipoprotein, LDL; Neutrophil extracellular traps, NETS; oxidized low-density lipoprotein, oxLDL; scavenger receptor, SR; T- helper 1 cell, Th1; vascular cell adhesion molecule-1, VCAM-1; vascular smooth muscle cell, VSMC. Cuthbert et al. Cells 2020, 9(11), 2453; https://doi.org/10.3390/cells9112453. Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

How do fatty streaks appear under the endothelial cells? LDL oxidized in the lipid monolayer and, through carbonylation of lysine side chains of the apoproteins in LDL, binds to "scavenger" receptors in macrophages, which have moved into the intima below the endothelial cell barrier. Scavenger receptors were first recognized to bind acetylated LDL (conversion of ε-amino groups of lysines, for example, to acetylated and uncharged derivatives). This mimics, to some degree, the carbonylation of the ε-amino groups to aldehydes, as shown in Figure \(\PageIndex{30}\).

Figure \(\PageIndex{30}\): Carbonylation and acetylation of lysine side chains

Either modification would make the apoprotein more acidic with a lower isoelectric point since positive lysine side chains are replaced with neutral derivatives. Assuming an asymmetric distribution of the negatively charged side chains (Asp and Glu) on the apoprotein, any pre-modification negative electrostatic potential surfaces would become more negative, enhancing binding to positive clusters displaying positive electrostatic potentials on scavenger receptors. Scavenger receptors often bind polyanions.

Many different classes (A-J) of scavenger receptor classes have been identified. One, class C, is only found in Drosophila. They bind a variety of polyanionic ligands and display broad binding specificity. Many in a single class have multiple names, which makes their designation even more confusing. They bind a diverse set of ligands, including those from bacteria and yeast (in a manner similar to pathogen-associated molecular patterns [PAMPs] in the innate immune system), as well as self- and modified-self ligands (such as oxidized LDL [oxLDL]). Once bound, the ligand and scavenger receptors are internalized by endocytosis to remove and degrade the bound ligand. They can also act in signaling pathways.

The members of the scavenger receptor family are designated as illustrated in this example, SR-F1.1, where S is Scavenger, R is Receptor, F is Class, 1 is Order in class, and 1 is an alternatively spliced form. Figure \(\PageIndex{31}\) shows domain structures of the different classes of scavenger receptors.

Figure \(\PageIndex{31}\): Domain structures of the different classes of scavenger receptors. Zani et al. Cells 2015, 4, 178-201. https://doi.org/10.3390/cells4020178. Creative Commons Attribution 4.0 International

A more detailed cartoon showing examples from a few different scavenger receptor classes involved in cardiovascular disease is shown in Figure \(\PageIndex{32}\).

Figure \(\PageIndex{32}\): Five different SR classes (A, B, E, G, J) are most closely linked to the initiation and progression of atherosclerosis. These molecules are potential biomarkers and therapeutic targets. Their structures are represented in the sketches above. Abbreviations: N-terminus, N; C-terminus, C. Cuthbert et al. ibid.

Modified LDL binds to several different scavenger receptors, including SR-A1 (also called SCARA1 or CD204), SR-A2 (also called MARCO), and SR-E1 (also called Lectin-like oxidized LDL receptor 1 or LOX-1), which binds oxidized and acetylated LDL.

Let's look in greater detail at two scavenger receptors that recognize oxLDL.

SR-A2 (MARCO)

This scavenger receptor is a trimer that binds oxLDL, polyanions, and pathogens. It has an extracellular domain (ectodomain) formed from three cysteine-rich monomers, so it's abbreviated SRCR. A five-stranded antiparallel β sheet and an α helix with a large loop covering it, while the dimer has a larger 8-stranded β-sheet. The polyanion ligands presumably bind to the receptors' surface with a positive (blue in figures) electrostatic potential associated with an arginine cluster. Crystal structures show that the protein also has a region of negative (red in figures) electrostatic potential, which is most likely involved in metal ion binding and in the self-association of monomers to form trimeric receptors.

Given the size of the oxidized LDL (250 Å in diameter), it would not be unexpected that oxLDL binding would promote the formation of clusters of the normal trimer scavenger receptor. This is illustrated in Figure \(\PageIndex{33}\).

Figure \(\PageIndex{33}\): Model for dimerization and oligomerization of trimeric MARCO molecules. Ojalaet al. JBC 282,16654-16666 (2007) DOI:https://doi.org/10.1074/jbc.M701750200. Creative Commons Attribution (CC BY 4.0)

Monomeric, dimeric, and oligomeric forms of SR-A2 (MARCO) are shown in the bottom part of the figure. The trimeric receptor molecules can form dimers and multimers by swapping domains. Multiple interactions would promote tighter binding of large ligands such as LDL and bacteria (0.2-2 μm diameter). The assembly could proceed to oligomer formation (the yellow molecule has swapped domains with three other molecules), resulting in a large surface capable of interacting with large ligands, such as modified LDL (250 Å in diameter) or bacteria (0.2-2 μm). The red and blue surfaces above the trimer represent the negative (red) and positive (blue) electrostatic surface potential of the oligomer.

SR-E1 (Lox1 or Lectin-like oxidized LDLR or Oxidized low-density lipoprotein receptor 1.

LOX-1 is expressed on macrophages, dendritic cells, endothelial cells, platelets, smooth muscle cells, and adipocytes. It binds oxLDL, some bacteria (through their negatively charged cell walls), and even apoptotic cells. Figure \(\PageIndex{34}\) shows an interactive iCn3D model of the extracellular C-type lectin-like domain of dimeric human Lox-1 (1YPQ)

Figure \(\PageIndex{34}\): Extracellular C-type lectin-like domain of dimeric human Lox-1 (1YPQ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...qGY4HBzPjjs7P7

A disulfide bridge connects the dimer in this structure. The two monomers are shown in gray spheres. They come together to form a heart-like structure. A series of arginines (with blue spheres for the surface Ns and labeled) is shown in the cradle of the heart shape. These most likely interact with the oxidized apoprotein B of the oxLDL. Other blue (N) and red (O) spheres near each other can form salt bridges and interact with the zwitterion heads of LDL surface lipids, such as phosphatidylcholine, sphingomyelin, or phosphatidylethanolamine.

Figure \(\PageIndex{35}\) shows an interactive iCn3D model of an AlphaFold predicted model of human oxidized LDL receptor - LOX (P78380)

Figure \(\PageIndex{35}\): . (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...rLoi7ZK4K3SsH8

The yellow spacefill indicates the transmembrane segment. Rotate the model along its long axis, and one face of the top domain shows a red (negative electrostatic potential) face, while the opposite side shows a blue (positive potential) face.

Oxidative Modification of DNA

Significant evidence suggests oxygen-free radicals are linked to aging and diseases. Mutations caused by hydroxylation reactions (presumably generated by hydroxyl free radicals, as shown above) can lead to cancer. A particularly nasty reaction is the insertion of a hydroxyl radical into DNA bases. Figure \(\PageIndex{36}\) shows the hydroxylation at position 8 of guanine to produce 8-oxy-G and at positions 5 and 6 in thymine.

Figure \(\PageIndex{36}\): Hydroxylation of G and T bases by the hydroxyl free radical

Mitochondrial DNA is more susceptible to oxidation than is nuclear DNA. The human mitochondrial genome is small (16.5 kb compared to the nuclear genome of 3 Gb) and encodes 13 protein subunits involved in respiration, 22 tRNAs, and two ribosomal RNAs. (The mitochondria presumably are vestiges of a bacterium that invaded an early cell and established a symbiotic relationship with the cell. There is an inverse correlation between oxidized mitochondrial DNA [8-oxoG] and the maximal life span of an organism, but this correlation is not observed with nuclear DNA. Presumably, the nuclear DNA is somewhat protected from oxidative damage because it is bound to histone proteins (which form nucleosome core particles with DNA) and to DNA repair enzymes. DNA repair enzymes encoded in the nucleus are found in the mitochondria, and mitochondrial DNA is packaged with mitochondrial transcription factor A (TFAM). Examination of human bladder, head and neck, and lung primary tumors reveals a high frequency of mitochondrial DNA mutations. In addition,  most cellular dioxygen use occurs in the mitochondria. Hence, this organelle probably encounters the highest concentration of toxic oxygen-reduction products. Recently, the crystal structure of an enzyme, adenine DNA glycosylase (MutY), that repairs 8-oxoG-modified DNA, has been determined in complex with the oxidatively damaged DNA. If not repaired, the 8-oxoG base pairs with adenine instead of cytosine, causing a GC-to-AT mutation during DNA replication.

Figure \(\PageIndex{37}\) shows an interactive iCn3D model of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase (1RRQ)

Figure \(\PageIndex{37}\): Adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase (1RRQ) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?4RiEc6TuqmexiW1r7

The protein MutY, which catalyzes the base excision and repair, is shown in gray. The DNA strands are shown in magenta and blue. 8-OxyG on the magenta strand is labeled 8OG7 and is shown in CPK-colored sticks. Its mismatched adenine base pair partner, labeled A18 on the blue strand, is shown in CPK-colored sticks. Notice its orientation is kinked away from the orientation in a canonical base pair. Key amino acid side chains (Thr49, Leu86, Tyr88, and Ser308) interacting with the 8-oxyG are shown in CPK-colored sticks, labeled.

Although oxidative damage in mitochondria clearly can promote premature aging, other independent mechanisms may also. Kujoth et al. developed a mouse model expressing a mutant form of mitochondrial DNA polymerase defective in proofreading activity. These mice displayed premature aging but showed no increased levels of oxidized mitochondrial lipids or hydroxylated G residues in mitochondrial DNA. They showed significant activation of a cytosolic enzyme called caspase-3, which, when active, triggers programmed cell death (apoptosis). This calcium-activated aspartic acid protease (with an active site Asp) is activated by binding mitochondrial cytochrome C that has "leaked" into the cytoplasm from its normal location in the intermembrane space in mitochondria. The process is usually associated with DNA damage (mutations, fragmentation) that would arise if the proofreading function of DNA polymerase were defective. This was indeed found in these mice.

Oxidative damage to biomolecules might not initiate aging and disease processes, but rather might be a secondary effect of other initiating events. Reversing or preventing oxidative damage might slow the progression of aging and disease. Aging is a complex feature of organisms and would be expected to have complex causes and biological effects. At the organismal level, aging has been studied in the roundworm C. elegans, which lives for only a few weeks. Genetic analyses can easily find gene alterations associated with premature aging. One hormonal system recently associated with aging in eukaryotes (and in C. elegans) involves the insulin and insulin-like growth factor I (IGF-1) signaling pathways, which regulate carbohydrate, lipid, and reproductive pathways in C. elegans. Mutations that reduce signaling through this pathway increase C. elegans lifespan. These mutations increase the activity of the DAF16 transcription factor, which upregulates the expression of many genes. In contrast, when exposed to insulin or IGF-1, wild-type organisms decrease DAF16 activity. Using DNA microarrays, investigators determined which DAF16-controlled genes were upregulated in mutant worms at midlife. These genes included, among others, peroxisomal and cytosolic catalase, Mn-superoxide dismutase, cytochrome P450, metallothionein-related Cd-binding protein, and heat shock proteins. In the next section, we will investigate the function of several of these gene products. Still, needless to say, they are all involved in cellular responses to stress, often involving dioxygen metabolites. The overexpression of mitochondrial catalase in mice increased their lifespan by 20%. Decreased levels of insulin-like growth factor also promote longevity in mice, further indicating that mechanisms beyond oxidative damage by ROS are involved in aging.

Beneficial Oxidation of Proteins: Oxidative Burst in Macrophages

There are cases in which oxidative damage to protein and lipids is desirable. One example involves the role of macrophages in the immune system, which eliminate foreign microorganisms. When macrophages recognize and engulf microbes, one mechanism deployed to kill the microorganism is oxidative damage. Stimulated macrophages undergo an oxidative burst, leading to increased oxygen consumption. One outcome of this is the generation of ROS. Activating the ROS-generating system can also kill the macrophage (which is OK).

In addition, immune function decreases with age. This probably also occurs due to ROS damage. Oxidative stress and irradiation also shorten telomeres at the end of chromosomes. They are enriched in guanine bases with many repeats (thousands) of TTAGGG sequences and, hence, are susceptible to oxidation. However, as you might expect, macrophages have developed mechanisms to limit ROS-mediated self-damage.

In the presence of ROS, the macrophage or mitochondrial kinase Mst1/2 is recruited to their respective membranes from the cytosol. The enzyme acts as a ROS sensor and "attenuator" by phosphorylating and stabilizing the protein Keap1, which binds to the transcription factor Nrf2 (also called NFE2L2). When bound to unphosphorylated Keap1, the transcription factor Nrf2 is a target for proteolysis. Keap1 phosphorylation prevents its binding to Nrf2. Free Nrf2 can then translocate to the nucleus, where it promotes transcription of antioxidant proteins such as glutamate-cysteine ligase catalytic subunit (Gclc), which catalyzes the first and rate-limiting step of glutathione (γ-glutamyl-cysteinyl-glycine) synthesis. These processes are illustrated in Figure \(\PageIndex{38}\).

Figure \(\PageIndex{38}\): A proposed working model for kinases Mst1/2 sense ROS and maintain cellular redox balance by modulating the stability of Nrf2. Wang, P., Geng, J., Gao, J. et al. Macrophage achieves self-protection against oxidative stress-induced aging through the Mst-Nrf2 axis. Nat Commun 10, 755 (2019). https://doi.org/10.1038/s41467-019-08680-6. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

Phagosomal or mitochondrial ROS release attracts Mst1/2 to the membrane of phagosome or mitochondrion from the cytosol and activates Mst1/2; Mst1/2
phosphorylate Keap1 to stabilize Nrf2 and regulate the expression of antioxidant enzymes to protect the cell against oxidative damage.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter traces the chemistry of dioxygen from its evolutionary origins to its molecular mechanisms of reactivity, toxicity, and biological management, integrating geochemical history with molecular biochemistry.

The rise of atmospheric oxygen was not inevitable — it required the evolution of cyanobacteria approximately 2.7–2.8 billion years ago, which were the first organisms capable of oxidizing water to produce dioxygen through photosynthesis. Prior to this, the atmosphere was reducing, with metals maintained in low oxidation states and life restricted to anaerobic pathways. The Great Oxidation Event (~2.4 billion years ago) caused a massive extinction of anaerobic organisms while simultaneously enabling the evolution of aerobic metabolism. A second oxygenation event at the end of the Gaskiers glaciation (~580 million years ago) raised deep-ocean oxygen to levels sufficient to support large multicellular animals, coinciding with the emergence of animal life.

The reactivity of dioxygen is best understood through molecular orbital theory. Ground-state dioxygen is a triplet diradical with two unpaired electrons in degenerate π* antibonding orbitals. Because typical organic molecules undergo two-electron oxidation steps with spin-paired electrons, reaction with triplet oxygen requires a spin flip — an energetically costly process (~25 kcal/mol) that accounts for dioxygen's kinetic sluggishness despite being thermodynamically favored as an oxidant. Singlet oxygen, produced by photosensitization or energy transfer, lacks this constraint and reacts far more readily with unsaturated organic molecules via ene reactions and Diels-Alder cycloadditions.

The stepwise one-electron reduction of dioxygen generates three progressively dangerous reactive oxygen species: superoxide (O₂•⁻), hydrogen peroxide (H₂O₂), and the hydroxyl radical (•OH). The hydroxyl radical is particularly destructive, reacting indiscriminately with whatever biomolecule it encounters. Organisms have evolved an interlocking network of enzymatic and non-enzymatic defenses. Superoxide dismutase (SOD) catalyzes the disproportionation of two superoxides to dioxygen and hydrogen peroxide via alternating Cu²⁺/Cu⁺ redox cycles; its efficiency approaches the diffusion limit, aided by an electrostatic field that steers the anionic superoxide toward the active site. Catalase then converts hydrogen peroxide to water and dioxygen via a heme-based mechanism, favoring H₂O₂ over water through hydrogen-bonding differences in a hydrophobic active-site channel. The tripeptide glutathione (γ-Glu-Cys-Gly) provides additional non-enzymatic scavenging of peroxides and alkyl radicals. Iron management through ferritin (intracellular storage) and transferrin (circulatory transport) prevents the Fenton reaction, in which Fe²⁺ generates the highly damaging hydroxyl radical from hydrogen peroxide. Ferritin's heavy chains oxidize Fe²⁺ to the less soluble Fe³⁺ via a dinuclear ferroxidase site, while light chains provide a nucleation surface for iron mineralization in the interior cavity.

Oxidative damage to proteins, lipids, and DNA underlies many diseases. Protein carbonylation — the irreversible conversion of lysine and other side chains to aldehydes — accumulates with age and correlates with protein misfolding, aggregation, and declining function. Mutations in Cu/Zn superoxide dismutase are associated with familial ALS, while elevated ROS and impaired catalase activity contribute to hair graying. Lipid peroxidation of unsaturated fatty acids generates reactive intermediates that oxidize apoprotein B in LDL; the resulting oxLDL is recognized by macrophage scavenger receptors (SR-A2/MARCO and SR-E1/LOX-1), driving foam cell formation and atherosclerotic plaque initiation. In DNA, hydroxyl radical attack generates 8-oxoguanine, which mispairs with adenine rather than cytosine, causing GC→AT mutations implicated in cancer; mitochondrial DNA is especially vulnerable given its proximity to the electron transport chain. Finally, in an adaptive context, macrophages deliberately generate ROS during oxidative bursts to kill engulfed pathogens, while protecting themselves through the Mst1/2–Keap1–Nrf2 signaling axis, which senses ROS and transcriptionally upregulates antioxidant proteins including the glutathione biosynthetic enzyme Gclc.