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Reactive Oxygen Species
& Antioxidant Systems

Explore how free radicals form in cells, why they cause oxidative damage, and the elegant enzymatic and non-enzymatic defenses that keep biology in balance.

Biochemistry

What Are Reactive Oxygen Species?

Reactive Oxygen Species (ROS) are chemically reactive molecules derived from molecular oxygen \( O_2 \). They contain unpaired electrons or unstable bonds, making them highly reactive with proteins, lipids, and DNA.

Superoxide
\( O_2^{\bullet-} \): first ROS formed when \( O_2 \) gains one electron
Hydrogen Peroxide
\( H_2O_2 \): not a radical, but a potent oxidizing agent
Hydroxyl Radical
\( OH^{\bullet} \): the most damaging ROS; reacts indiscriminately

Sources of ROS Production

Mitochondrial
ETC
 NADPH Oxidase Xanthine Oxidase Cytochrome P450 UV Radiation Pollutants Ionizing Radiation ENDOGENOUS EXOGENOUS ROS

Sequential Reduction of \( O_2 \)

Molecular oxygen is reduced one electron at a time, producing increasingly reactive intermediates before reaching water:

\( O_2 \)
\( +e^- \)
\( O_2^{\bullet-} \)
\( +e^-+2H^+ \)
\( H_2O_2 \)
\( +e^-+H^+ \)
\( OH^{\bullet} \)
\( +e^-+H^+ \)
\( 2\,H_2O \)
 Stable Most Reactive Stable

Targets of Oxidative Damage

Lipid Peroxidation
ROS attack polyunsaturated fatty acids in membranes, forming malondialdehyde (MDA) and disrupting membrane fluidity
Protein Oxidation
Amino acid side chains (especially Cys, Met, His) are oxidized, causing misfolding and loss of enzymatic activity
DNA Damage
Guanine is oxidized to 8-oxo-dG, causing \( G \to T \) transversion mutations if unrepaired

The Fenton & Haber-Weiss Reactions

Transition metals catalyze the conversion of less reactive ROS into the devastating hydroxyl radical:

Enzymatic Antioxidant Defenses

Superoxide Dismutase (SOD)
\( 2\,O_2^{\bullet-} + 2H^+ \longrightarrow H_2O_2 + O_2 \)
 Cu/Zn-SOD (cytosol), Mn-SOD (mito) Catalase
\( 2\,H_2O_2 \longrightarrow 2\,H_2O + O_2 \)
 Located in peroxisomes Glutathione Peroxidase (GPx)
\( H_2O_2 + 2\,\text{GSH} \longrightarrow 2\,H_2O + \text{GSSG} \)
 Selenium-dependent (Se-Cys)

The Glutathione Redox Cycle

2 GSH GSSG GPx Glutathione Reductase
\( H_2O_2 \)
\( 2\,H_2O \)
\( \text{NADPH} \longrightarrow \text{NADP}^+ \)
 via Pentose Phosphate Pathway GSH/GSSG ratio: key redox indicator Normal cytosol: GSH:GSSG ≈ 100:1

Non-Enzymatic Antioxidants

Vitamin E
Lipid-soluble tocopherol that breaks lipid peroxidation chains in membranes by donating an H atom
Vitamin C
Water-soluble ascorbate that regenerates vitamin E and directly scavenges ROS in the cytosol
Uric Acid
Scavenges \( OH^{\bullet} \) and peroxynitrite; accounts for ~50% of plasma antioxidant capacity
Coenzyme Q10
Lipid-soluble electron carrier in the ETC that also functions as a membrane antioxidant

Enzymatic vs Non-Enzymatic

Enzymatic Non-Enzymatic
Mechanism Catalytic conversion of ROS Direct scavenging / radical quenching
Recyclable? Yes: enzyme is not consumed Sometimes (Vit E by Vit C)
Specificity High: each enzyme targets specific ROS Broader range of targets
Location Cytosol, mitochondria, peroxisomes Membranes (E, CoQ) or cytosol (C, GSH)
Examples SOD, Catalase, GPx Vit C, Vit E, Uric acid, CoQ10
Regulation Gene expression (Nrf2 pathway) Dietary intake

The Oxidative Stress Balance

BALANCE ROS Production
\( O_2^{\bullet-}, H_2O_2, OH^{\bullet} \)
 Antioxidants SOD, CAT, GPx, GSH OXIDATIVE STRESS ROS > Antioxidants REDUCTIVE STRESS Antioxidants > ROS

The Nrf2-Keap1 Pathway

Cells have a master transcriptional regulator that senses oxidative stress and activates antioxidant gene expression:

1
Normal conditions: Nrf2 is bound to Keap1 in the cytoplasm and targeted for ubiquitin-mediated degradation
2
Oxidative stress: ROS modify Keap1 cysteine residues, releasing Nrf2
3
Nuclear translocation: Free Nrf2 enters the nucleus and binds Antioxidant Response Elements (AREs)
4
Gene activation: Transcription of SOD, catalase, GPx, glutathione synthetase, and heme oxygenase-1 increases

How to Analyze an ROS Problem

1
Identify the ROS: Which species is involved? (\( O_2^{\bullet-} \), \( H_2O_2 \), \( OH^{\bullet} \))
2
Trace the source: Is the origin endogenous (ETC, NADPH oxidase) or exogenous (UV, toxin)?
3
Determine the defense: Which antioxidant handles this ROS? (SOD for \( O_2^{\bullet-} \), catalase/GPx for \( H_2O_2 \))
4
Predict the consequence: If the defense fails, what macromolecule is damaged and how?
5
Check the signaling role: Is some ROS needed for normal function (e.g., immune respiratory burst)?

Example 1: SOD Deficiency

A researcher knocks out the Mn-SOD gene in a mouse model. Predict the biochemical consequences.

1
Identify ROS: \( O_2^{\bullet-} \) will accumulate because Mn-SOD is its primary scavenger in mitochondria
2
Downstream effects: Less \( H_2O_2 \) is produced from SOD, BUT superoxide reacts with NO to form peroxynitrite (\( \text{ONOO}^- \))
3
Damage: Mitochondrial DNA and ETC proteins suffer oxidative damage, reducing ATP production
4
Phenotype: Severe cardiomyopathy, neurodegeneration, and neonatal lethality in homozygous knockouts

Example 2: Iron Overload

A patient with hereditary hemochromatosis has excess free iron (\( Fe^{2+} \)) in the liver. Explain the oxidative consequences.

\( Fe^{2+} \)
 +
\( H_2O_2 \)
 Fenton
\( OH^{\bullet} \)
\( Fe^{3+} \)
 Lipid peroxidation DNA damage
Excess \( Fe^{2+} \) drives continuous Fenton chemistry → liver fibrosis → cirrhosis

Example 3: Neutrophil Respiratory Burst

During infection, neutrophils deliberately generate massive amounts of ROS to kill bacteria. Trace the pathway:

1
NADPH oxidase is activated on the phagosome membrane, producing \( O_2^{\bullet-} \)
2
SOD converts \( O_2^{\bullet-} \) to \( H_2O_2 \)
3
Myeloperoxidase (MPO) uses \( H_2O_2 + Cl^- \) to produce hypochlorous acid (\( \text{HOCl} \)): a powerful antimicrobial agent
4
\( \text{HOCl} \) destroys bacterial proteins and membranes within the phagosome

Common Mistakes

✕ "All ROS Are Bad"
ROS at low levels serve as signaling molecules (e.g., \( H_2O_2 \) in growth factor signaling) and are essential for immune defense
✕ Confusing SOD and Catalase
SOD converts \( O_2^{\bullet-} \to H_2O_2 \). Catalase converts \( H_2O_2 \to H_2O \). They are sequential, not interchangeable
✕ "More Antioxidants = Better"
Megadose antioxidant supplements can cause reductive stress and may interfere with chemotherapy, exercise adaptation, and immune function

Summary: ROS and Their Defenses

ROS Primary Defense Cofactor / Location
\( O_2^{\bullet-} \) SOD (Cu/Zn or Mn) Cu, Zn (cytosol) / Mn (mito)
\( H_2O_2 \) Catalase Fe-heme / peroxisomes
\( H_2O_2 \) Glutathione Peroxidase Se (selenocysteine) / cytosol
Lipid radicals Vitamin E (tocopherol) Membranes
Various radicals Vitamin C (ascorbate) Cytosol (water-soluble)
\( OH^{\bullet} \) No direct enzyme Prevention: sequester Fe (ferritin)

Question 1 of 5

Which enzyme directly converts superoxide (\( O_2^{\bullet-} \)) into hydrogen peroxide (\( H_2O_2 \))?

Question 2 of 5

In the Fenton reaction, which transition metal ion reacts with \( H_2O_2 \) to produce the hydroxyl radical?

Question 3 of 5

Glutathione peroxidase requires which trace element as part of its active site?

Question 4 of 5

A patient with Chronic Granulomatous Disease (CGD) has a defective NADPH oxidase. Which of the following is the most direct consequence?

Question 5 of 5

Which non-enzymatic antioxidant regenerates vitamin E after it has neutralized a lipid radical in the membrane?