Glutathione Peroxidase

Glutathione peroxidase (GPx) is a family of antioxidant enzymes that play a central role in protecting cells and organisms from oxidative damage. These enzymes catalyse the reduction of harmful peroxides, including hydrogen peroxide and lipid hydroperoxides, thereby preventing oxidative stress–induced damage to lipids, proteins, and nucleic acids. Glutathione peroxidases are widely distributed in living organisms and are particularly important in aerobic life, where reactive oxygen species are continuously generated as by-products of normal metabolism.
The biological significance of glutathione peroxidase lies in its close integration with the cellular glutathione system, one of the most important redox-buffering and detoxification mechanisms in biology. Through this system, GPx enzymes help maintain redox homeostasis and preserve the structural and functional integrity of cellular and subcellular membranes.

Biochemical Function and Catalytic Reaction

The principal biochemical function of glutathione peroxidase is the reduction of peroxides using reduced glutathione (GSH) as an electron donor. The most common reaction catalysed by GPx is the conversion of hydrogen peroxide into water, thereby neutralising a potentially harmful oxidant. In addition, many GPx isoforms reduce lipid hydroperoxides to their corresponding alcohols, preventing the propagation of lipid peroxidation within biological membranes.
The overall reaction can be represented as:

• 2 GSH + H₂O₂ → GSSG + 2 H₂O

In this reaction, GSH represents reduced glutathione, while GSSG denotes oxidised glutathione (glutathione disulfide). The enzyme’s catalytic mechanism involves a highly reactive selenocysteine residue at the active site. Hydrogen peroxide oxidises the selenol group (R–SeH) to form a selenenic acid intermediate (R–SeOH). This intermediate is subsequently reduced back to its active form through two sequential reactions with glutathione molecules, generating GSSG as a by-product.
The oxidised glutathione produced during this process is then recycled back to its reduced form by the enzyme glutathione reductase, using nicotinamide adenine dinucleotide phosphate (NADPH) as a reducing agent. This completes the glutathione redox cycle and allows continuous detoxification of peroxides.

Isozymes and Gene Family

Glutathione peroxidase is not a single enzyme but a family of related isozymes encoded by different genes. These isozymes differ in their tissue distribution, cellular localisation, substrate specificity, and physiological roles. In humans, eight distinct glutathione peroxidase isoforms (GPX1–GPX8) have been identified.

• GPX1 is the most abundant and widely distributed isoform, found primarily in the cytoplasm of nearly all mammalian tissues. Its preferred substrate is hydrogen peroxide, making it a key component of general cellular antioxidant defence.
• GPX2 is expressed mainly in the gastrointestinal tract and also has extracellular activity, suggesting a role in protecting the intestinal epithelium from oxidative stress.
• GPX3 is an extracellular enzyme that is particularly abundant in blood plasma, where it contributes to systemic antioxidant protection.
• GPX4 displays a high specificity for lipid hydroperoxides and is capable of reducing complex membrane-bound lipid peroxides. Although expressed at relatively low levels, it is present in almost all mammalian cells and plays a critical role in membrane integrity and cell survival.
• Other isoforms, including GPX5, GPX6, GPX7, and GPX8, exhibit more specialised expression patterns and functions, some of which are still under investigation.

Structural Characteristics

Structurally, glutathione peroxidase enzymes show notable diversity within the family. GPX1, GPX2, and GPX3 are typically homotetrameric proteins, consisting of four identical subunits. In contrast, GPX4 is a monomeric enzyme, a structural feature that is closely linked to its ability to interact with membrane lipids.
Most mammalian glutathione peroxidases are selenium-containing enzymes, incorporating selenocysteine at their active site. Selenium is essential for their catalytic efficiency, and its availability directly influences GPx activity. GPX6 is a selenoprotein in humans but has cysteine-containing homologues in rodents, illustrating evolutionary variation within the family.
Because cellular and subcellular membranes are particularly vulnerable to oxidative damage, the antioxidant protective system provided by glutathione peroxidase is critically dependent on adequate selenium nutrition.

Role in Oxidative Stress and Cellular Protection

Reactive oxygen species such as hydrogen peroxide are generated continuously during normal metabolic processes, especially within mitochondria. At low levels, these molecules can act as signalling agents, but excessive accumulation leads to oxidative stress, damaging lipids, proteins, and DNA.
Glutathione peroxidase functions as a first-line defence against such damage by rapidly removing peroxides before they can initiate chain reactions of lipid peroxidation. GPX4 is particularly important in this context, as it directly reduces lipid hydroperoxides within biological membranes, thereby preventing membrane destabilisation and cell death.
Through its interaction with glutathione reductase and NADPH, the GPx system is tightly linked to cellular metabolism and energy status, integrating antioxidant defence with broader metabolic regulation.

Evidence from Animal Models

Genetically modified animal models have provided valuable insights into the physiological importance of different glutathione peroxidase isoforms. Mice lacking GPX1 are generally viable, display normal lifespans, and show no gross developmental abnormalities. This suggests that GPX1 is not strictly essential for survival under normal conditions. However, these mice develop early-onset cataracts and exhibit defects in muscle satellite cell proliferation, highlighting a protective role under specific physiological stresses.
GPX1-deficient mice also demonstrate increased susceptibility to noise-induced hearing loss, with elevated auditory brainstem response thresholds compared with control animals. In contrast, mice lacking GPX2 or GPX3 also develop normally, indicating functional redundancy or compensation by other antioxidant systems.
By comparison, GPX4 is indispensable. Knockout mice lacking GPX4 die during early embryonic development, underscoring its critical role in cellular viability and membrane protection. Interestingly, some evidence suggests that reduced, but not absent, GPX4 activity may be associated with increased lifespan in mice, pointing to complex interactions between oxidative stress and ageing.

Discovery and Historical Background

Glutathione peroxidase was first discovered in 1957 by Gordon C. Mills. This discovery represented a major advance in understanding enzymatic antioxidant defence mechanisms and highlighted the biological importance of selenium, which was later recognised as an essential trace element due to its role in selenoproteins such as GPx.
Subsequent research expanded the concept of glutathione peroxidase from a single enzyme to a multifunctional family with diverse physiological roles across tissues and organisms.

Methods for Measuring Enzyme Activity

The activity of glutathione peroxidase is commonly measured using spectrophotometric assays. One widely used method couples the GPx-catalysed reduction of peroxides to the action of glutathione reductase, allowing the oxidation of NADPH to NADP⁺ to be monitored as a decrease in absorbance.
Alternative approaches involve measuring residual reduced glutathione after the reaction, often using Ellman’s reagent. Various hydroperoxides, such as hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide, may be used as substrates. Additional methods employ reagents such as CUPRAC for spectrophotometric detection or o-phthalaldehyde for fluorescence-based assays.

Clinical and Medical Significance

Altered glutathione peroxidase activity has been associated with a range of clinical conditions. Reduced serum GPx levels have been reported in individuals with vitiligo, suggesting a link between impaired antioxidant defence and depigmentation disorders. Lower plasma GPx activity has also been observed in patients with type 2 diabetes and macroalbuminuria, correlating with the progression of diabetic nephropathy.
In neurological disorders, glutathione peroxidase activity has been found to be decreased in patients with relapsing–remitting multiple sclerosis. Genetic polymorphisms affecting GPx and other antioxidant enzymes, such as superoxide dismutase, have been implicated in susceptibility to conditions including coeliac disease. Additionally, GPx activity is known to decrease in cases of copper deficiency, particularly in the liver and plasma.