Key findings
- X-ray work on glutathione reductase crystals resolved two bound molecules in different conformations, one V-shaped and one extended, with the zwitterionic glutamyl end as the most tightly held part and a mixed disulfide forming with Cys58 in the reduced enzyme. [1]
- Copper-catalyzed oxidation followed biphasic kinetics, consuming one mole of oxygen per four moles of thiol with minimal hydroxyl radical in the first phase and producing hydrogen peroxide and hydroxyl radical at an increased oxygen consumption rate in the second, so the molecule can act as a prooxidant. [2]
- A glutaredoxin-1 fused redox-sensitive fluorescent protein equilibrated specifically with the glutathione couple and resolved nanomolar changes in the oxidized disulfide against a millimolar background of the reduced thiol, on a scale of seconds to minutes. [3]
- A cryo-electron microscopy structure of an ABCC family transporter bound to the oxidized disulfide shows the two halves of that substrate held differently, one in a tight pocket and one in a loose pocket. [4]
Identity and structure
- Oxidation state
- The reduced thiol and the oxidized disulfide are different chemical species and are resolved separately in the cited structural and imaging work [3][4]
- Binding determinant
- In the reductase complex the zwitterionic glutamyl end dominates binding, and every conjugate and derivative tested bound through interactions at that same site [1]
- Biosynthesis
- The final step is catalysed by glutathione synthetase, whose ATP-binding site spans two domains and differs from the classic mononucleotide-binding fold [5]
- Metal sensitivity
- Copper forms a catalytic complex at roughly one copper per two molecules and drives the thiol to oxidize [2]
- Form as supplied
- Dry crystalline or lyophilized powder
Mechanism as studied
The reductase crystallography shows the disulfide exchange geometry directly. Two bound molecules occupy the site in different conformations, and in the reduced enzyme one of them forms a mixed disulfide intermediate with the active site cysteine, with further structural changes accompanying reduction. [1]
Copper chemistry explains how a reductant becomes a radical source. The kinetic and spectroscopic data point to a catalytic copper complex and two parallel routes in the first phase, superoxide dependent and peroxide dependent, followed by a superoxide independent second phase that produces hydrogen peroxide and hydroxyl radical. [2]
Peroxidase chemistry runs through a selenocysteine site in a shallow surface depression. In a catalytically active cysteine substituted variant the catalytic triad sits at a flat impression extending into a basic surface patch, and the enzyme lacks the surface loop that limits active site access in other isoforms. [6]
Research findings
Structural
- System
- Crystals of glutathione reductase soaked with the tripeptide, related molecules, two redox compounds and lipoate, at 0.3 nm resolution
- Measured
- Bound ligand models after crystallographic refinement, the residues forming the binding site, the geometry of disulfide exchange, and the structural changes on enzyme reduction
- Reported
- One bound molecule adopts a V-shaped conformation and a second an extended one, with the zwitterionic glutamyl end most tightly bound and dominating the binding of all conjugates and derivatives tested. In the reduced enzyme a mixed disulfide forms with Cys58. Lipoate occupies a defined site with its disulfide exposed to that cysteine, and a second affinity region was found in a cavity at the dimer interface with no known functional role. [1]
Analytical
- System
- Solutions of the thiol with catalytic copper under physiological conditions
- Measured
- Oxygen consumption, thiol depletion, spectroscopic signatures and hydroxyl radical detection across the reaction time course
- Reported
- Kinetics were biphasic. The first phase consumed one mole of oxygen per four moles of thiol with minimal hydroxyl radical. The second phase consumed excess oxygen at an increased rate with significant hydrogen peroxide and hydroxyl radical production. The data indicate a catalytic complex of one copper per two molecules of the thiol. [2]
In vitro
- System
- Living cells expressing a fusion of human glutaredoxin-1 to a redox-sensitive green fluorescent protein, targeted to different subcellular compartments
- Measured
- Equilibration of the sensor with the glutathione redox couple, the redox potential in each compartment, and the response to growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation
- Reported
- The fusion equilibrated specifically and in real time with the glutathione couple, unlike the unfused sensor whose specificity was undefined and whose response was slow. It detected nanomolar changes in the oxidized disulfide against a millimolar background of the reduced thiol over seconds to minutes and followed redox changes under each of the tested conditions. [3]
Structural
- System
- Yeast Cadmium Factor 1, an ABCC family transporter, resolved by cryo-electron microscopy with the oxidized disulfide bound rather than the reduced thiol, with cellular survival assays under high cadmium and molecular dynamics simulations alongside
- Measured
- The substrate binding mode, the contacts made by each half of the bound disulfide, and the binding determinants tested by survival assay and simulation
- Reported
- The two halves of the bound substrate are held differently, one binding tightly in the substrate pocket and the other sitting in a loose pocket. The authors show the two halves of the site working together to drive substrate selectivity, which they relate to the transporter handling both the oxidized molecule and its heavy metal conjugates. [4]
Structural
- System
- The catalytically active U46C variant of human phospholipid hydroperoxide glutathione peroxidase-4, so the resolved protein carries a cysteine in place of the native selenocysteine, at 1.55 angstrom resolution
- Measured
- Overall fold, the position of the catalytic triad and the surrounding surface, the effect of multiple triad mutations, and the residues involved in enzyme polymerization
- Reported
- The protein is monomeric with four alpha-helices and seven beta-strands and lacks the surface exposed loop that limits active site access in other isoforms, which the authors link to its broad substrate range. The catalytic triad sits at a flat surface impression next to a basic patch, triad mutations confirmed its functional importance, and polymerization involved C46, C10 and C66 and was prevented by reductants. [6]
Structural
- System
- Escherichia coli glutathione synthetase crystallized at pH 7.5, the optimal catalytic condition, compared with its own structure at pH 6.0 and with 294 other known protein structures
- Measured
- Interdomain movement between the two pH conditions, the resulting polar contacts, and the arrangement of secondary structure elements at the ATP-binding site against other proteins
- Reported
- The central domain moves toward the N-terminal domain almost as a rigid body at the higher pH, creating new interdomain and intersubunit polar interactions that stabilize the dimer. The ATP-binding site spans two domains and is built from two antiparallel beta-sheets, unlike the classic mononucleotide-binding fold, and resembles only three of the compared enzymes. [5]
Handling for in-vitro work
- Trace metal control
- Copper at catalytic amounts drives oxidation of the thiol with hydrogen peroxide and hydroxyl radical formation, so trace metal in buffers changes the outcome [2]
- Oxygen exposure
- Oxygen is consumed stoichiometrically during the first phase of metal catalysed oxidation, so solutions left open to air do not stay in the reduced state [2]
- Storage
- Dry powder at -20 °C, dark and dry; solutions prepared fresh and kept cold
Open questions
- The cited structures resolve the molecule bound to enzymes and to a transporter; none of them characterizes a supplied preparation, whose oxidation state and purity need separate analytical confirmation.
- The reductase crystallography found a second affinity region at the dimer interface with no functional role assigned in the cited work.
- The peroxidase structure used a cysteine variant in place of the native selenocysteine, so the geometry around the catalytic residue is that of the variant.
- The prooxidant behaviour reported under copper catalysis is not reconciled in the cited set with the antioxidant role assumed elsewhere, and the balance depends on metal availability.
A clinical literature on this tripeptide exists and concerns finished formulations; it is out of scope for a research material profile.
Lot records
Check the record for the exact material you order. A published paper and a batch certificate answer different questions.
- RV-24-0042-2 ↗Glutathione · 99.60% HPLC2026-09-22
- RV-24-0042-1 ↗Glutathione · 99.60% HPLC2026-09-16
References
- Karplus PA, Pai EF, Schulz GE A crystallographic study of the glutathione binding site of glutathione reductase at 0.3-nm resolution. European journal of biochemistry. 1989.
- Kachur AV, Koch CJ, Biaglow JE Mechanism of copper-catalyzed oxidation of glutathione. Free radical research. 1998.
- Gutscher M, Pauleau AL, Marty L, et al. Real-time imaging of the intracellular glutathione redox potential. Nature methods. 2008.
- Soong TH, Hotze CF, Raghav D, et al. Structural Basis for Oxidized Glutathione Recognition by Yeast Cadmium Factor 1. Journal of the American Chemical Society. 2025.
- Matsuda K, Mizuguchi K, Nishioka T, et al. Crystal structure of glutathione synthetase at optimal pH: domain architecture and structural similarity with other proteins. Protein engineering. 1996.
- Scheerer P, Borchert A, Krauss N, et al. Structural basis for catalytic activity and enzyme polymerization of phospholipid hydroperoxide glutathione peroxidase-4 (GPx4). Biochemistry. 2007.
Publication records fetched from PubMed on 2026-09-20. Profile text reviewed 2026-09-20.