NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Glutathione Reductase

Glutathione (GSH) is a ubiquitous low molecular weight thiol tripeptide containing glutamate, cysteine, and glycine (Glu-Cys-Gly) discovered by Hopkins, Hunter and Eagles in the 1920s . Nucleophylic properties making glutatione as a major component of redox buffer in the cells. Glutathione-dependent enzymes such as glutatione peroxidases (GPXs), glutaredoxins (GRXs) and glutatione S-transferases (GSTs) are typical for most living organisms. The intracellular concentration of GSH is in ranges ofapproximately 0.1 to 15 mM and GSH/GSSH ratio depends on cellular compartment. GSH work reducing agent for disulfides non-enzymatically or enzymatically (in glutaredoxing cycle). GSSG work as major thiol modifying agent in glutatione S-transferases cycle [ 1, 2, 3, 4, 5 ].

Glutatione Reductase (GR) is enzyme maintaining the cellular reduced GSH pool by catalyzing the reduction of GSSG to GSH. Glutatione reductase was purified from yeast in 1955 by Racker and NADPH was confirmed as the electron donor. In 1963, Mapson and Isherwood confirmed that Glutatione Reductase use FAD and a thiol-group for activity [ 4, 6 ].

GSSG + NADPH + H+ → 2GSH + NADP+;

Hydrogen peroxide scavenging in Glutathione Peroxidase (GPx)/Glutahione Reductase (GR) cycle.

Glutatione reductase is a flavoenzyme of the pyridine nucleotide-disulfide oxidoreductase family characterized by NADPH:GSSG oxidoreductase activity. This family of oxidoreductases includes trypanothione reductases, dihydrolipoamide dehydrogenase, mercuric ion reductase and the animal type of thioredoxin reductases [ 4, 6, 7, 8, 9, 10 ]. Recent studies shown that animal thioredoxin reductases (TTRs) are evolved from glutatione reductases by C-terminal extention. Moreover, potein sequenses of GR and TRR are very similat to each other [ 11 ].

Phylogenetic tree for thioredoxin reductases any glutatione reductases. The tree demonstrate evolutional separation of prokaryotic and eukaryotic glutathione reductases (GR) and the origin of large thioredoxin reductases [ 11 ].

GR-isoforms from prokaryotes and eukaryotes are homodimers of ~ 110 kDa]. Each subunit contains an FAD-binding site. Each GSSG-binding site is formed by both subunits, thus GR is only functional as a homodimer. The structure and amino acid sequence of different GR-isoforms are extremely conserved in the course of evolution [ 12 ]. There are 106 solved Glutathione Reductase structures in Protein Data Bank at this time (September 2015).

Crystal structure of yeast Saccharomyces cerevisiae Glutathione Reductase Glutathione is shown in pink color and FAD is shown in blue color.

Considering GR kinetics data, Massey and Williams suggested a ping-pong mechanism for yeast glutathione reductase [ 13 ]. Glutathione reductase contains two essential cysteine residues (proximal and interchange Cys) that may form a disulfide bond. The disulfide bond is close to a histidine residue. NADPH reduces the flavin to FADH- which subsequently shuttles an electron pair to the proximal cysteine residue. At the end of the reductive half-reaction, NADP+ dissociates from the two-electron reduced enzyme and is replaced by another molecule of NADPH. After oxidized glutathione binding, its disulfide is attacked by the interchange Cys residue in half reduced form of GR, resulting in the formation of an intermolecular disulfide bond. The nucleophilic attack could be accelerated owing to the deprotonation of the interchange Cys thiol group by His. Once the first GSH molecule has left the active site, the intermolecular disulfide bond is attacked by the thiolate of proximal cysteine residue resulting in oxidized form of GR. The thiolate of remaining second GSH molecule could again be protonated by His again [ 4, 13, 14, 15, 16, 17, 18 ]. Finally GR requires FAD, two essential cysteines, an activated histidine for acid-base catalysis as well as several other conserved residues for substrate binding.

Proposed model of Glutathione Reductase catalysis.

Yeast glutathione reductase knockout strains are viable, but more sensitive to oxidative stress and to have higher GSSG levels in the cytosol and in the mitochondrial matrix [ 19, 20 ]. E. coli glutathione reductase knockout strains have not demonstrated any phenotype and GSSG levels are comparable with wild type strain [ 21 ]. Homozygous glutathione reductase deficiency in humans have no any visable phenotype as well. Thus, glutatione reductase function is not essential for major aerobic model organisms including humans and this observation allow to suggest that such organisms have an alternative strategy to maintain sufficient level of GSH and a physiological GSH/GSSG ratio [ 22, 23 ].


Methods for Measurement of Glutathione Reductase Activity

1. Decrease in absorbance caused by the oxidation of NADPH. The oxidation of NADPH to NADP+ in catalitic cyle of glutathione reductase is accompanied by a decrease in absorbance at 340 nm. Since The rate of decrease in the A340 is directly proportional to the glutathione reductase activity in the sample [ 24, 25, 26 ].

2. Reduction of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) (DTNB). Glutathione reductase reduces GSSG to GSH, which reacts with DTNB to generate TNB (yellow color). Thes reaction can be monitored at 405 nm. The assay can detect 0.1 - 40 mU/ml GR in various samples [ 26 27, 28 ].

DTNB calorimetric glutatione reductase assay [ 29 ].

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