The role of glutathione(GSH) is to remove accumulated reactive oxygen species which may damage cells. During this process, its thiol side chain is oxidised and two glutathione molecules are connected by a disulphide bond to form a dimer(GSSG). In order to regenerate glutathione the disulphide bond has to be broken, In human cells, this is done by glutathione reductase(GR).
Glutathione reductase is a dimer that contains two identical subunits. It requires one NADP and one FAD as the cofactors. The active site is located in the linkage between two subunits. The NADPH is involved in the generation of FADH-. In the active site, there are two cysteine residues besides the FAD cofactor and are used to break the disulphide bond during the catalytic reaction. NADPH is bound by three positively charged residues: Arg-218, His-219 and Arg-224.
The catalytic process starts when the FAD is reduced by NADPH to accept one electron and from FADH. It then attacks the disulphide bond formed between 2 cysteine residues, forming one SH bond and a single S group. This S group will act as a nucleophile to attack the disulphide bond in the oxidised glutathione(GSSG), breaking it and forming a cysteine-SG complex. The first SG anion is released and then receives one proton from adjacent SH group and from the first glutathione monomer. Next the adjacent S group attack disulphide bond in cysteine-SG complex and release the second SG anion. It receives one proton in solution and forms the second glutathione monomer.