Although lipid peroxidation associated with oxidative stress can result in cellular death, sub-lethal lipid peroxidation can gradually resolve with return to the pre-exposure state. We have shown that resolution of lipid peroxidation is greatly delayed in lungs or cells that are null for peroxiredoxin 6 (Prdx6) and that both the phospholipase A₂ and the GSH peroxidase activities of Prdx6 are required for a maximal rate of recovery. Like other peroxiredoxins, Prdx6 can reduce H₂O₂ and short chain hydroperoxides, but in addition can directly reduce phospholipid hydroperoxides. This study evaluated the relative role of these two different peroxidase activities of Prdx6 in the repair of peroxidized cell membranes. The His26 residue in Prdx6 is an important component of the binding site for phospholipids. Thus, we evaluated the lungs from H26A-Prdx6 expressing mice and generated H26A-Prdx6 expressing pulmonary microvascular endothelial cells (PMVEC) by lentiviral infection of Prdx6 null cells to compare with wild type in the repair of lipid peroxidation. Isolated lungs and PMVEC were exposed to tert-butyl hydroperoxide and mice were exposed to hyperoxia (> 95% O₂). Assays for lipid peroxidation in wild type control and mutant lungs and cells showed ~4-fold increase at end-exposure. Control lungs and cells showed gradual resolution during a post-exposure recovery period. However, there was no recovery from lipid peroxidation by H26A-Prdx6 lungs or PMVEC. These studies confirm an important role for Prdx6 in recovery from membrane lipid peroxidation and indicate that reduction of H₂O₂ or short chain hydroperoxides does not play a role in the recovery process.

The expression of the phospholipase A₂ activity (aiPLA₂) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA₂ assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-oxidized substrate. However, the addition of glutathione (GSH) to the assay medium significantly increased aiPLA₂ activity at cytosolic pH, while oxidized GSH (GSSG) and several other thiols had no effect. By mass spectroscopy (ESI MS), the addition of GSH to Prdx6 paradoxically led to oxidation of its conserved Cys47 residue to a sulfinic acid. The effect of GSH on PLA₂ activity was abolished by incubation under anaerobic conditions, confirming that auto-oxidation of the protein was the mechanism for the GSH effect. Analysis by circular dichroism (CD) and tryptophan fluorescence showed alterations of the protein structure in the presence of GSH. Independently of GSH, the oxidation of Prdx6 by exposure to H₂O₂ or the presence of oxidized phospholipid as substrate also significantly increased aiPLA₂ activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA₂ activity at pH 7 without effect on the activity of the enzyme at pH 4.

The expression of the phospholipase A₂ activity (aiPLA₂) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA₂ assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-oxidized substrate. However, the addition of glutathione (GSH) to the assay medium significantly increased aiPLA₂ activity at cytosolic pH, while oxidized GSH (GSSG) and several other thiols had no effect. By mass spectroscopy (ESI MS), the addition of GSH to Prdx6 paradoxically led to oxidation of its conserved Cys47 residue to a sulfinic acid. The effect of GSH on PLA₂ activity was abolished by incubation under anaerobic conditions, confirming that auto-oxidation of the protein was the mechanism for the GSH effect. Analysis by circular dichroism (CD) and tryptophan fluorescence showed alterations of the protein structure in the presence of GSH. Independently of GSH, the oxidation of Prdx6 by exposure to H₂O₂ or the presence of oxidized phospholipid as substrate also significantly increased aiPLA₂ activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA₂ activity at pH 7 without effect on the activity of the enzyme at pH 4.