The functioning of the N, K-ATPase depends on the redox status of cells and its activity is inhibited by oxidative stress and hypoxia. We previously found that redox sensitivity of the Na,K-ATPase is mediated by glutathionylation of the alpha-subunit. An increase in the level of glutathionylation of cysteine residues in the Na,K-ATPase alpha-subunit under stressful conditions leads to a decrease in the activity of the enzyme and a change in its receptor function. The structure of the Na,K-ATPase undergoes significant conformational changes during functioning. The effects of enzyme conformation on its ability to undergo glutathionylation are not clear. Here we show that the highest level of glutathionylation in the alpha-subunit of Na,K-ATPase is achieved in the El (Nat-induced) conformation. The transition of the Na,K-ATPase to the E2 (K-induced) conformation leads to a decrease in the efficiency of glutathionylation. The lowest efficiency of Na,K-ATPase glutathionylation was observed in the E2P and E2P ouabain states. According to molecular modelling data, the maximum number of cysteine residues available for glutathionylation are present in the El P conformation. In the E2P conformation, the main functional cysteine residues (Cys204, Cys242, Cys452, and Cys456) are buried from the solvent, which makes them inaccessible for glutathionylation. Thus, the efficiency of alpha-subunit glutathionylation depends on enzyme conformation, which is altered by bound ligands and proteins. A shift in the El/E2 equilibrium towards prevalence of El can lead to better access for the relevant ligands and proteins to the binding site located in the Na,K-ATPase alpha-subunit. Na,K-ATPase. (C) 2019 Elsevier Inc. All rights reserved.