Glutathione peroxidase (GPx) family proteins, including GPx1-8, have a crucial role in the antioxidative defense system in intracellular redox homeostasis. They catalytically recycle reactive oxygen species, mainly hydrogen peroxide, using glutathione as a reducing agent (Toppo et al. 2009; Brigelius-Flohé and Maiorino 2013; Deponte 2013). Among them, GPx4 is a unique antioxidant enzyme that directly reduces complex hydroperoxides such as peroxidized phospholipids produced in the cell membrane (Maiorino et al. 2012). GPx4 has recently attracted much attention as a key regulator of lipid peroxidation-dependent regulated cell death, termed ferroptosis (Yang et al. 2014). In addition to the antioxidative function described above, GPx4 is known to be involved in several biological events, i.e., it functions as a structural protein in spermatogenesis and a regulator of gene expression (Ursini et al. 1999). Recent studies have demonstrated that some drug-resistant cancer cells are dependent on the enzymatic activity of GPx4 to maintain the intracellular redox state to suppress ferroptosis (Bayır et al. 2020; Harris and DeNicola 2020; Jiang et al. 2021). Therefore, GPx4 is also attracting attention as a target molecule for cancer therapy.
Three splice variants of GPx4 that have different intracellular localizations have been reported: cytoplasmic GPx4 (cGPx4), mitochondrial GPX4 (mGPx4), and sperm nuclear GPx4 (snGPx4) (Brigelius-Flohé and Maiorino 2013). The crystal structures of cGPx4 and mGPx4 have been reported for the wild type, various mutants, and complexes with inhibitors (Scheerer et al. 2007; Janowski et al. 2016; Sakamoto et al. 2017; Borchert et al. 2018; Moosmayer et al. 2021; Liu et al. 2022). These structural studies demonstrated that GPx4 forms a catalytic center with selenocysteine (Sec73 in the case of mGPx4) and the surrounding glutamine (Gln108), tryptophan (Trp163), and asparagine (Asn164) residues. However, the details of the functions, such as the interaction with the substrate and the regulation of the activity, are not yet fully understood. The backbone NMR resonance assignments of GPx4 have been reported by Labrecque and Fuglestad (2021) (Labrecque and Fuglestad 2021). In this study, we report the nearly complete backbone and side chain resonance assignments for human mGPx4 (Cys29-Phe197) containing eight mutations (Cys29Ser, Cys37Ala, Cys64Ser, Sec73Cys, Cys93Arg, Cys102Ser, Cys134Glu, and Cys175Val) (hereafter referred to as GPx4mu). We also report the secondary structure and the dynamics of the main chain using the assignments. These results will provide an important basis for the determination of the first solution structure of GPx4 and will aid in obtaining detailed information on interactions with substrates, cofactors, and inhibitors.