Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides.

Glutathione peroxidase (GPx) is a widespread protein superfamily found in many organisms throughout all kingdoms of life. Although it was initially thought to use only glutathione as reductant, recent evidence suggests that the majority of GPxs have specificity for thioredoxin. We present a thorough in silico analysis performed on 724 sequences and 12 structures aimed to clarify the evolutionary, structural, and sequence determinants of GPx specificity. Structural variability was found to be limited to only two regions, termed oligomerization loop and functional helix, which modulate both reduced substrate specificity and oligomerization state. We show that mammalian GPx-1, the canonic selenocysteine-based tetrameric glutathione peroxidase, is a recent “invention” during evolution. Contrary to common belief, cysteine-based thioredoxin-specific GPx, which we propose the TGPx, are both more common and more ancient. This raises interesting evolutionary considerations regarding oligomerization and the use of active-site selenocysteine residue. In addition, phylogenetic analysis has revealed the presence of a novel member belonging to the GPx superfamily in Mammalia and Amphibia, for which we propose the name GPx-8, following the present numeric order of the mammalian GPxs.

In GPxs, the redox-active Se or S, is at hydrogen bonding distance from Gln and Trp residues that contribute to catalysis. From sequence homology of >400 sequences and modeling of the DmGPx as a paradigm, Asn136 emerged as a fourth essential component of the active site. Mutational substitution of Asn136 by His, Ala, or Asp results in a dramatic decline of specific activity. Kinetic analysis indicates that k(+1), the rate constant for the oxidation of the enzyme, decreases by two to three orders of magnitude, whereas the reductive steps characterized by k'(+2) are less affected. Accordingly, MS/MS analysis shows that in Asn136 mutants, the peroxidatic Cys45 stays largely reduced also in the presence of a hydroperoxide, whereas in the wild-type enzyme, it is oxidized, forming a disulfide with the resolving Cys. Computational calculation of pK(a) values indicates that the residues facing the catalytic thiol, Asn136, Gln80, and, to a lesser extent Trp135, contribute to the dissociation of the thiol group, Asn136 being most relevant. These data disclose that the catalytic site of GPxs has to be redrawn as a tetrad, including Asn136, and suggest a mechanism accounting for the extraordinary catalytic efficiency of GPxs.

A nuclear variant of phospholipid-hydroperoxide glutathione peroxidase (PHGPx, GPx-4) was considered to be derived from alternative pre-mRNA splicing in testis and to regulate sperm maturation. The genomic sequence of rat gpx-4 was established and investigated in respect to expression into the cytosolic, mitochondrial, and nuclear forms of PHGPx. In silico analysis suggested the presence of two distinct promoter regions, the upstream one leading to transcripts translating into cPHGPx or mPHGPx and the downstream one yielding nPHGPx. The promoter activity of both regions was verified by luciferase-based reporter constructs in A7r5 and H9c2 cells. The data reveal that the formation of nPHGPx is due to alternative transcription and not to alternative splicing. Transcripts encoding nPHGPx were most abundant in testis although not restricted to this organ. This observation points to a general role of the nuclear PHGPx variant in regulating cell division.