Dissecting the role of selenothiol- versus thiol-based catalysis using the model enzyme glutathione peroxidase 4 (GPx4)

Selenocysteine (Sec) is the 21st amino acid. Unlike other amino
acids incorporation of Sec into proteins is more complex by far as
it is encoded by the opal stop codon UGA. A complex and highly
sophisticated incorporation mechanism is required in order to
assure the correct co-translational incorporation of Sec into the
nascent polypeptide chain. Even after years of intensive research
the question as to why selenoproteins are important for mammalian
life has not been fully understood. This study aimed to provide
answers to this question using gluthathione peroxidase 4 (GPx4) as
a model enzyme. It has been shown that GPx4 is essential for early
embryogenesis, neuro- and retina-protection and male fertility in
mice. But the role of selenothiol- versus thiol-based GPx4
catalysis in cells and mice, as well as the role of the catalytic
tetrad in GPx4 catalysis and the subcellular localisation of the
different GPx4 isoforms and their impact on cell protection have
remained unclear. Therefore, a series of mutant variants of GPx4
were generated by site-directed mutagenesis, stably expressed in
tamoxifen-inducible GPx4 knockout cells (Seiler et al., Cell Metab
2008), and functionally analysed. GPx4 mutant variants included
those of the peroxidatic Sec, the catalytic tetrad, the
mitochondrial leader sequence (Mls) and the non-peroxidatic
cysteines. These studies revealed that Cys can functionally replace
Sec in the active centre of GPx4 to a large extent in cells,
whereas some amino acids of the catalytic site are necessary for
GPx4 function. None of the non-peroxidatic Cys were shown to be
required for the cell-protective function of GPx4, ruling out a
possible resolving Cys in the catalytic cycle of GPx4 unlike
homologues in other organisms that use a resolving Cys. The fuction
of a resolving Cys is to start a nucleophilic attack upon the
intermolecular bond between the peroxidatic Cys and the substrate
in order to create an intramolecular disulfidbride. Apparently
subcellular localization of GPx4 in the
extramitochondrial/cytosolic compartment is crucial for its cell
death preventing qualities as overexpression of mitochondrial GPx4
in the knockout cells failed to rescue cell death induced by
endogenous GPx4 disruption.