Why selenium rather than sulphur catalysis in peroxidases? More we learn, less we understand it

Hydrogen peroxide is both a byproduct of oxygen utilization during respiration and a physiological oxidant, build up to faith pathogens and act as a chemical signal in signal transduction cascades. Irrespective of its final use, hydrogen peroxide has also to be quickly removed to prevent collateral damage. A pivotal enzyme for this is the Selenocysteine (Sec)-containing enzyme Glutathione Peroxidase (GPx1), the first discovered of a family encompassing to date eight mammalian proteins. The amazing catalytic efficiency is provided by the reactivity with hydrogen peroxide of the selenol at the active site, in turn attributed to its low pKa. This redox reaction is indeed much faster than the enzyme substrate interaction, giving rise to unusual non-saturation kinetics where the calculated rate constant for the oxidation of the active site selenol (k+1) is around 108 M-1 sec-1. This peculiar reactivity is assumed similar for the other SecGPxs, although careful kinetic data are available only for GPx1 and 4. However, in the last years, new available information challenged the notion that the peculiar reactivity of GPxs can only be obtained when the redox moiety is selenol. First, a large number of GPxs have been identified in all living kingdoms, where the highly conserved active site contains a Cys residue in place of the redox-active Sec. As suggested by kinetic analysis of the Drosophila melanogaster variant (DmGPx), these CysGPxs are predicted only marginally less reactive with the hydroperoxide than the SecGPxs. Similarly, in peroxiredoxins, the oxidation of the peroxidatic Cys by hydrogen peroxide takes place with a k+1 in the range of 107 M-1 sec-1. Thus, apparently, when properly activated, a thiol can substitute for a selenol without affecting fast reduction of hydroperoxides. This raises the issue of the actual relevance of having a Sec residue in the active site. Aiming to better define the constraints for Se or S activation at the active site of GPxs, we obtained an updated picture of the active site of these enzymes by analyzing more than 700 structures for sequence homology and molecular modeling. Data were implemented with activity measurement and kinetic analysis of the DmGPx as a model, where some conserved residues were substituted by site directed mutagenesis. Our results suggest that the thiol or the selenol is activated by H bonding to the nitrogens of three strongly conserved residues, namely Gln 80, Trp 135 and Asn 136, while computational calculation of pKa and kinetic analysis, suggest that the pKa of the redox moiety is less relevant than H bonding. The deduced reaction mechanism suggests that, instead, the polarization of the peroxidic substrate and protonation of the hydroxyl-leaving group play a major role in accelerating the peroxidatic reaction. In conclusion, the previous notion that in nature Se is preferred to S just for a much faster reduction of hydroperoxides is not convincing anymore. Moreover, further complexity is added from phylogenetic analysis. While the ancestor of all GPxs is a CysGPx, and substitution of active site Cys with Sec is a relatively recent acquisition, for some GPxs an unexpected shift back to Cys is apparently taking place. In conclusion, from post-genomic acquisitions, unraveling the actual relevance about the use of Se rather than S in GPx catalysis, far from being clarified, appears more complex than before.