The Dual Role of Active Site Hydroxylated Residue in Peroxiredoxin Sulfinylation Catalysis.

Peroxiredoxins (Prx) are ubiquitous Cys peroxidases regulated by sulfinylation, a modification that occurs when the sulfenic acid generated on the catalytic Cys by peroxide reduction reacts with a second molecule of peroxide. In the Prx1 family, sulfinylation sensitivity is controlled by competition between a structural transition from a fully folded (FF) to locally unfolded (LU) conformation and the chemical step of sulfinylation. The initial peroxide reduction relies on a conserved catalytic hydroxylated residue that allows peroxide optimal activation. This study aimed at investigating the role of this catalytic residue in sulfinylation. Sulfenate attack on peroxide was favored by one order of magnitude when a catalytic Thr was present, for yeast cytosolic Prx1-type enzymes, human Prx1 and yeast mitochondrial Prx, a Prx6-type enzyme. Furthermore, pKa determination supported the notion of electrostatic interaction between the catalytic hydroxyl and sulfenate intermediate. Finally, FF-LU transition kinetics was faster with a catalytic Thr, supporting that the hydroxyl group proximity to the nascent sulfenate group also promotes the FF-LU transition. We identify a major mechanism that activates sulfinylation in hyperoxidation-sensitive Prxs from the Prx1 and Prx6 families. Furthermore, we show that the catalytic hydroxylated residue holds a dual role in regulating hyperoxidation sensitivity, by activating the sulfinylation reaction, while also promoting the competing FF to LU transition, thus acting as an important regulatory determinant. The present work sets the basis for investigating other instances of Cys proteins regulated by sulfinylation, a modification increasingly recognized in cell redox regulation and signaling. 00, 000-000.