9 - Dissecting the Mechanism of Prx2-Mediated H2O2 Sensing

Abstract

Peroxiredoxins (Prx) are a large family of thiol-dependent peroxidases that reduce hydroperoxides at expense of thioredoxin or other disulfide reductases. Human Prx2 is well expressed in the cytosol, a 2-Cys typical Prx. The first reaction in the catalytic cycle, the peroxidatic cysteine with H 2 O 2 , is extraordinary fast (10 8 M -1 s -1 ) placing Prx as preferential target for intracellular H 2 O 2 . Oxidation to sulfenic acid of the reactive Cys inside the decameric reduced protein results in conformational changes at the active site needed for the attack of the resolving Cys to form a disulfide. This conformational transition needed for catalysis imposes a kinetic pause that can result in overoxidation of the active-site Cys before disulfide formation, a modification that has been linked to redox signaling pathways. Among mammalian Prx, Prx2 is particularly sensitive to hyperoxidation and sensitivity to hyperoxidation is linked to the rate of disulfide formation. By stopped-flow fluorimetry we characterize the different kinetic steps in the catalysis at increasing levels of H 2 O 2 and at different pHs. Our results confirm the first step in the cycle, reaction with H 2 O 2 is fast, and step 2, resolution, is the rate limiting step. The peroxidatic cys is acidic, pKa = 4.8 but it is not the only factor responsible for the outstanding rate of H 2 O 2 reduction. The reactivity of the groups involved in second step (resolving thiol and peroxidatic sulfenic) are not affected by changes in the pKas (8.0 and 7.0, respectively) but rather determined by conformational changes, from the so-called fully-folded FF to the locally-unfolded LU conformation.