Determination of kinetic constants of the peroxiredoxins reduction by thioredoxins through fluorescence changes in different redox states

Abstract

2-Cys Peroxiredoxins (2-Cys Prx) are antioxidant enzymes able to decompose a variety of hydroperoxides (R-OOH) using a cysteine residue, named peroxidation cysteine (CP-SH), which is oxidized to cysteine sulfenic acid (CP-SOH) in the peroxide decomposition process. 2-Cys Prx possesses a second cysteine, (resolving cysteine - CR) that forms a disulfide bond during the catalytic cycle with CP. The disulfide is frequently reduced by the enzyme thioredoxin (Trx). Additionally, to Prx reduction, the Trx enzymes are involved in diverse biological processes such as cell growth, inhibition of apoptosis, transcriptional activation, and DNA synthesis. Initial studies involving steady-state kinetics indicated that the 2-Cys Prx had lower rates of decomposition of hydroperoxides, 104-5 M-1s-1, however new approaches involving changes in Prx fluorescence or competitive kinetics with HRP revealed that these enzymes have a reactivity over hydroperoxides so higher as 107-8 M-1s-1. In high oxidative stress, the CP can over oxidize, forming overoxidized species such as cysteine sulfinic acid (CP-SO2H) and sulfonic (CP-SO3H). In eukaryotes, CP overoxidation of causes the loss of peroxidase activity, since these species are not reduced by Trx. However, overoxidation alters the quaternary structure of Prx, resulting in the formation of high molecular weight structures with molecular chaperone properties. In this context, the relationship of Prx with Trx appears to have high importance since in Dtrx eukaryotic lineages the oligomerization of Prx is highly compromised. In Saccharomyces cerevisiae, there are two isoforms of cytosolic Prx (Tsa1 and Tsa2) which are very similar and are highly related to human isoforms Prx1 and Prx2 (67% identity and 77% similarity). Tsa1 and Tsa2 exhibit chaperone activity and both enzymes are efficiently reduced by the cytosolic thioredoxin, Trx1, and Trx2, which share high similarity in their primary structures (78% identity and 89% similarity). Recent studies involving competitive kinetic with HRP revealed that Tsa1 Tsa2 and enzymes present in the composition rates of H2O2 and NOO-of 107-8 M-1s-1. Additionally, it has been shown that Trx1 and Trx2 are efficiently reduced by the TrxR1 107 M-1s-1. However, the determination ok kinetic contents of the thioredoxin-dependent peroxidase activity of Prx enzymes using the Trx system (NADPH®TrxR®Trx ®Prx®R-OOH) reveals low rates of peroxide decomposition (104-5 M-1s-1). In this context, the reduction rates for Trx1 or Trx2 appear to be limiting for the reduction of Prx, however, no study till the present determined the specific rates of disulfide reduction of Tsa1 and Tsa2 by Trx1 or Trx2. Additionally, we have recently demonstrated that E50 and R146 residues of Tsa1 are related to the interaction with Trx, since mutants Tsa1E50A and Tsa1R146Q presented a very noteworthy decrease of Trx-dependent peroxidase, using the Trx system however it was not possible to quantify accurately the decay rates reduction. This project aims to determine the rates of Tsa1 and Tsa2 and mutant TsaE50A and TsaR146Q reduction by Trx1 and Trx2, by means of intrinsic fluorescence differences of Prx and mutants using Trx1W29F and Trx2W30F through the stopped flow. We also will perform efforts to determine the rates of reduction of Prx1 and Prx2 by human Trx (Trx2W30F). Since the rates of reduction of Prx by Trx are related to maintenance of redox homeostasis and also is related to the switch of peroxidase®chaperone function, we believe that the results of this project can provide important contributions to a better understanding of the 2-Cys Prx biology enzymes. (AU)