The typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sulfenic acid. Sulfiredoxin was originally proposed to operate by covalent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-reducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of ∼2 min^–1, for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoichiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thiosulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway.

Bryophytes produce rare and bioactive compounds with a broad range of therapeutic potential, and many species are reported in ethnomedicinal uses. However, only a few studies have investigated their potential as natural anti-inflammatory drug candidate compounds. The present study investigates the anti-inflammatory effects of thirty-two species of bryophytes, including mosses and liverworts, on Raw 264.7 murine macrophages stimulated with lipopolysaccharide (LPS) or recombinant human peroxiredoxin (hPrx1). The 70% ethanol extracts of bryophytes were screened for their potential to reduce the production of nitric oxide (NO), an important pro-inflammatory mediator. Among the analyzed extracts, two moss species significantly inhibited LPS-induced NO production without cytotoxic effects. The bioactive extracts of Dicranum majus and Thuidium delicatulum inhibited NO production in a concentration-dependent manner with IC₅₀ values of 1.04 and 1.54 µg/mL, respectively. The crude 70% ethanol and ethyl acetate extracts were then partitioned with different solvents in increasing order of polarity (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol). The fractions were screened for their inhibitory effects on NO production stimulated with LPS at 1 ng/mL or 10 ng/mL. The NO production levels were significantly affected by the fractions of decreasing polarity such as n-hexane and diethyl ether ones. Therefore, the potential of these extracts to inhibit the LPS-induced NO pathway suggests their effective properties in attenuating inflammation and could represent a perspective for the development of innovative therapeutic agents.