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 ϳ2 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.

Sulfiredoxin catalyzes the ATP-dependent reduction of overoxidized eukaryotic 2-Cys peroxiredoxin PrxSOA growing number of studies have shown the importance of the versatility of Cys redox biochemistry in the regulation of various cellular processes such as catalysis, metal binding, or signal transduction (1). The typical eukaryotic 2-Cys-peroxiredoxins (Prx) 3 represent a family of proteins that exemplifies these mechanisms, as these thiol peroxidases have been described under six redox states of the essential Cys residue, from ϪII to ϩIV. For example, the Cys under reduced, disulfide, and sulfenic forms (oxidation states ϪII, ϪI, and 0, respectively) is involved in the catalytic mechanism of Prx as peroxidase enzymes (2-5), the disulfide and sulfenic states in their function as redox sensor (6, 7), and the sulfinic (ϩII) and sulfonic (ϩIV) states in possible chaperon-like function (8, 9). Cysteine under the sulfinic state ϩII (PrxSO 2 ) is formed by a mechanism of escape of the reactive sulfenic acid intermediate during the catalytic peroxidase cycle (5, 10). This overoxidation constitutes a post-translational modification that is thought to afford a regulation mechanism between these different functions depending on the oxidative stress conditions. In addition, Prxs are also subject to other post-translational modifications by phosphorylation and N-acetylation (11,12).In contrast to Cys under ϪI and 0 oxidation states, the Cys under sulfinate state is not reducible by cellular thiols such as glutathione and thioredoxin (Trx). Therefore, regulation of Prx functions is dependent on a sulfinyl reductase referred to as sulfiredoxin (Srx), which catalyzes the ATP-dependent reduction of PrxSO 2 into sulfenic Prx (PrxSOH) (13-15). Although the sestrin family of human proteins was initially proposed to possess this activity, the Srxs appear to be the only enzymes with sulfinyl reductase activity (16). Recent mechanistic studies on Srx from Saccharomyces cerevisiae and human origin support a mechanism in which the sulfinic moiety of the PrxSO 2 substrate is first activated by formation of an anhydride bond with the ␥-phosphate of ATP, leading to a phosphoryl sulfinic intermediate, followed by attack of Srx catalytic Cys, which results in a thiosulfinate intermediate PrxSO-SSrx (oxidation state of Prx Cys ϩI) (Fig. 1) (17-19). Such a mechanism implies the recycling of Srx into the reduced form.In the case of S. cerevisiae Srx, one product of the reaction with PrxSO 2 and ATP in the absence of added reductant was a monomeric form of Srx oxidized under a disulfide state (17). In addition, Trx was shown to act as a reductant in the catalytic cycle at a rate that is not limiting compared with the rate of the first steps of the reaction (17). Thus, several questions have to be addressed regarding the mechanism of the recycling process of Srx. First, what is the mechanism of the recycling of Srx from S. cerevisiae?

Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.

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