Thomas J. Jönsson

Oxidation state governs structural transitions in peroxiredoxin II that correlate with cell cycle arrest and recovery.

Inactivation of eukaryotic 2-Cys peroxiredoxins (Prxs) by hyperoxidation has been proposed to promote accumulation of hydrogen peroxide (H2O2) for redox-dependent signaling events. We examined the oxidation and oligomeric states of PrxI and -II in epithelial cells during mitogenic signaling and in response to fluxes of H2O2. During normal mitogenic signaling, hyperoxidation of PrxI and -II was not detected. In contrast, H2O2-dependent cell cycle arrest was correlated with hyperoxidation of PrxII, which resulted in quantitative recruitment of ∼66- and ∼140-kD PrxII complexes into large filamentous oligomers. Expression of cyclin D1 and cell proliferation did not resume until PrxII-SO2H was reduced and native PrxII complexes were regenerated. Ectopic expression of PrxI or -II increased Prx-SO2H levels in response to oxidant exposure and failed to protect cells from arrest. We propose a model in which Prxs function as peroxide dosimeters in subcellular processes that involve redox cycling, with hyperoxidation controlling structural transitions that alert cells of perturbations in peroxide homeostasis.

Structure of the sulphiredoxin-peroxiredoxin complex reveals an essential repair embrace

Typical 2-Cys peroxiredoxins (Prxs) have an important role in regulating hydrogen peroxide-mediated cell signalling. In this process, Prxs can become inactivated through the hyperoxidation of an active site Cys residue to Cys sulphinic acid. The unique repair of this moiety by sulphiredoxin (Srx) restores peroxidase activity and terminates the signal. The hyperoxidized form of Prx exists as a stable decameric structure with each active site buried. Therefore, it is unclear how Srx can access the sulphinic acid moiety. Here we present the 2.6 Å crystal structure of the human Srx–PrxI complex. This complex reveals the complete unfolding of the carboxy terminus of Prx, and its unexpected packing onto the backside of Srx away from the Srx active site. Binding studies and activity analyses of site-directed mutants at this interface show that the interaction is required for repair to occur. Moreover, rearrangements in the Prx active site lead to a juxtaposition of the Prx Gly-Gly-Leu-Gly and Srx ATP-binding motifs, providing a structural basis for the first step of the catalytic mechanism. The results also suggest that the observed interactions may represent a common mode for other proteins to bind to Prxs.

Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins

The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 microM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.

Reduction of cysteine sulfinic acid in peroxiredoxin by sulfiredoxin proceeds directly through a sulfinic phosphoryl ester intermediate.

Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cys-SO}_{2}^{-}\) \end{document}. This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the γ-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8Å resolution crystal structure of Srx in complex with ATP and Mg2+. This structure reveals the role of the Mg2+ ion to position the γ-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Prx-SO}_{2}^{-}\) \end{document} species, support a mechanism where phosphorylation of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Prx-SO}_{2}^{-}\) \end{document} is the first chemical step.

The Peroxiredoxin Repair Proteins

Sulfiredoxin and sestrin are cysteine sulfinic acid reductases that selectively reduce or repair the hyperoxidized forms of typical 2-Cys peroxiredoxins within eukaryotes. As such these enzymes play key roles in the modulation of peroxide-mediated cell signaling and cellular defense mechanisms. The unique structure of sulfiredoxin facilitates access to the peroxiredoxin active site and novel sulfur chemistry.

Hydroxyapatite chromatography: altering the phosphate-dependent elution profile of protein as a function of pH.

Hydroxyapatite is a form of calcium phosphate thathaslongbeenusedinthechromatographicseparationofproteinsandDNA[1].Hydroxyapatiteisbestknownasacrystallinematerialbutisnowavailableinarangeofce-ramicderivativesthatarevastlysuperiorintermsofflowrate, stability, and reproducibility over many cycles ofuse.Thesedevelopmentshaveledtoarenewedinterestintheuseofthismediawithuniqueseparationproperties.Thisreportaimstofurtherextendthe usefulnessofhy-droxyapatiteforthepurificationofproteins.The adsorption of proteins to hydroxyapatite iscomplicated because it involves both anionic and cat-ionicexchange.TheCa

Identification of Intact Protein Thiosulfinate Intermediate in the Reduction of Cysteine Sulfinic Acid in Peroxiredoxin by Human Sulfiredoxin

The reversible oxidation of the active site cysteine in typical 2-Cys peroxiredoxins (Prx) to sulfinic acid during oxidative stress plays an important role in peroxide-mediated cell signaling. The catalytic retroreduction of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Prx}\mathrm{-}\mathrm{SO}_{2}^{-}\) \end{document} by sulfiredoxin (Srx) has been proposed to proceed through two novel reaction intermediates, a sulfinic phosphoryl ester and protein-based thiosulfinate. Two scenarios for the repair mechanism have been suggested that differ in the second step of the reaction. The attack of Srx or GSH on the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Prx}\mathrm{-}\mathrm{SO}_{2}\mathrm{PO}_{3}^{2-}\) \end{document} intermediate would result in either the formation of Prx-Cys-S(=O)–S-Cys-Srx or the formation of Prx-Cys-S(=O)–S-G thiosulfinates, respectively. To elucidate the mechanism of Prx repair, we monitored the reduction of human \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{PrxII}\mathrm{-}\mathrm{SO}_{2}^{-}\) \end{document} using rapid chemical quench methodology and electrospray ionization time-of-flight mass spectrometry. An 18O exchange study revealed that the Prx sulfinic acid phosphoryl ester is rapidly formed and hydrolyzed (k = 0.35 min–1). Furthermore, we observed the exclusive formation of a thiosulfinate linkage between Prx and Srx (k = 1.4 min–1) that collapses to the disulfide-bonded Srx-Prx species (k = 0.14 min–1). Thus, the kinetic and chemical competences of the first two steps in the Srx reaction have been demonstrated. It is clear, however, that GSH may influence thiosulfinate formation and that GSH and Srx may play additional roles in the resolution of the thiosulfinate intermediate.

Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N‐terminal domain (NTD) with two contiguous thioredoxin folds but only one redox‐active disulfide (within the sequence ‐Cys129‐His‐Asn‐Cys132‐). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase‐like segment of AhpF to AhpC, the peroxiredoxin component of the two‐protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in‐house X‐ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129–Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be ∼5.1, a relatively low pKa given its redox potential (∼265 mV) compared with most members of the thioredoxin family.

Protein Engineering of the Quaternary Sulfiredoxin·Peroxiredoxin Enzyme·Substrate Complex Reveals the Molecular Basis for Cysteine Sulfinic Acid Phosphorylation

Oxidative stress can damage the active site cysteine of the antioxidant enzyme peroxiredoxin (Prx) to the sulfinic acid form, Prx-SO2−. This modification leads to inactivation. Sulfiredoxin (Srx) utilizes a unique ATP-Mg2+-dependent mechanism to repair the Prx molecule. Using selective protein engineering that involves disulfide bond formation and site-directed mutagenesis, a mimic of the enzyme·substrate complex has been trapped. Here, we present the 2.1 Å crystal structure of human Srx in complex with PrxI, ATP, and Mg2+. The Cys52 sulfinic acid moiety was substituted by mutating this residue to Asp, leading to a replacement of the sulfur atom with a carbon atom. Because the Srx reaction cannot occur, the structural changes in the Prx active site that lead to the attack on ATP may be visualized. The local unfolding of the helix containing C52D resulted in the packing of Phe50 in PrxI within a hydrophobic pocket of Srx. Importantly, this structural rearrangement positioned one of the oxygen atoms of Asp52 within 4.3 Å of the γ-phosphate of ATP bound to Srx. These observations support a mechanism where phosphorylation of Prx-SO2− is the first chemical step.

Mutant AhpC Peroxiredoxins Suppress Thiol-Disulfide Redox Deficiencies and Acquire Deglutathionylating Activity

The bacterial peroxiredoxin AhpC, a cysteine-dependent peroxidase, can be converted through a single amino acid insertion to a disulfide reductase, AhpC*, active in the glutathione and glutaredoxin pathway. Here we show that, whereas AhpC* is inactive as a peroxidase, other point mutants in AhpC can confer the in vivo disulfide reductase activity without abrogating peroxidase activity. Moreover, AhpC* and several point mutants tested in vitro exhibit an enhanced reductase activity toward mixed disulfides between glutathione and glutaredoxin (Grx-S-SG), consistent with the in vivo requirements for these components. Remarkably, this Grx-S-SG reductase activity relies not on the peroxidatic cysteine but rather on the resolving cysteine that plays only a secondary role in the peroxidase mechanism. Furthermore, putative conformational changes, which impart this unusual Grx-S-SG reductase activity, are transmissible across subunits. Thus, AhpC and potentially other peroxiredoxins in this widespread family can elaborate a new reductase function that alleviates disulfide stress.