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Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2.

ABSTRACT: Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H₂O₂ and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H₂O₂ reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H₂O₂. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.

SUBMITTER: Peskin AV

PROVIDER: S-EPMC8049995 | biostudies-literature | 2021 Jan-Jun

REPOSITORIES: biostudies-literature

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Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2.

Peskin Alexander V AV Meotti Flavia C FC Kean Kelsey M KM Göbl Christoph C Peixoto Albert Souza AS Pace Paul E PE Horne Christopher R CR Heath Sarah G SG Crowther Jennifer M JM Dobson Renwick C J RCJ Karplus P Andrew PA Winterbourn Christine C CC

The Journal of biological chemistry 20210101

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H<sub>2</sub>O<sub>2</sub> and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulf ...[more]

PMID: 33667550

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Project description:Hydrogen peroxide (H2O2) plays a crucial role in cell signaling in response to physiological and environmental perturbations. H2O2 can oxidize typical 2-Cys peroxiredoxin (PRX) first into a sulfenic acid, which resolves into a disulfide that can be reduced by thioredoxin (TRX)/TRX reductase (TR). At high levels, H2O2 can also hyperoxidize sulfenylated PRX into a sulfinic acid that can be reduced by sulfiredoxin (SRX). Therefore, PRX, TRX, TR, and SRX (abbreviated as PTRS system here) constitute the coupled sulfenylation and sulfinylation cycle (CSSC), where certain oxidized PRX and TRX forms also function as redox signaling intermediates. Earlier studies have revealed that the PTRS system is capable of rich signaling dynamics, including linearity, ultrasensitivity/switch-like response, nonmonotonicity, circadian oscillation, and possibly, bistability. However, the origins of ultrasensitivity, which is fundamentally required for redox signal amplification, have not been adequately characterized, and their roles in enabling complex nonlinear dynamics of the PTRS system remain to be determined. Through in-depth mathematical modeling analyses, here we revealed multiple sources of ultrasensitivity that are intrinsic to the CSSC, including zero-order kinetic cycles, multistep H2O2 signaling, and a mechanism arising from diminished H2O2 removal at high PRX hyperoxidation state. The CSSC, structurally a positive feedback loop, is capable of bistability under certain parameter conditions, which requires embedding multiple sources of ultrasensitivity identified. Forming a negative feedback loop with cytosolic SRX as previously observed in energetically active cells, the mitochondrial PTRS system (where PRX3 is expressed) can produce sustained circadian oscillations through supercritical Hopf bifurcations. In conclusion, our study provided novel quantitative insights into the dynamical complexity of the PTRS system and improved appreciation of intracellular redox signaling.

Dataset Information

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2.

Publications

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2.

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