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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 . 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.

Prdx1 Interacts with ASK1 upon Exposure to H2O2 and Independently of a Scaffolding Protein

“…The reason behind this is likely to be purely technical: BiFC simply cannot capture small differences in reaction rates. The slightly slower k on of the resolving cysteine mutant could be a consequence of minor local structural changes by which the efficiency of reactivity of C P is partially being compromised, as reported recently for Prdx2 [51]. The reaction of the resolving cysteine Prdx1 mutant with urate hydroperoxide was also lower than for the WT [61].…”

“…This Prdx1 variant was less sensitive to hyperoxidation compared to Prdx1 WT. Peskin et al [51] also noticed this in some of the resolving cysteine mutants of Prdx2, where they showed that the rate of hyperoxidation was dramatically decreased compared to Prdx2 WT [51].…”

Effects of Serine or Threonine in the Active Site of Typical 2-Cys Prx on Hyperoxidation Susceptibility and on Chaperone Activity

“…It has been shown by SEC and TEM that the AhpC from Enterococcus faecalis, a naturally Ser-Prx, is a decamer independent of the redox state [43], similarly to yeast Tsa2 [3]. Thus, our results showed that the presence of Thr or Ser in the catalytic triad is a relevant factor related to the redox sensitive oligomerization, but additional features also interfere with the quaternary assembly of typical 2-Cys Prx [43,45,61].…”

“…In fact, our data indicated that, in yeast enzymes (displaying the GGLG/YF motifs), the differences in hyperoxidation are more pronounced between Ser-Prx and Thr-Prx. Accordingly, Peskin and colleagues (2021) [61] showed that substitution of human Prx2 C R by amino acids that disrupts the C-terminal structure is able to affect the enzyme activity, indicating an interconnection between the C-terminal region and the active site microenvironment in eukaryotes. These data indicate that even regions located far from the active site can exert effect in the 2-Cys Prx activity.…”

The Human 2-Cys Peroxiredoxins form Widespread, Cysteine-Dependent- and Isoform-Specific Protein-Protein Interactions

“…It is difficult to unambiguously exclude that proteins no longer bind to the used peroxiredoxin mutants due to structural changes other than loss of the cysteine thiol. For the resolving cysteine mutants at least, a recent study shows that the cysteine to serine mutation has only a limited effect on the rate of oxidation of the peroxidatic cysteine in PRDX2 [32]. Characterization of the functional consequences of specific peroxiredoxinbased interactions is outside the scope of this study.…”