Fig. 3

TriPer’s responsiveness to H₂O₂ in a thiol-oxidizing environment. a Traces of time-dependent changes to the excitation ratio of HyPer (orange squares), TriPer (blue spheres), and TriPer mutant lacking its peroxidatic cysteine (TriPer ^C199S, black diamonds) following the introduction of oxidized PDI (8 μM, empty squares, spheres, and diamonds) or diamide (general oxidant, 2 mM, filled squares and spheres). b Ratiometric traces (as in a) of HyPer and TriPer sequentially exposed to oxidized PDI (8 μM) and H₂O₂ (2 μM). c Ratiometric traces (as in a) of HyPer or TriPer exposed to H₂O₂ (4 μM) followed by DTT (2.6 mM). The excitation spectra of the reaction phases (1–4) are analyzed in Additional file 3: Figure S3C. d Schema of TriPer oxidation pathway. Oxidants drive the formation of the optically distinct (high R^488/405) C_P199-C_R208 (C_Peroxidatic, C_Resolving) disulfide, which re-equilibrates with an optically inert (low R^488/405) C_D187-C_R208 (C_Divergent, C_Resolving) disulfide in a redox relay [44–46] imposed by the three-cysteine system. The pool of TriPer with a reduced peroxidatic C_P199 thus generated is available to react with H₂O₂, forming a sulfenic intermediate. Resolution of this intermediate in trans shifts TriPer to a new, optically indistinct low R^488/405 state, depleting the original optically distinct (high R^488/405) intermolecular disulfide C_P199-C_R208. This accounts for the biphasic response of TriPer to H₂O₂ (Fig. 2b) and for its residual responsiveness to H₂O₂ after oxidation by PDI (b of this figure)