Fig. 7: Stereoselective oxidation of a target protein by cis-WOOH.

a Representative (n=6) immunoblot showing the effects of endogenous cis-WOOH formation on the redox states PKG1α, Prx2, and Prx4 in mesenteric resistance arteries isolated from LPS-treated mice after one-minute infusion of vehicle or l-Trp (8 mM) via the jugular vein. a–e Summary data of experiments performed in (a): l-Trp significantly increased dimerization of PKG1α (p = 0.0173) (b) without altering the thiol redox state of Prx2 (c) or Prx4 (d). Data in b, c, and d, are shown as mean ± SEM (n = 6 independent experiments) and statistical analysis performed using two-tailed Mann–Whitney tests with *p ≤ 0.05. e Purified recombinant PKG1α (3.3 µM) was incubated with 200 µM cis-WOOH or H₂O₂ for 5 min in the presence of 200 µM tris(2-carboxyethyl)phosphine (TCEP) in 25 mM phosphate buffer pH 7.0 at 25 °C. At the specified time points, an aliquot was collected, and the reaction stopped by the addition of 125 mM maleimide. PKG1α redox state (proportion monomer and dimer to total) was assessed using non-reducing SDS-PAGE and silver staining. f Percentage of PKG1α monomer and dimer of total PKG1α was determined by densitometry for the samples analyzed in (e). g Model of stereospecific hindrance of reductive inactivation of cis-WOOH facilitating downstream target protein oxidation. Accordingly, H₂O₂ is required for but does not itself cause, oxidation of target signaling proteins. Rather, H₂O₂ is utilized by IDO1 in the presence of Prx to form an amino acid-derived hydroperoxide in a stereospecific manner, i.e., preferential formation of cis- over trans-WOOH. cis-WOOH and trans-WOOH differently bind to and react with Prx and downstream protein targets that may limit exposure of the hydroperoxide moiety of cis-WOOH to the catalytic cysteine residue in Prx. If so, this impedes Prx oxidation/cis-WOOH reduction, thereby facilitating cis-WOOH reaction with downstream target proteins such as PKG1α. Interaction with Prx2 leads to comparatively enhanced reduction of trans-WOOH, thereby impeding reaction of trans-WOOH with target proteins. We propose that this stereospecific evasion of reduction by cis-WOOH allows for greater spatial regulation of redox signaling such as inter-cellular signaling, and in cells with high Prx concentrations. See Discussion for further details. Uncropped blots and source data are provided as a Source data file.