Extended Data Fig. 5: Role of ¹O₂ in IDO1–H₂O₂-mediated oxidation of Trp to cis-WOOH and Trp-induced arterial relaxation.

a, LC–MS/MS analysis of cis-WOOH (circles) and trans-WOOH (squares) following exposure of Trp to Rose Bengal and O₂ and light for the time indicated. b, Formation of cis- and trans-WOOH during oxidation of Trp by Rose Bengal and O₂ and light (RB) and IDO1–H₂O₂. c, Inhibition of IDO1–H₂O₂-mediated cis-WOOH formation by different pharmacological IDO1 inhibitors (all tested at 1 mM), with results expressed as the percentage of cis-WOOH formed in presence versus absence of inhibitor (control). d, IDO1-mediated formation of Trp-derived metabolites with bolus addition of H₂O₂ in Ar or O₂-flushed buffer. Metabolite yield is expressed as percentage of that observed in the reaction under air. e, Trp-induced relaxation of mouse abdominal aortas pretreated with mouse recombinant IFN-γ and vehicle (Ctrl), polyethylene glycol (PEG) or PEG-catalase (PEG-Cat). f, Fluorescence changes upon addition of H₂O₂ to SOSG in the presence of human serum albumin (HSA, red), or IDO1 without (green) and with Trp (black). g, LC–MS/MS chromatograms of the reaction mixture of IDO1 and EAS after incubation without (top) and with H₂O₂ (bottom). h, Fluorescence changes upon addition of H₂O₂ to SOSG in the presence of either IDO1 ± inactivated carbon monoxide-releasing molecule-A1 (iCORM-A1, green), IDO1 pretreated with CORM-A1 (red), urate (blue), or DEANO (black). i, NIR emission upon addition to H₂O₂ of IDO1 pretreated with CO gas. j, Formation of cis- and trans-WOOH during oxidation of Trp by horseradish peroxidase (HRP)–H₂O₂, myeloperoxidase (MPO)–H₂O₂ and purified IDO2–H₂O₂. k, Trp-induced relaxation of pre-constricted abdominal aortas from wild-type, Ido1^−/− or Ido1^−/−Ido2^−/− mice. l, Effect of cis-norbixin (100 μM) on relaxation of IFN-γ-pretreated mouse abdominal aorta induced by Trp or the nitric oxide donor DEANO. In b, c and f, reactions were initiated by the addition of H₂O₂–Trp and the mixtures incubated for 5–15 min at 25 °C before products were quantified by LC–MS/MS. Data shown are mean ± s.e.m. of three (a–d, j, k, relaxation to DEANO in l) or five (e, relaxation to Trp in l) independent experiments, or representative data (f, h) of three independent experiments. Ep, epacadostat; NLG, NLG919; RB, Rose Bengal. *P = 0.05 compared with absence of inhibitor (c) or air control (d), one-tailed Mann–Whitney test. The specific P values indicated were derived using a one-sided Mann–Whitney (l) and a Kruskal–Wallis test with Dunn’s multiple comparison (e).