Extended Data Fig. 9: Proposed mechanism for formation of ¹O₂ by IDO1 plus H₂O₂ and chemical synthesis of epacadostat.

a, Proposed mechanism for formation of ¹O₂ by IDO1 plus H₂O₂. Reaction of Fe³⁺-haem IDO1 with H₂O₂ results in formation of the two-electron oxidized form, compound I, consisting of a Fe⁴⁺=O iron centre and a porphyrin cation radical (not shown). The latter is transferred to an amino acid side chain of IDO1, such as Trp (not shown) or—more likely—a Tyr such as Y126 or Y353, which are close to the active-site haem in IDO1⁶¹. The IDO1 tyrosyl radical may then engage in the formation of fluorescent di-tyrosine crosslinks or H-abstraction reactions leading to protein fragmentation according to established pathways⁶⁰. Alternatively, a resonance form of the IDO1 tyrosyl radical may add to molecular oxygen, resulting in the formation of a quinone peroxy radical at the para position (for example, shown on R1-Tyr-R2) or the ortho position (for example, shown on R6-Tyr-R7). The resulting intermediate quinone peroxy radical may then participate in inter- or intra-residue peroxy radical combination, the latter subsequent to additional H-abstraction and oxidation reactions and formation of a putative intra-residue di-peroxy radical. For inter-residue peroxy radical combination to occur, it is hypothesized that the haem of IDO1 catalyses the decomposition of the hydroperoxide to an alkoxy radical. In the scheme, this reaction is combined with oxygen addition to the alpha carbon radical. The resulting tetroxides will decay with release of ¹O₂ according to the Russell mechanism²⁶. The observed stereospecific formation of cis-WOOH by IDO1–H₂O₂–l-Trp suggests formation of ¹O₂ close to the active site haem rather than release into the bulk phase, and may therefore be more consistent with the intra-residue peroxy radical combination pathway. For clarity, the R1–R7 labels represent the continuation of the peptide backbone. In the case of the inter-residue peroxy radical combination, it is envisioned that peroxy radicals on two different Tyr residues within the same IDO1 molecule combine. b, Chemical synthesis of epacadostat.