Syun-Ru Yeh

Human tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two important targets in cancer immunotherapy. Extensive research has led to a large number of potent IDO inhibitors; in addition, 52 structures of IDO in complex with inhibitors with a wide array of chemical scaffolds have been documented. In contrast, progress in the development of TDO inhibitors has been limited. Only four structures of TDO in complex with competitive inhibitors that compete with the substrate L-tryptophan for binding to the active site have been reported to date. Here we systematically evaluated the structures of TDO in complex with competitive inhibitors with three types of pharmacophores, imidazo-isoindole, indole-tetrazole, and indole-benzotriazole. The comparative assessment of the protein-inhibitor interactions sheds new light into the structure-based design of enzyme-selective inhibitors.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable O_H state, and a reductive phase, in which O_H is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if O_H is allowed to relax to the resting oxidized state (O), a redox equivalent to O_H, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a₃ iron and Cu_B in the active site of the O state, like those in the O_H state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O_H, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide insights into the proton translocation mechanism of CcO.

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) composed of a copper atom (Cu_B) and a heme a₃ iron, where O₂ binds and is reduced to water. CO is a versatile O₂ surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a₃ iron leads to a metastable intermediate (Cu_B-CO), where CO is bound to Cu_B, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a₃ iron and moved to a temporary binding site midway between the Cu_B and the heme a₃ iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a₃ iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.

Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu _B ) and a heme a ₃ iron, where O ₂ binds and is reduced to water. CO is a versatile O ₂ surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a ₃ iron leads to a metastable intermediate (Cu _B -CO), where CO is bound to Cu _B , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a ₃ iron and moved to a temporary binding site midway between the Cu _B and the heme a ₃ iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a ₃ iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

Cytochrome c oxidase (C c O) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis ¹ . The full turnover of the C c O reaction involves an oxidative phase, in which the reduced enzyme ( R ) is oxidized by molecular oxygen to the metastable oxidized O _H state, and a reductive phase, in which O _H is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes ² . However, if O _H is allowed to relax to the resting oxidized state ( O ), a redox equivalent to O _H , its subsequent reduction to R is incapable of driving proton translocation ^2,3 . How the O state structurally differs from O _H remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX) ⁴ , we show that the heme a ₃ iron and Cu _B in the active site of the O state, like those in the O _H state ^5,6 , are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three Cu _B ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O _H , where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of C c O.

Mammalian nitric oxide synthase (NOS) mediates the two‐step O₂‐dependent oxidative degradation of arginine, and has been linked to a medley of disease situations in humans. Nonetheless, its exact mechanism of action still remains unclear. This work presents the first NOS model system where biologically proposed heme superoxo and peroxo intermediates are assessed as active oxidants against oxime substrates. Markedly, heme peroxo intermediates engaged in a bioinspired oxime oxidation reaction pathway, converting oximes to ketones and nitroxyl anions (NO⁻). Detailed thermodynamic, kinetic, and mechanistic interrogations all evince a rate‐limiting step primarily driven by the nucleophilicity of the heme peroxo moiety. Coherent with other findings, ¹⁸O and ¹⁵N isotope substitution experiments herein suffice compelling evidence toward a detailed mechanism, which draw close parallels to one of the enzymatic proposals. Intriguingly, recent enzymatic studies also lend credence to these findings, and several relevant reaction intermediates have been observed during NOS turnover.

Mammalian nitric oxide synthase (NOS) mediates the two‐step O₂‐dependent oxidative degradation of arginine, and has been linked to a medley of disease situations in humans. Nonetheless, its exact mechanism of action still remains unclear. This work presents the first NOS model system where biologically proposed heme superoxo and peroxo intermediates are assessed as active oxidants against oxime substrates. Markedly, heme peroxo intermediates engaged in a bioinspired oxime oxidation reaction pathway, converting oximes to ketones and nitroxyl anions (NO⁻). Detailed thermodynamic, kinetic, and mechanistic interrogations all evince a rate‐limiting step primarily driven by the nucleophilicity of the heme peroxo moiety. Coherent with other findings, ¹⁸O and ¹⁵N isotope substitution experiments herein suffice compelling evidence toward a detailed mechanism, which draw close parallels to one of the enzymatic proposals. Intriguingly, recent enzymatic studies also lend credence to these findings, and several relevant reaction intermediates have been observed during NOS turnover.