title: Single-molecule spectroscopy exposes hidden states in an enzymatic electron relay.
abstract: The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles.
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Iris
authors.initials: I
authors.lastname: Grossman
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Haim
authors.initials: H
authors.lastname: Yuval Aviram
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Gad
authors.initials: G
authors.lastname: Armony
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Amnon
authors.initials: A
authors.lastname: Horovitz
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Hagen
authors.initials: H
authors.lastname: Hofmann
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Gilad
authors.initials: G
authors.lastname: Haran
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Deborah
authors.initials: D
authors.lastname: Fass
mesh_terms: D004579:Electron Transport; D031541:Fluorescence Resonance Energy Transfer; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism*; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C548687:quiescin sulfhydryl oxidase, Trypanosoma brucei