title: Enzyme structure captures four cysteines aligned for disulfide relay.
abstract: Thioredoxin superfamily proteins introduce disulfide bonds into substrates, catalyze the removal of disulfides, and operate in electron relays. These functions rely on one or more dithiol/disulfide exchange reactions. The flavoenzyme quiescin sulfhydryl oxidase (QSOX), a catalyst of disulfide bond formation with an interdomain electron transfer step in its catalytic cycle, provides a unique opportunity for exploring the structural environment of enzymatic dithiol/disulfide exchange. Wild-type Rattus norvegicus QSOX1 (RnQSOX1) was crystallized in a conformation that juxtaposes the two redox-active di-cysteine motifs in the enzyme, presenting the entire electron-transfer pathway and proton-transfer participants in their native configurations. As such a state cannot generally be enriched and stabilized for analysis, RnQSOX1 gives unprecedented insight into the functional group environments of the four cysteines involved in dithiol/disulfide exchange and provides the framework for analysis of the energetics of electron transfer in the presence of the bound flavin adenine dinucleotide cofactor. Hybrid quantum mechanics/molecular mechanics (QM/MM) free energy simulations based on the X-ray crystal structure suggest that formation of the interdomain disulfide intermediate is highly favorable and secures the flexible enzyme in a state from which further electron transfer via the flavin can occur.
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Yair
authors.initials: Y
authors.lastname: Gat
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vardi-Kilshtain
authors.forename: Iris
authors.initials: I
authors.lastname: Grossman
authors.forename: Dan Thomas
authors.initials: DT
authors.lastname: Major
authors.forename: Deborah
authors.initials: D
authors.lastname: Fass
mesh_terms: D000818:Animals; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011789:Quantum Theory; D051381:Rats; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004220:Disulfides; D013879:Thioredoxins; C525178:quiescin Q6 sulfhydyl oxidase 1, rat; D003545:Cysteine
keywords: X-ray crystallography; cis-proline; enzyme mechanism; flavin adenine dinucleotide; quantum mechanics/molecular mechanics; thioredoxin fold