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The widespread thioredoxin superfamily enzymes typically share the following features: a characteristic α-β fold, the presence of a Cys-X-X-Cys (or Cys-X-X-Ser) redox-active motif, and a proline in the cis configuration abutting the redox-active site in the tertiary structure. The Cys-X-X-Cys motif is at the solvent-exposed amino terminus of an α-helix, allowing the first cysteine to engage in nucleophilic attack on substrates, or substrates to attack the Cys-X-X-Cys disulfide, depending on whether the enzyme functions to reduce, isomerize, or oxidize its targets. We report here the X-ray crystal structure of an enzyme that breaks many of our assumptions regarding the sequence-structure relationship of thioredoxin superfamily proteins. The yeast Protein Disulfide Isomerase family member Eps1p has Cys-X-X-Cys motifs and proline residues at the appropriate primary structural positions in its first two predicted thioredoxin-fold domains. However, crystal structures show that the Cys-X-X-Cys of the second domain is buried and that the adjacent proline is in the trans, rather than the cis isomer. In these configurations, neither the "active-site" disulfide nor the backbone carbonyl preceding the proline is available to interact with substrate. The Eps1p structures thus expand the documented diversity of the PDI oxidoreductase family and demonstrate that conserved sequence motifs in common folds do not guarantee structural or functional conservation.

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., (© 2014 The Protein Society.)