Going Through the Barrier: Coupled Disulfide Exchange Reactions Promote Efficient Catalysis in Quiescin Sulfhydryl Oxidase [Protein Structure and Folding]

December 30th, 2013 by Israel, B. A., Kodali, V. K., Thorpe, C.

The Quiescin-sulfhydryl oxidase (QSOX) family of enzymes generate disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CxxC motif of the thioredoxin domain is comparatively oxidizing (E°' of -144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CxxC disulfide in the ERV domain of TbQSOX is strongly reducing (E°' of -273 mV) representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E°' of -153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed-disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the TRX domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequences. Mixed-disulfide bond formation is accompanied by the generation of a charge-transfer complex with the flavin cofactor. This provides thermodynamic coupling between the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm, and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.
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