How thioredoxin can reduce a buried disulphide bond

Area of Science:

• Biochemistry
• Enzymology
• Protein redox biology

Background:

• Arsenate reductase (ArsC) from Staphylococcus aureus catalyzes arsenate reduction to arsenite.
• ArsC utilizes three redox-active cysteine residues (Cys10, Cys82, Cys89) in its catalytic cycle.
• Oxidized ArsC can form inter-cysteine disulfide bridges, impacting enzyme activity and stability.

Purpose of the Study:

• To investigate the interaction mechanism between thioredoxin and oxidized arsenate reductase (ArsC).
• To elucidate how thioredoxin facilitates the reduction of ArsC, particularly under conditions of double oxidation.
• To understand the role of specific cysteine residues and disulfide bridges in ArsC's redox regulation.

Main Methods:

• Studied the interaction between thioredoxin and Staphylococcus aureus pI258 arsenate reductase (ArsC).
• Analyzed the formation and accessibility of disulfide bridges (Cys82-Cys89 and Cys10-Cys15) in ArsC.
• Investigated the role of an intramolecular Cys10-Cys82 disulfide switch in the reduction process.

Main Results:

• Thioredoxin reduces ArsC by interacting with a looped-out redox helix, targeting the Cys82-Cys89 disulfide.
• A second, buried disulfide bridge (Cys10-Cys15) forms in the active site, hindering thioredoxin access.
• An intramolecular Cys10-Cys82 disulfide switch is essential for reducing the buried Cys10-Cys15 bridge.

Conclusions:

• Thioredoxin selectively reduces ArsC when the redox helix is looped out.
• The formation of a buried active-site disulfide bridge may protect nucleophilic cysteines from irreversible oxidation.
• A unique intramolecular disulfide switch mechanism enables full reduction of double-oxidized ArsC by thioredoxin.