Native state fluctuations in a peroxiredoxin active site match motions needed for catalysis

Area of Science:

• Biochemistry
• Enzymology
• Redox Biology

Background:

• Peroxiredoxins are essential enzymes involved in peroxide detoxification and redox signaling.
• Their catalytic mechanism involves a peroxidatic cysteine (Cₚ) undergoing oxidation and disulfide formation with a resolving cysteine, often coupled with conformational changes.

Purpose of the Study:

• To investigate the dynamics and conformational states of Xanthomonas campestris peroxiredoxin Q.
• To determine if a catalytically relevant local unfolding equilibrium exists in the enzyme's resting state.

Main Methods:

• Nuclear magnetic resonance (NMR) spectroscopy was employed.
• Chemical exchange saturation transfer (CEST) was used to probe protein dynamics.

Main Results:

• NMR analysis revealed 26 residues in the active site region of peroxiredoxin Q are in dynamic exchange.
• A high-energy, locally unfolded state exists in equilibrium (2.5% population) with the resting Cₚ-thiolate form, with an exchange rate of 72 s⁻¹.
• Faster motions indicate active site instability potentially linked to Cₚ-sulfenate formation.

Conclusions:

• This study unequivocally establishes a catalytically relevant local unfolding equilibrium in the Cₚ-thiolate form of peroxiredoxin Q.
• The observed active site dynamics and instability likely facilitate the enzyme's catalytic cycle, including peroxide reduction and redox signaling regulation.