Observing Confined Local Oxygen-induced Reversible Thiol/Disulfide Cycle with a Protein Nanopore

Area of Science:

• Biochemistry
• Chemical Physics
• Nanotechnology

Background:

• Thiol-based redox regulation is crucial in biological systems.
• The role of local oxygen in protein disulfide bond dynamics remains poorly understood due to limited analytical tools.
• Protein confinement effects on redox reactions are largely unexplored.

Purpose of the Study:

• To investigate how local oxygen concentration influences the reversible thiol/disulfide cycle within a confined protein environment.
• To develop and utilize single-molecule techniques for real-time monitoring of redox reactions.
• To elucidate the mechanisms by which nanoconfinement affects oxygen-mediated redox processes.

Main Methods:

• Utilized a protein nanopore system within a glove box to precisely control local oxygen levels.
• Employed single-molecule sensing to monitor the thiol/disulfide cycle in real-time.
• Performed kinetic calculations to analyze reaction mechanisms.

Main Results:

• Nanoconfinement within protein nanopores enhances the frequency of effective reactant collisions, facilitating the disulfide formation and cleavage cycle.
• Local oxygen molecules within nanopores demonstrate a strong oxidation capability.
• Kinetic analysis revealed that negatively charged residues near reactive sites promote proton-involved, oxygen-induced disulfide cleavage under confinement.

Conclusions:

• Confined local oxygen exhibits significant oxidation potential, impacting thiol-disulfide redox dynamics.
• Nanoconfinement modulates the kinetics of redox reactions by increasing effective collision frequency.
• The findings suggest a critical role for confined oxygen in cellular redox signaling and enzymatic reactions.