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

Disulfide bonds play an important role in thiol-based redox regulation. However, owing to the lack of analytical tools, little is known about how local O mediates the reversible thiol/disulfide cycle under protein confinement. In this study, a protein-nanopore inside a glove box is used to control local O for single-molecule reaction, as well as a single-molecule sensor for real-time monitoring of the reversible thiol/disulfide cycle. The results demonstrate that the local O molecules in protein nanopores could facilitate the redox cycle of disulfide formation and cleavage by promoting a higher fraction of effective reactant collisions owing to nanoconfinement. Further kinetic calculations indicate that the negatively charged residues near reactive sites facilitate proton-involved oxygen-induced disulfide cleavage under protein confinement. The unexpectedly strong oxidation ability of confined local O may play an essential role in cellular redox signaling and enzyme reactions.