Electrochemical Kinetic Fingerprinting of Single-Molecule Cooridations in the Confined Nanopores

Abstract

Metal centers are essential for enzyme catalysis, stabilizing the active site, facilitating electron transfer, and maintaining the structure through coordination with amino acids. In this study, K238H-AeL nanopores with histidine sites were designed for the first time as single-molecule reactors for the measurement of single-molecule coordination reactions. The coordination mechanism of Au(Ⅲ) with histidine and glutamate in nano-confined biological nanopores was explored. Specifically, Au(Ⅲ) interacts with the nitrogen (N) atom in the histidine imidazole ring of the K238C-AeL nanopore and the oxygen (O) atom in glutamate to form a stable K238H-Au-Cl2 complex. The formation mechanism of this complex was further validated through single-molecule nanopore analysis, mass spectrometry, and molecular dynamics simulations. By introducing histidine and glutamic acid into different positions within the nanopore revealed that the formation of the histidine-Au coordination bond in the confined space requires a distance within 2.5 Å between the ligand and the central metal atom. By analyzing the association and dissociation rates of single Au(Ⅲ) ions under the applied voltages, it was found that a confined nanopore increased the bonding rate of Au(Ⅲ)-Histidine coordination reactions by around 105 times compared to the bulk solution, and the optimal voltage for single-molecule coordination., providing valuable insights for designing reaction pathways in electrochemical catalysis. This research revealed a novel mechanism for metal coordination and amino acid residues in protein nanoconfined space, highlighting the dynamic interactions between metal ions and amino acid residues and the importance of the confined effect, providing insights for developing efficient, eco-friendly electrocatalytic nanomaterials.