Electrochemical kinetic fingerprinting of single-molecule coordinations in confined nanopores

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 as single-molecule reactors for the measurement of single-molecule coordination reactions. The coordination mechanism of Au(III) with histidine and glutamate in biological nanopore confined space was explored. Specifically, Au(III) interacts with the nitrogen (N) atom in the histidine imidazole ring of the K238H-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. Introducing histidine and negative charge amino acids with carboxyl group into different positions within the nanopore revealed that the formation of the histidine-Au coordination bond in the confined space requires a suitable distance between the ligand and the central metal atom. By analyzing the association and dissociation rates of the single Au(III) ion under the applied voltages, it was found that a confined nanopore increased the bonding rate constant of Au(III)-histidine coordination reactions by around 10-100 times compared to that in the bulk solution and the optimal voltage for single-molecule. Therefore, nanopore techniques for tracking single-molecule reactions could offer valuable insights into designing metalloenzymes in metal-catalyzed organic reactions.