Real-time electrical monitoring of enzymatic catalytic dynamics at the single-molecule level

Monitoring enzyme structural dynamics is essential for elucidating catalytic mechanisms, yet transient conformational fluctuations on microsecond-to-millisecond timescales remain challenging to resolve with conventional techniques. Here, we investigate the catalytic dynamics of cytochrome P450 1A1 (CYP1A1) during benzo[a]pyrene (BaP) metabolism by measuring single-molecule protein conductance. We show that catalysis-induced α-helix structural rearrangements, together with redox transitions of the heme center, modulate charge-transport efficiency. A negative correlation between BaP concentration and conductance enables construction of a kinetic model, yielding an apparent Michaelis constant of 24.2-43.2 μM. Real-time conductance measurements resolve four distinct conductance states associated with catalytic intermediates, which are further assigned using metabolic intermediates as substrates. These results provide insight into competing detoxification and activation pathways of BaP metabolism. This work establishes protein conductance as a generalizable platform for probing transient enzymatic dynamics and kinetics at the single-molecule level.