Molecular Mechanisms of Weak Electric Field-Regulated Nitrate Transport Mediated by Nrt Protein.

The transmembrane transport of NO3- is a key step in microbial nitrogen uptake and transformation, critically influencing nitrogen cycling efficiency. However, the molecular mechanism by which external electric fields regulate nitrate transporter function remains unclear. Here, molecular dynamics simulations were used to construct Nrt transport systems under different transmembrane electric fields, and the results were experimentally validated using non-invasive micro-test technology (NMT). The results show that in the constructed systems, a -10 mV/Å electric field significantly enhances NO3- transmembrane transport, reducing its passage time through the NrtB channel by ∼2.8-fold relative to the no-field condition. In contrast, under a +10 mV/Å electric field, NO3- remains trapped near the binding site without effective transport. Mechanistic analysis reveals that the negative electric field strengthens NrtA-NO3- interactions, optimizes interfacial electrostatics, and improves the conformational adaptability of the NrtB channel, thereby lowering the transport energy barrier, whereas the positive electric field has the opposite effect. NMT measurements further confirm that NO3- influx was strongest under negative-potential treatment, followed by the open-circuit and positive-potential treatments, consistent with simulation results. This study elucidates the molecular mechanism of electric field-regulated NO3- transport, providing a theoretical basis for optimizing nitrogen transformation in bioelectrochemical systems under weak electric fields.