Interaction between Na⁺ ion and carboxylates of the PomA-PomB stator unit studied by ATR-FTIR spectroscopy

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. The PomA-PomB stator complex of Vibrio alginolyticus couples Na⁺ influx to torque generation in this supramolecular motor, but little is known about how Na⁺ associates with the PomA-PomB complex in the energy conversion process. Here, by means of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, we directly observed binding of Na⁺ to carboxylates in the PomA-PomB complex, including the functionally essential residue Asp24. The Na⁺ affinity of Asp24 is estimated to be ∼85 mM, close to the apparent K_m value from the swimming motility of the cells (78 mM). At least two other carboxylates are shown to be capable of interacting with Na⁺, but with somewhat lower affinities. We conclude that Asp24 and at least two other carboxylates constitute Na⁺ interaction sites in the PomA-PomB complex. This work reveals features of the Na⁺ pathway in the PomA-PomB Na⁺ channel by using vibrational spectroscopy.