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 Km 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.
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