Mechanistic insights into Na+ pumping by KR2: Distinct roles of Asp102 and Asn112 coupled with retinal distortion in two O intermediates

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven Na + pump. In the initial state, a sodium ion does not bind near the protonated retinal Schiff base (PRSB) due to electrostatic repulsion, and is instead taken up during formation of the O intermediate in the photocycle. Previous cryogenic and time-resolved crystallographic studies showed that Na + can be coordinated by either Asn112 or Asp252 near the PRSB. In addition, KR2 forms a pentamer in which each protomer binds a Na + at Asp102 located at the interfacial region, although the functional relevance of this site has remained unclear. Here, we used time-resolved Fourier-transform infrared spectroscopy to clarify the Na + translocation mechanism. Four states—K, L/M, and two O (O 1 and O 2 ) states—were spectrally resolved and characterized using site-directed mutants and isotopically labeled retinal analogs. C–C stretching vibrations indicated that O 1 and O 2 contain 13- cis and all- trans retinal configurations, respectively. The C=O stretching band of Asn112 was most intense in the O 1 state, consistent with close Na + interaction at this residue. Retinal hydrogen out-of-plane vibrational modes further revealed enhanced torsion around the C 11 , C 12 , and C 15 positions specifically in O 1 . Importantly, the decay of the O 2 intermediate was markedly slowed at high Na + concentration in the D102N mutant, demonstrating that Na + rebinding at Asp102 facilitates Na + release along the exit pathway during the O 2 intermediate. These findings provide a unified mechanistic model in which coordinated retinal distortion and site-specific ion interactions drive directional Na + pumping through the two O intermediates of KR2.