Abstract: 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 (O1 and O2) states—were spectrally resolved and characterized using site-directed mutants and isotopically labeled retinal analogs. C–C stretching vibrations indicated that O1 and O2 contain 13-cis and all-trans retinal configurations, respectively. The C=O stretching band of Asn112 was most intense in the O1 state, consistent with close Na+ interaction at this residue. Retinal hydrogen out-of-plane vibrational modes further revealed enhanced torsion around the C11, C12, and C15 positions specifically in O1. Importantly, the decay of the O2 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 O2 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.
Tomida et al. (Sun,) studied this question.