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 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 protonated retinal Schiff base. 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, suggesting that Na(+) rebinding at Asp102 may facilitate 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.