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 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 (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, suggesting that Na⁺ rebinding at Asp102 may facilitate 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.