The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy

Abstract

Krokinobacter rhodopsin 2 (KR2) is a recently discovered light-driven Na⁺ pump that holds significant promise for application as a neural silencer in optogenetics. KR2 transports Na⁺ (in NaCl solution) or H⁺ (in larger cation solution, e.g. in CsCl) during its photocycle. Here, we investigate the photochemistry of KR2 with the recently developed watermarked, baseline-free femto- to submillisecond transient stimulated Raman spectroscopy (TSRS), which enables us to investigate retinal chromophore dynamics in real time with high spectral resolution over a large time range. We propose a new photocycle from femtoseconds to submilliseconds: J (formed in ∼200 fs) → K (∼3 ps) → K/L₁ (∼20 ps) → K/L₂ (∼30 ns) → L/M (∼20 μs). KR2 binds a Na⁺ ion that is not transported on the extracellular side, of which the function is unclear. We demonstrate with TSRS that for the D102N mutant in NaCl (with Na⁺ unbound, Na⁺ transport) and for WT KR2 in CsCl (with Na⁺ unbound, H⁺ transport), the extracellular Na⁺ binding significantly influences the intermediate K/L/M state equilibrium on the photocycle, while the identity of the transported ion, Na⁺ or H⁺, does not affect the photocycle. Our findings will contribute to further elucidation of the molecular mechanisms of KR2.