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

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/L 1 (20 ps) K/L 2 (30 ns) L/M (20 s). 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. The femtosecond-to-submillisecond photocycle of sodium ion pump rhodopsin KR2 was revealed through watermarked stimulated Raman spectroscopy.