Dual Photoisomerization on Distinct Potential Energy Surfaces in a UV-Absorbing Rhodopsin

UV-absorbing rhodopsins are essential for UV vision and sensing in all kingdoms of life. Unlike the well-known visible-absorbing rhodopsins, which bind a protonated retinal Schiff base for light absorption, UV-absorbing rhodopsins bind an unprotonated retinal Schiff base. Thus far, the photoreaction dynamics and mechanisms of UV-absorbing rhodopsins have remained essentially unknown. Here, we report the complete excited- and ground-state dynamics of the UV form of histidine kinase rhodopsin 1 (HKR1) from eukaryotic algae, using femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy, covering time scales from femtoseconds to milliseconds. We found that energy-level ordering is inverted with respect to visible-absorbing rhodopsins, with an optically forbidden low-lying S excited state that has Ag symmetry and a higher-lying UV-absorbing S state of Bu symmetry. UV-photoexcitation to the S state elicits a unique dual-isomerization reaction: first, C13═C14 - isomerization occurs during S-S evolution in <100 fs. This very fast reaction features the remarkable property that the newly formed isomer appears in the excited state rather than in the ground state. Second, C15═N16 - isomerization occurs on the S-S evolution to the ground state in 4.8 ps. We detected two ground-state unprotonated retinal photoproducts, 13-/15- (all-) and 13-/15-, after relaxation to the ground state. These isomers become protonated in 58 μs and 3.2 ms, respectively, resulting in formation of the blue-absorbing form of HKR1. Our results constitute a benchmark of UV-induced photochemistry of animal and microbial rhodopsins.