Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

Department of Physics and Astronomy Faculty of Science Vrije Universiteit Amsterdam Amsterdam 1081 HV The Netherlands

Department of Physics University of Ioannina Ioannina Gr 45110 Greece

Faculty of Life Sciences Institute for Biology Experimental Biophysics Humboldt Universität zu Berlin Berlin D 10115 Germany

The Extreme Light Infrastructure ERIC Dolní Břežany 252 41 Czech Republic

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Ernst O. P., et al. , Microbial and animal rhodopsins: Structures, functions, and molecular mechanisms. Chem. Rev. 114, 126–163 (2014). PubMed PMC

Govorunova E. G., Sineshchekov O. A., Li H., Spudich J. L. “Microbial rhodopsins: Diversity, mechanisms, and optogenetic applications” in Annual Review of Biochemistry, R. D. Kornberg, Ed. (Annual Reviews, 2017), vol. 86, pp. 845–872. PubMed PMC

Zhang F., et al. , The microbial opsin family of optogenetic tools. Cell 147, 1446–1457 (2011). PubMed PMC

Hochbaum D. R., et al. , All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014). PubMed PMC

Luck M., et al. , A photochromic histidine kinase rhodopsin (HKR1) that is bimodally switched by ultraviolet and blue light. J. Biol. Chem. 287, 40083-90 (2012). PubMed PMC

Mukherjee S., Hegemann P., Broser M., Enzymerhodopsins: Novel photoregulated catalysts for optogenetics. Curr. Opin. Struct. Biol. 57, 118–126 (2019). PubMed

Rozenberg A., et al. , Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels. Nat. Struct. Mol. Biol. 29, 592–603 (2022). PubMed

Herdean A., et al. , A voltage-dependent chloride channel fine-tunes photosynthesis in plants. Nat. Commun. 7, 11654 (2016). PubMed PMC

Chien L. T., Zhang Z. R., Hartzell H. C., Single Cl- channels activated by Ca2+ in Drosophila S2 cells are mediated by bestrophins. J. Gen. Physiol. 128, 247–259 (2006). PubMed PMC

Mukherjee A., et al. , Thylakoid localized bestrophin-like proteins are essential for the CO2 concentrating mechanism of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U.S.A. 116, 16915–16920 (2019). PubMed PMC

Burlacot A., et al. , Alternative photosynthesis pathways drive the algal CO2-concentrating mechanism. Nature 605, 366–371 (2022). PubMed

Broser M., et al. , NeoR, a near-infrared absorbing rhodopsin. Nat. Commun. 11, 5682 (2020). PubMed PMC

Broser M., et al. , Experimental assessment of the electronic and geometrical structure of a near-infrared absorbing and highly fluorescent microbial rhodopsin. J. Phys. Chem. Lett. 14, 9291–9295 (2023). PubMed

Sugiura M., et al. , Unusual photoisomerization pathway in a near-infrared light absorbing enzymerhodopsin. J. Phys. Chem. Lett. 13, 9539–9543 (2022). PubMed

Oda K., et al. , Crystal structure of the red light-activated channelrhodopsin Chrimson. Nat. Commun. 9, 3949 (2018). PubMed PMC

Urmann D., et al. , Photochemical properties of the red-shifted channelrhodopsin chrimson. Photochem. Photobiol. 93, 782–795 (2017). PubMed

Weissleder R., Ntziachristos V., Shedding light onto live molecular targets. Nat. Med. 9, 123–128 (2003). PubMed

Vierock J., et al. , BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons. Nat. Commun. 12, 4527 (2021). PubMed PMC

Strother J. A., et al. , The emergence of directional selectivity in the visual motion pathway of Drosophila. Neuron 94, 168–182.e10 (2017). PubMed

Braiman M., Mathies R., Resonance raman evidence for an all-trans to 13-cis isomerization in the proton-pumping cycle of bacteriorhodopsin. Biochemistry 19, 5421–5428 (1980). PubMed

Braiman M., Mathies R., Resonance raman-spectra of bacteriorhodopsins primary photoproduct–Evidence for a distorted 13-cis retinal chromophore. Proc. Natl. Acad. Sci. U.S.A. 79, 403–407 (1982). PubMed PMC

Edman K., et al. , High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle. Nature 401, 822–826 (1999). PubMed

Nogly P., et al. , Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser. Science 361, eaat0094 (2018). PubMed

Kovacs G. N., et al. , Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin. Nat. Commun. 10, 3177 (2019). PubMed PMC

Kukura P., McCamant D. W., Mathies R. A., Femtosecond stimulated Raman spectroscopy. Ann. Rev. Phys. Chem. 58, 461–488 (2007). PubMed

Hontani Y., et al. , Dual photoisomerization on distinct potential energy surfaces in a UV-absorbing rhodopsin. J. Am. Chem. Soc. 142, 11464–11473 (2020). PubMed PMC

Andrikopoulos P. C., et al. , Femtosecond-to-nanosecond dynamics of flavin mononucleotide monitored by stimulated Raman spectroscopy and simulations. Phys. Chem. Chem. Phys. 22, 6538–6552 (2020). PubMed

Hontani Y., et al. , Molecular origin of photoprotection in Cyanobacteria probed by watermarked femtosecond stimulated raman spectroscopy. J. Phys. Chem. Lett. 9, 1788–1792 (2018). PubMed PMC

Kloz M., Weissenborn J., Polivka T., Frank H. A., Kennis J. T. M., Spectral watermarking in femtosecond stimulated Raman spectroscopy: Resolving the nature of the carotenoid S-star state. Phys. Chem. Chem. Phys. 18, 14619–14628 (2016). PubMed

Vivancos J. M. A., et al. , Unraveling the excited-state dynamics and light-harvesting functions of Xanthophylls in light-harvesting complex II using femtosecond stimulated raman spectroscopy. J. Am. Chem. Soc. 142, 17346–17355 (2020). PubMed PMC

Hontani Y., et al. , The femtosecond-to-second photochemistry of red-shifted fast-closing anion channelrhodopsin PsACR1. Phys. Chem. Chem. Phys. 19, 30402–30409 (2017). PubMed

Hontani Y., et al. , Reaction dynamics of the chimeric channelrhodopsin C1C2. Sci. Rep. 7, 7217 (2017). PubMed PMC

Hontani Y., et al. , The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy. Phys. Chem. Chem. Phys. 18, 24729–24736 (2016). PubMed

Hontani Y., et al. , Strong pH-dependent near-infrared fluorescence in a microbial rhodopsin reconstituted with a red-shifting retinal analogue. J. Phys. Chem. Lett. 9, 6469–6474 (2018). PubMed PMC

Hontani Y., et al. , Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome. Commun. Chem. 4, 3 (2021). PubMed PMC

Kajimoto K., et al. , Transient resonance raman spectroscopy of a light-driven sodium-ion-pump rhodopsin from indibacter alkaliphilus. J. Phys. Chem. B 121, 4431–4437 (2017). PubMed

Smith S. O., et al. , Chromophore structure in bacteriorhodopsins-o640 photointermediate. Biochemistry 22, 6141–6148 (1983).

Smith S. O., et al. , Vibrational analysis of the all-trans-retinal chromophore in light-adapted bacteriorhodopsin. J. Am. Chem. Soc. 109, 3108–3125 (1987).

Curry B., Broek A., Lugtenburg J., Mathies R., Vibrational analysis of all-trans-retinal. J. Am. Chem. Soc. 104, 5274–5286 (1982).

Eyring G., Curry B., Broek A., Lugtenburg J., Mathies R., Assignment and interpretation of hydrogen out-of-plane vibrations in the resonance raman-spectra of rhodopsin and bathorhodopsin. Biochemistry 21, 384–393 (1982). PubMed

Berera R., van Grondelle R., Kennis J. T. M., Ultrafast transient absorption spectroscopy: Principles and application to photosynthetic systems. Photosynthesis Res. 101, 105–118 (2009). PubMed PMC

van Stokkum I. H. M., Larsen D. S., van Grondelle R., Global and target analysis of time-resolved spectra. Biochim. Biophys. Acta 1657, 82–104 (2004). PubMed

Kennis J. T. M., et al. , Ultrafast protein dynamics of bacteriorhodopsin probed by photon echo and transient absorption spectroscopy. J. Phys. Chem. B 106, 6067–6080 (2002).

van Stokkum I. H. M., et al. , (Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system. Photochem. Photobiol. 82, 380–388 (2006). PubMed

Rouhani S., et al. , Crystal structure of the D85S mutant of bacteriorhodopsin: Model of an O-like photocycle intermediate. J. Mol. Biol. 313, 615–628 (2001). PubMed

Lenz M. O., Woerner A. C., Glaubitz C., Wachtveitl J., Photoisomerization in proteorhodopsin mutant D97N. Photochem. Photobiol. 83, 226–231 (2007). PubMed

Scholz F., Bamberg E., Bamann C., Wachtveitl J., Tuning the primary reaction of channelrhodopsin-2 by imidazole, pH, and site-specific mutations. Biophys. J. 102, 2649–2657 (2012). PubMed PMC

Van Stokkum I. H. M., et al. , Reaction dynamics in the chrimson channelrhodopsin: Observation of product-state evolution and slow diffusive protein motions. J. Phys. Chem. Lett. 14, 1485–1493 (2023). PubMed PMC

McCamant D. W., Kukura P., Mathies R. A., Femtosecond stimulated Raman study of excited-state evolution in bacteriorhodopsin. J. Phys. Chem. B 109, 10449–10457 (2005). PubMed PMC

Smith S. O., Pardoen J. A., Lugtenburg J., Mathies R. A., Vibrational analysis of the 13-cis-retinal chromophore in dark-adapted bacteriorhodopsin. J. Phys. Chem. 91, 804–819 (1987).

Yi A., et al. , Structural changes in an anion channelrhodopsin: Formation of the K and L Intermediates at 80 K. Biochemistry 56, 2197–2208 (2017). PubMed PMC

Ogren J. I., et al. , Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas Algae. Biochemistry 54, 377–388 (2015). PubMed PMC

Nakamizo Y., et al. , Low-temperature Raman spectroscopy of sodium-pump rhodopsin from Indibacter alkaliphilus: Insight of Na+ binding for active Na+ transport. Phys. Chem. Chem. Phys. 23, 2072–2079 (2021). PubMed

Fujisawa T., Kiyota H., Kikukawa T., Unno M., Low-temperature raman spectroscopy of halorhodopsin from natronomonas pharaonis: Structural discrimination of blue-shifted and red-shifted photoproducts. Biochemistry 58, 4159–4167 (2019). PubMed

Fujisawa T., et al. , Low-temperature Raman spectroscopy reveals small chromophore distortion in primary photointermediate of proteorhodopsin. Febs Lett. 592, 3054–3061 (2018). PubMed

Herbst J., Heyne K., Diller R., Femtosecond infrared spectroscopy of bacteriorhodopsin chromophore isomerization. Science 297, 822–825 (2002). PubMed

Shim S., Dasgupta J., Mathies R. A., Femtosecond time-resolved stimulated raman reveals the birth of bacteriorhodopsin’s J and K intermediates. J. Am. Chem. Soc. 131, 7592–7597 (2009). PubMed

Kuhne J., et al. , Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2. Proc. Natl. Acad. Sci. U.S.A. 116, 9380–9389 (2019). PubMed PMC

Wand A., Friedman N., Sheves M., Ruhman S., Ultrafast photochemistry of light-adapted and dark-adapted bacteriorhodopsin: Effects of the initial retinal configuration. J. Phys. Chem. B 116, 10444–10452 (2012). PubMed

Altoe P., Cembran A., Olivucci M., Garavelli M., Aborted double bicycle-pedal isomerization with hydrogen bond breaking is the primary event of bacteriorhodopsin proton pumping. Proc. Natl. Acad. Sci. U.S.A. 107, 20172–20177 (2010). PubMed PMC

Gozem S., Luk H. L., Schapiro I., Olivucci M., Theory and simulation of the ultrafast double-bond lsomerization of biological chromophores. Chem. Rev. 117, 13502–13565 (2017). PubMed

Sen S., Kar R. K., Borin V. A., Schapiro I., Insight into the isomerization mechanism of retinal proteins from hybrid quantum mechanics/molecular mechanics simulations. Wiley Interdisc. Rev. Comput. Mol. Sci. 12, e1562 (2022).

Logunov S. L., ElSayed M. A., Redetermination of the quantum yield of photoisomerization and energy content in the K-intermediate of bacteriorhodopsin photocycle and its mutants by the photoacoustic technique. J. Phys. Chem. B 101, 6629–6633 (1997).

Birge R. R., et al. , Revised assignment of energy-storage in the primary photochemical event in bacteriorhodopsin. J. Am. Chem. Soc. 113, 4327–4328 (1991).

Barneschi L., et al. , On the fluorescence enhancement of arch neuronal optogenetic reporters. Nat. Commun. 13, 6432 (2022). PubMed PMC

Freedman K. A., Becker R. S., Comparative investigation of the photoisomerization of the protonated and unprotonated normal-butylamine schiff-bases of 9-cis-retinals, 11-cis-retinals, 13-cis-retinals. and all-trans-retinals. J. Am. Chem. Soc. 108, 1245–1251 (1986).

Hayashi S., Tajkhorshid E., Schulten K., Molecular dynamics simulation of bacteriorhodopsin’s photoisomerization using ab initio forces for the excited chromophore. Biophys. J. 85, 1440–1449 (2003). PubMed PMC

Ren Z., Photoinduced isomerization sampling of retinal in bacteriorhodopsin. PNAS Nexus 1, pgac103 (2022). PubMed PMC

Pedraza-González L., Cignoni E., D’Ascenzi J., Cupellini L., Mennucci B., How the pH controls photoprotection in the light-harvesting complex of mosses. J. Am. Chem. Soc. 145, 7482–7494 (2023). PubMed PMC

Kennis J. T. M., Multiple Retinal Isomerizations during the Early Phase of the Bestrhodopsin Photoreaction. Research Data. 10.34894/TXUZJX. Deposited 26 February 2024. PubMed DOI PMC

Retinal to Retinal Energy Transfer in a Bistable Microbial Rhodopsin Dimer

Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction