Retinal to Retinal Energy Transfer in a Bistable Microbial Rhodopsin Dimer

Abstract

Neorhodopsin (NeoR) is a newly discovered fungal bistable rhodopsin that reversibly photoswitches between UV- and near-IR absorbing states denoted NeoR₃₆₇ and NeoR₆₉₀, respectively. NeoR₃₆₇ represents a deprotonated retinal Schiff base (RSB), while NeoR₆₉₀ represents a protonated RSB. Cryo-EM studies indicate that NeoR forms homodimers with 29 Å center-to-center distance between the retinal chromophores. UV excitation of NeoR₃₆₇ takes place to an optically allowed S3 state of 1B_u⁺ symmetry, which rapidly converts to a low-lying optically forbidden S1 state of 2A_g^- symmetry in 39 fs, followed by a multiexponential decay to the ground state on the 1-100 ps time scale. A theoretically predicted nπ* (S2) state does not get populated in any appreciable transient concentration during the excited-state relaxation cascade. We observe an intradimer retinal to retinal excitation energy transfer (EET) process from the NeoR₃₆₇ S1 state to NeoR₆₉₀, in competition with photoproduct formation. To quantitatively assess the EET mechanism and rate, we experimentally addressed and modeled the EET process under varying NeoR₃₆₇-NeoR₆₉₀ photoequilibrium conditions and determined the EET rate at (200 ps)^-1. The NeoR₃₆₇ S1 state shows a weak stimulated emission band in the near-IR around 700 nm, which may result from mixing with an intramolecular charge-transfer (ICT) state, enhancing the transition dipole moment of the S1-S0 transition and possibly facilitating the EET process. We suggest that EET may bear general relevance to the function of bistable multiwavelength rhodopsin oligomers.

Figure 1

Absorption spectra showing the progressive photoconversion of the red-absorbing state (NeoR₆₉₀) into the near UV-absorbing state at 367 nm (NeoR₃₆₇) by red illumination. Total illumination time for each spectrum as indicated, illumination was at 690 nm at 13 mW/cm². (Figure adapted from, available under a CC-BY-4.0 license. Copyright 2020 M. Broser et al.).

Scheme 1. Energy Level Ordering in Protonated RSB (Left Panel) and Unprotonated RSB (Right Panel)

Figure 2

Evolution-associated difference spectra (EADS) (A) and Decay-associated difference spectra (DADS) (C) estimated with a sequential or parallel analysis of NeoR₃₆₇ upon excitation at 360 nm. (B,D) Representative traces of data (gray) and fit (black dashed). Note that the time axis is linear until 10 ps, and logarithmic afterward. The estimated lifetimes are 39 fs (black), 0.94 (red), 10 (blue), 93 ps (green), 1.0 ns (magenta) and long-lived (cyan). For presentation purposes the black EADS has been divided by 2. (E) Time-gated spectrum at 100 ps of NeoR₆₉₀* with direct excitation at 650 nm; (F) FSRS EADS of the NeoR₃₆₇ S1 state.

Figure 3

Homodimeric arrangement of the NeoR sample as derived from CryoEM. Helices are numbered according to canonical rhodopsins (H1–H7), with the preceding extra helix (H0) found for enzymerhodopsins. Right: representative CryoEM micrograph and selected 2D-class averages.

Figure 4

(A) Transient absorption of NeoR in five conditions monitored at 711 nm upon excitation at 360 nm. Key: experiment 1 (magenta), 2 (green), 3 (cyan), 4 (orange), 5 (gray). Black, red, blue, dark green and purple lines indicate the target analysis fit. (B,C) Dimer fractions (with color key from A) and schematic depiction of the target analysis kinetic schemes for (i) UV–UV dimers; (ii) Red–Red dimers; (iii) UV-Red dimers. Color key: gray S3, blue hot S1, red S1, purple S1′, green P460 photoproduct; black NeoR₆₉₀* excited state. For case (i), transitions from hot S1, S1 and S1′ to S0 have not been depicted. In case (ii), excitation occurs to a higher excited state NeoR**, which rapidly relaxes to the lowest excited state NeoR*. NeoR** is not further considered in the target analysis. (D) Populations of the states with the color code indicated in (C) that follow from the target analysis under the 5 excitation conditions. Line type key: 1 (dotted), 2 (dot dot dashed), 3 (dot dashed), 4 (dashed), experiment 5 (solid). (E) SADS estimated from the target analysis, with color code identical to panel (D). Note that the SADS of S1 and S1′ are assumed identical and are indicated in red. For presentation purposes the black NeoR₆₉₀* SADS has been divided by 2, and the S3 SADS has been omitted in panel (E). Both axes in panel (D) are partially linear, and partially logarithmic.