Absorption and Emission Spectroscopic Investigation of Thermal Dynamics and Photo-Dynamics of the Rhodopsin Domain of the Rhodopsin-Guanylyl Cyclase from the Nematophagous Fungus Catenaria anguillulae

The rhodopsin-guanylyl cyclase from the nematophagous fungus Catenaria anguillulae belongs to a recently discovered class of enzymerhodopsins and may find application as a tool in optogenetics. Here the rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. The absorption cross-section spectrum and excitation wavelength dependent fluorescence quantum distributions of CaRh samples were determined (first absorption band in the green spectral region). The thermal stability of CaRh was studied by long-time attenuation measurements at room temperature (20.5 °C) and refrigerator temperature of 3.5 °C. The apparent melting temperature of CaRh was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 62 ± 2 °C). The photocycle dynamics of CaRh was investigated by sample excitation to the first inhomogeneous absorption band of the CaRhda dark-adapted state around 590 nm (long-wavelength tail), 530 nm (central region) and 470 nm (short-wavelength tail) and following the absorption spectra development during exposure and after exposure (time resolution 0.0125 s). The original protonated retinal Schiff base PRSBall-trans in CaRhda photo-converted reversibly to protonated retinal Schiff base PRSBall-trans,la1 with restructured surroundings (CaRhla1 light-adapted state, slightly blue-shifted and broadened first absorption band, recovery to CaRhda with time constant of 0.8 s) and deprotonated retinal Schiff base RSB13-cis (CaRhla2 light-adapted state, first absorption band in violet to near ultraviolet spectral region, recovery to CaRhda with time constant of 0.35 s). Long-time light exposure of light-adapted CaRhla1 around 590, 530 and 470 nm caused low-efficient irreversible degradation to photoproducts CaRhprod. Schemes of the primary photocycle dynamics of CaRhda and the secondary photocycle dynamics of CaRhla1 are developed.


Introduction
The nematophagous fungus Catenaria anguillulae is a facultative endoparasite of free living and plant parasitic nematodes ( [1,2] and references therein). Searches of the genome assembly of Catenaria anguillulae found the presence of a rhodopsin-guanylyl cyclase gene fusion [3]. This guanylyl

Fluorescence Behavior of CaRh
Fluorescence emission quantum distributions EF(λ) of the fresh centrifuged CaRh sample used in Figure 1 at excitation wavelengths in the region from λF,exc = 540 to 260 nm are displayed in Figure 2 with sub-sets (a)-(f). The corresponding fluorescence quantum yield curve in the range from λF,exc = 560 to 260 nm is shown in Figure 3.

Fluorescence Behavior of CaRh
Fluorescence emission quantum distributions E F (λ) of the fresh centrifuged CaRh sample used in Figure 1 at excitation wavelengths in the region from λ F,exc = 540 to 260 nm are displayed in Figure 2 with sub-sets (a)-(f). The corresponding fluorescence quantum yield curve in the range from λ F,exc = 560 to 260 nm is shown in Figure 3.   Over the broad inhomogeneous S 0 -S 1 absorption band the fluorescence quantum distributions change their spectral shapes and magnitudes.
The fluorescence lifetimes (lifetimes of emitting states) of the considered transitions i = I-V are determined by the fluorescence quantum yields φ F,i and the radiative lifetimes τ rad,i according to The radiative lifetimes of the transitions are given by the Strickler-Berg relation [10][11][12] τ rad,i = n a,i λ where n a.i and n F,i are the mean refractive indices in the absorption band region and the fluorescence region of the considered transitions, respectively (determined by the water solvent), λ F,i = E F,i (λ)λ 3 dλ/ E F,i (λ)dλ 1/3 are the mean fluorescence wavelengths, and σ a,i = [σ a,i (λ)/λ]dλ are the absorption band cross-section strengths of the considered transitions. In Table 1 approximate and estimated values of absorption wavelength positions λ a,i , peak fluorescence emission wavelengths λ F,max,i , fluorescence spectral half-widths ∆ ν F,i (FWHM), mean fluorescence wavelengths λ F,i , mean refractive indices n a,i and n F,i , fluorescence quantum yields φ F,i , absorption band cross-section strengths of considered bands σ a,i , radiative lifetimes τ rad,i , and Strickler-Berg based fluorescence lifetimes τ F,i of the transitions i = I-V are collected. The S 0 -S 1 absorption band cross-section strength σ a of the retinal species PRSB is σ a = σ a,I = σ a,I I = (3.3 ± 0.3) × 10 −17 cm 2 . It was determined from Figure S2 using σ a = λ≥440 nm [σ a (λ)/λ]dλ. The S 0 -S 2 and S 0 -S 3 absorption band cross-section strengths σ a,I I I and σ a,IV are thought to be roughly a factor of ten smaller than the S 0 -S 1 absorption band cross-section strength considering the smaller absorption cross-section peak heights and the smaller inhomogeneous broadening. The value of σ a,V was determined using the absorption cross-section spectrum of Trp, i.e., σ V = σ Trp = λ≥240 nm σ Trp (λ)/λ dλ (from [13], incoherent independent emission of each Trp residue in the protein). The fluorescence lifetime τ F,I of the long-wavelength part of the inhomogeneous S 0 -S 1 transition is found to be τ F,I ≈ 88 fs. The fluorescence lifetime τ F,II of the short-wavelength part of the inhomogeneous S 0 -S 1 transition is determined to be τ F,II ≈ 305 fs. These short fluorescence lifetimes agree with barrier-less first excited state twist to funnel positions (conical intersection [14]) where fast internal conversion from the excited state S 1 potential energy surface to the S 0 ground-state potential energy surface occurs (twisted internal conversion [15]) with partial transfer to products (photo-isomers) and partial recovery to the initial educt conformations [15]. The inhomogeneous S 0 -S 1 absorption band and different short-wavelength and long-wavelength excitation S 1 -S 0 fluorescence emission bands indicate different retinal relaxation dynamics caused by the surrounding inhomogeneous opsin protein arrangement.
The higher excited-state S 2 -S 0 (III) (τ F,III ≈ 7.4 ps) and S 3 -S 0 (IV) (τ F,IV ≈ 13.7 ps) picosecond fluorescence lifetimes indicate activation-barrier-slowed-down S 2 and S 3 potential energy surface relaxation twists to conical intersections with internal conversion to the S 0 potential energy surface.
The apoprotein (V) fluorescence behavior is determined by Trp emission (fluorescence quantum yield φ F,V ≈ 0.045, fluorescence lifetime τ F,V ≈ 448 ps). The fluorescence efficiency is reduced and the fluorescence lifetime is shortened by Förster-type energy transfer from apoprotein to retinal [16,17]. The fluorescence quantum yield of Trp outside the protein in aqueous solution is φ F = 0.13 [18][19][20].

Thermal CaRh Behavior
The CaRh apparent melting temperature and the temporal attenuation behavior at the fixed temperatures of 3.5 and 20.5 • C were studied.

Apparent CaRh Melting Temperature
The thermal protein stability of CaRh was studied by stepwise sample heating up to 69.2 • C, then cooling down and thereby measuring the attenuation coefficient spectra development [6,21]. The apparent protein melting temperature was derived from the onset of strong light attenuation in the transparency region of CaRh with inverse wavelength power dependence of α s (λ) ∝ λ −γ (γ ≤ 4) [7].
The situation is displayed in Figure 4a. The applied heating-cooling cycle is shown by the right inset in Figure 4a. The main part of Figure 4a shows attenuation coefficient spectra of CaRh at fixed temperatures during sample heating up and cooling down. In the transparency region (λ > 640 nm) the attenuation coefficient (light scattering) increased during heating up. Strong onset of light scattering was observed for ϑ = 64.6 • C. Between ϑ = 64.6 • C and ϑ = 69.2 • C the light attenuation at λ = 700 nm increased more than a factor of 20 within a time interval of 10 min. The increase in light scattering is due to protein unfolding and concurring aggregation [21]. The attenuation continued to increase during sample cooling, showing that the protein heat denaturation was irreversible. Only due to sample centrifugation at ϑ = 4 • C (4400 rpm for 30 min), the light attenuation was strongly reduced due to sedimentation of aggregated CaRh.
The attenuation coefficient development at λ = 750 nm versus temperature during heating and cooling is shown in the left inset of Figure 4a. The temperature position of steep onset of light attenuation gives the apparent protein melting temperature ϑ m . A value of ϑ m = 62 ± 2 • C is determined. At ϑ m the protein begins to unfold (denature) quickly with time which causes a quick light-scattering increase with time (the expression "protein melting" is synonymously used to the expression "protein denaturation" [22]).
Concurrent with the sample heating CaRh retinal conversion occurred from protonated retinal Schiff base (PRSB) with peak absorption at λ max = 541 nm to unprotonated retinal Schiff base (RSB) with peak absorption at λ max = 384 nm. This change is seen by looking to the attenuation coefficient spectra in the main part of Figure 4a. It is more clearly worked out in the main part of Figure 4b where the absorption coefficient spectra development deprived from the scattering contributions is displayed. The inset in Figure 4b shows the development of α a (541 nm) and α a (380 nm) versus temperature. Above ϑ = 50 • C strong conversion of PRSB to RSB is observed.
The absorption cross-section spectrum of CaRh with the retinal cofactor converted from PRSB to RSB is shown by the dotted curve in the top part of Figure S2 (PRSB contribution at ϑ = 64.6 • C is subtracted and the resulting spectrum is normalized to 100% RSB content). The long-wavelength absorption cross-section tail for λ > 450 nm is thought to be caused by the presence of some released free protonated retinal Schiff base [23].
The absorption cross-section spectrum of CaRh with the retinal cofactor converted from PRSB to RSB is shown by the dotted curve in the top part of Figure S2 (PRSB contribution at ϑ = 64.6 °C is subtracted and the resulting spectrum is normalized to 100% RSB content). The long-wavelength absorption cross-section tail for λ > 450 nm is thought to be caused by the presence of some released free protonated retinal Schiff base [23].
The fluorescence quantum distribution of the deprotonated retinal Schiff base RSB of the heat-denatured and centrifuged CaRh sample was determined for fluorescence excitation at λF,exc = 360 nm. The obtained spectrum is shown in Figure S3. The fluorescence emission peaks at ≈ 550 nm. The fluorescence quantum yield is φF = (1.5 ± 0.2) × 10 −3 . A radiative lifetime of τrad = 7.17 ns, and a fluorescence lifetime of τF = 10.8 ± 1.5 ps are calculated using Equations (1)    The attenuation coefficient development α(λ) at ϑ = 3.5 ± 0.5 °C with time is displayed in Figure 5. The light scattering contribution αs(λ) to the attenuation coefficient spectra is small as is seen in the long-wavelength transparency region of λ > 640 nm. The attenuation coefficient spectra in the absorption region decrease in height with storage time at 3.5 °C. This attenuation reduction is attributed to CaRh aggregate cluster compactization with storage time (loosely packed globules with small volume fill factor densify to tightly packed globules) [24]. The apparent absorption cross-section per molecule decreases because of specific surface reduction of the aggregates (for  The attenuation coefficient development α(λ) at ϑ = 3.5 ± 0.5 • C with time is displayed in Figure 5. The light scattering contribution α s (λ) to the attenuation coefficient spectra is small as is seen in the long-wavelength transparency region of λ > 640 nm. The attenuation coefficient spectra in the absorption region decrease in height with storage time at 3.5 • C. This attenuation reduction is attributed to CaRh aggregate cluster compactization with storage time (loosely packed globules with small volume fill factor densify to tightly packed globules) [24]. The apparent absorption cross-section per molecule decreases because of specific surface reduction of the aggregates (for detailed discussion of aggregation dependent absorption reduction see reference [24]).

Temporal Absorption Development of CaRh at 20.5 °C
The attenuation coefficient development α(λ) at ϑ = 20.5 ± 1 °C with time is displayed in Figure 6. In the main part attenuation coefficient spectra at various storage times are shown. At start the light scattering contribution is negligible. Up to t = 144 h the light scattering increased as is seen in the transparency region of λ > 660 nm. This scattering increase indicates some slow protein denaturation at room temperature. For t > 144 h the light attenuation in the transparency region decreased with time likely due to sedimentation of aggregated (denaturated) protein. The attenuation of the main absorption band around 540 nm decreased with time due to protein aggregation (mainly absorption reduction due to protein aggregate cluster compactization [24]) and due to conversion of PRSB to RSB. The attenuation in the region between 300 and 460 nm increases with time during the first 144 h because of increasing light scattering and conversion of PRSB to RSB. For t >144 h some attenuation reduction is seen likely due to protein aggregate sedimentation. In the dominant apoprotein absorption region around 280 nm the aggregation dependent absorption coefficient reduction is mainly compensated by aggregation dependent light scattering increase.

Temporal Absorption Development of CaRh at 20.5 • C
The attenuation coefficient development α(λ) at ϑ = 20.5 ± 1 • C with time is displayed in Figure 6. In the main part attenuation coefficient spectra at various storage times are shown. At start the light scattering contribution is negligible. Up to t = 144 h the light scattering increased as is seen in the transparency region of λ > 660 nm. This scattering increase indicates some slow protein denaturation at room temperature. For t > 144 h the light attenuation in the transparency region decreased with time likely due to sedimentation of aggregated (denaturated) protein. The attenuation of the main absorption band around 540 nm decreased with time due to protein aggregation (mainly absorption reduction due to protein aggregate cluster compactization [24]) and due to conversion of PRSB to RSB.
The attenuation in the region between 300 and 460 nm increases with time during the first 144 h because of increasing light scattering and conversion of PRSB to RSB. For t >144 h some attenuation reduction is seen likely due to protein aggregate sedimentation. In the dominant apoprotein absorption region around 280 nm the aggregation dependent absorption coefficient reduction is mainly compensated by aggregation dependent light scattering increase. In the inset of Figure 6, the temporal attenuation coefficient development at λ = 541 nm (S0-S1 absorption peak of PRSB), λ = 380 nm (higher excited-state absorption of PRSB and S0-S1 absorption of formed RSB), and λ = 280 nm (dominant apoprotein Trp and Tyr absorption) is depicted. The ratio α(λ,t)/α(λ,t = 0) is plotted. As described above, α(541 nm) decreased with time due to absorption reduction by aggregate compactization, PRSB conversion to RSB, and final aggregate sedimentation. α(380 nm) increased with time during the first 144 h due to dominant light scattering increase over aggregation dependent absorption coefficient reduction and because of PRSB conversion to RSB. For t > 144 h the decrease of α(380 nm) is thought to be due to protein aggregate sedimentation. α(280 nm) increased slightly within the first 120 h due to slightly dominating attenuation increase by scattering over absorption reduction by aggregate compactization. For t > 144 h the decrease of α(280 nm) is thought to be due to protein aggregate sedimentation. The attenuation coefficient α(700 nm) in the transparency region of CaRh is caused by light scattering. The increase of α(700 nm) during the first 144 h is due to growth of aggregate size. The slight decrease for t > 144 h is due to beginning protein aggregate sedimentation.

Photo-Excitation Dynamics of CaRh
The photocycle dynamics and the photo-degradation dynamics of CaRh samples were studied. In the photocycle experiments the samples were excited for a short time interval and attenuation coefficient spectra were measured before, during, and after light exposure. Temporal absorption changes during light exposure and after light exposure were recorded at fixed probe wavelengths with a time resolution of tres = 0.0125 s (time step interval). For photo-degradation studies the samples were excited in several repetitions over long time periods with short recovery periods in In the inset of Figure 6, the temporal attenuation coefficient development at λ = 541 nm (S 0 -S 1 absorption peak of PRSB), λ = 380 nm (higher excited-state absorption of PRSB and S 0 -S 1 absorption of formed RSB), and λ = 280 nm (dominant apoprotein Trp and Tyr absorption) is depicted. The ratio α(λ,t)/α(λ,t = 0) is plotted. As described above, α(541 nm) decreased with time due to absorption reduction by aggregate compactization, PRSB conversion to RSB, and final aggregate sedimentation. α(380 nm) increased with time during the first 144 h due to dominant light scattering increase over aggregation dependent absorption coefficient reduction and because of PRSB conversion to RSB. For t > 144 h the decrease of α(380 nm) is thought to be due to protein aggregate sedimentation. α(280 nm) increased slightly within the first 120 h due to slightly dominating attenuation increase by scattering over absorption reduction by aggregate compactization. For t > 144 h the decrease of α(280 nm) is thought to be due to protein aggregate sedimentation. The attenuation coefficient α(700 nm) in the transparency region of CaRh is caused by light scattering. The increase of α(700 nm) during the first 144 h is due to growth of aggregate size. The slight decrease for t > 144 h is due to beginning protein aggregate sedimentation.

Photo-Excitation Dynamics of CaRh
The photocycle dynamics and the photo-degradation dynamics of CaRh samples were studied. In the photocycle experiments the samples were excited for a short time interval and attenuation coefficient spectra were measured before, during, and after light exposure. Temporal absorption changes during light exposure and after light exposure were recorded at fixed probe wavelengths with a time resolution of t res = 0.0125 s (time step interval). For photo-degradation studies the samples were excited in several repetitions over long time periods with short recovery periods in between. In the photocycle and the photo-degradation experiments, the samples were excited at three different spectral positions around 590 nm (with Thorlabs LED 590 nm), 530 nm (with Thorlabs LED 530 nm), and 470 nm (with Thorlabs LED 470 nm). The spectral distributions of the light emitting diodes are indicated in Figure 7.  For the absorption coefficient spectra presented in Figure 7a the CaRh sample was excited with Thorlabs LED 530 nm. The spectral light distribution of the LED 530 nm is included in Figure 7a. The input excitation intensity was Iexc = 226 mW cm −2 and the duration of light exposure was texc = 12 s. Within texc = 3 s a new absorption band (this band is named Rh-365) in the violet and near ultraviolet spectral region was formed and the original first absorption band (named Rh-541) in the green spectral region was lowered, slightly blue shifted and spectrally broadened (this shifted band is named Rh-527). After texc = 12 s the excitation was switched off, and the sample recovery was observed over a time range of 102 s. After excitation light switch-off the absorption coefficient spectrum recovered dominantly back to the situation before light exposure. A complete recovery did not occur because of some permanent photo-product formation.
The photocycle behavior of CaRh in the case of sample excitation with Thorlabs LED 590 nm and Thorlabs LED 470 nm are displayed in Figure 7b,c, respectively. The qualitative behavior was similar to the excitation with Thorlabs LED 530 nm (Figure 7a).

Photocycle Dynamics of CaRh
The photocycle results for excitation with LED 530 nm, LED 590 nm and LED 470 nm are presented in Figures 7-9 and Figure S4.
For the absorption coefficient spectra presented in Figure 7a the CaRh sample was excited with Thorlabs LED 530 nm. The spectral light distribution of the LED 530 nm is included in Figure 7a. The input excitation intensity was I exc = 226 mW cm −2 and the duration of light exposure was t exc = 12 s. Within t exc = 3 s a new absorption band (this band is named Rh-365) in the violet and near ultraviolet spectral region was formed and the original first absorption band (named Rh-541) in the green spectral region was lowered, slightly blue shifted and spectrally broadened (this shifted band is named Rh-527). After t exc = 12 s the excitation was switched off, and the sample recovery was observed over a time range of 102 s. After excitation light switch-off the absorption coefficient spectrum recovered dominantly back to the situation before light exposure. A complete recovery did not occur because of some permanent photo-product formation.
The photocycle behavior of CaRh in the case of sample excitation with Thorlabs LED 590 nm and Thorlabs LED 470 nm are displayed in Figure 7b,c, respectively. The qualitative behavior was similar to the excitation with Thorlabs LED 530 nm (Figure 7a).
The temporal attenuation coefficient development of the investigated CaRh samples at λ pr = 550 nm before, during, and after photo-excitation with LED 530 nm, LED 590 nm and LED 470 nm for various excitation intensities I exc is shown in Figure 8a. The situation for λ pr = 370 nm is shown in Figure 8b. Light excitation occurred in the time range of 0 ≤ t ≤ 3 s for LED 530 nm and in the time range of 0 ≤ t ≤ 5 s for LED 590 nm and LED 470 nm. The steepness of attenuation coefficient changes at the start of light excitation increased with excitation intensity. Attenuation coefficient plateaus are formed. The attenuation coefficient changes approach limits with increasing excitation intensity (complete conversion of the dark-adapted CaRh to the light-adapted CaRh, see also Supplementary Material S5 with Figure S4a,b). After excitation light switch-off the attenuation coefficients recovered nearly fully back to the situation before light exposure.
In order to gain information on the photocycle dynamics of CaRh (see Discussion below) the temporal attenuation coefficient development of CaRh was measured in the probe wavelength region from λ pr = 300 to 630 nm in steps of 10 nm. The sample was exposed using LED 590 nm with I exc = 66.7 mW cm −2 over a time range of t exc between 30 and 40 s. The attenuation coefficient development before, during and after exposure was followed over a time range of 150 s The temporal attenuation coefficient development of the investigated CaRh samples at λpr = 550 nm before, during, and after photo-excitation with LED 530 nm, LED 590 nm and LED 470 nm for various excitation intensities Iexc is shown in Figure 8a. The situation for λpr = 370 nm is shown in Figure 8b. Light excitation occurred in the time range of 0 ≤ t ≤ 3 s for LED 530 nm and in the time range of 0 ≤ t ≤ 5 s for LED 590 nm and LED 470 nm. The steepness of attenuation coefficient changes at the start of light excitation increased with excitation intensity. Attenuation coefficient plateaus are formed. The attenuation coefficient changes approach limits with increasing excitation intensity (complete conversion of the dark-adapted CaRh to the light-adapted CaRh, see also Supplementary Material S5 with Figure S4a,b). After excitation light switch-off the attenuation coefficients recovered nearly fully back to the situation before light exposure.
In order to gain information on the photocycle dynamics of CaRh (see Discussion below) the temporal attenuation coefficient development of CaRh was measured in the probe wavelength region from λpr = 300 to 630 nm in steps of 10 nm. The sample was exposed using LED 590 nm with Iexc = 66.7 mW cm −2 over a time range of texc between 30 and 40 s. The attenuation coefficient development before, during and after exposure was followed over a time range of 150 s.

Photo-Degradation Dynamics of CaRh
Continued CaRh sample excitation with LED 590 nm, LED 530 nm, or LED 470 nm after light-adapted state formation caused photo-induced CaRh photo-degradation (CaRh Prod photoproduct formation).
This situation is shown in Figure 10 for long-time exposure of samples with LED 530 nm (top part, I exc = 226 mW cm −2 ), LED 590 nm (middle part, I exc = 69.2 mW cm −2 ), and LED 470 nm (bottom part, I exc = 187.1 mW cm −2 ). The inhomogeneous S 0 -S 1 absorption band of CaRh in the light-adapted state (Rh-527) decreased with exposure time and the absorption in the violet and near ultraviolet spectral range increased and changed its shape due to photoproduct (CaRh Prod ) formation.
The permanent spectral changes due to long-time exposure of CaRh samples are seen in Figure 11a where attenuation coefficient spectra of dark-adapted CaRh samples are shown before light exposure (t exc = 0) and in cases of recovery in the dark after continued exposure in repeated intervals of 1000 s. For λ > 430 nm the decrease of absorption of the S 0 -S 1 transition of Rh-541 due to photo-degradation is seen, and for λ < 430 nm absorption changes due to photo-product formation (CaRh Prod ) are seen. This situation is shown in Figure 10 for long-time exposure of samples with LED 530 nm (top part, Iexc = 226 mW cm −2 ), LED 590 nm (middle part, Iexc = 69.2 mW cm −2 ), and LED 470 nm (bottom part, Iexc = 187.1 mW cm −2 ). The inhomogeneous S0-S1 absorption band of CaRh in the light-adapted state (Rh-527) decreased with exposure time and the absorption in the violet and near ultraviolet spectral range increased and changed its shape due to photoproduct (CaRhProd) formation.
The permanent spectral changes due to long-time exposure of CaRh samples are seen in Figure  11a where attenuation coefficient spectra of dark-adapted CaRh samples are shown before light exposure (texc = 0) and in cases of recovery in the dark after continued exposure in repeated intervals of 1000 s. For λ > 430 nm the decrease of absorption of the S0-S1 transition of Rh-541 due to photo-degradation is seen, and for λ < 430 nm absorption changes due to photo-product formation (CaRhProd) are seen. The attenuation coefficient spectra development of the photoproducts is seen in Figure 11b where the attenuation coefficient contribution of dark-adapted CaRh in Figure 11a is subtracted. In the three cases of excitation with LED 530 nm, LED 590 nm and LED 470 nm the formation of (at least) four photoproducts, Ret520, Ret405, Ret380 and Ret335, is revealed. Their absorption peaks are at ≈ 520 nm (Ret520), ≈ 405 nm (Ret405), ≈ 380 nm (Ret380), and ≈ 335 nm (Ret335). Ret520 is thought to be a The attenuation coefficient spectra development of the photoproducts is seen in Figure 11b where the attenuation coefficient contribution of dark-adapted CaRh in Figure 11a is subtracted. In the three cases of excitation with LED 530 nm, LED 590 nm and LED 470 nm the formation of (at least) four photoproducts, Ret 520 , Ret 405 , Ret 380 and Ret 335 , is revealed. Their absorption peaks are at ≈ 520 nm (Ret 520 ), ≈ 405 nm (Ret 405 ), ≈ 380 nm (Ret 380 ), and ≈ 335 nm (Ret 335 ). Ret 520 is thought to be a protonated retinal Schiff base form. It may have lost its proper covalent binding to the opsin protein.
Ret 520 absorbs in the spectral region of the LED 590 nm, LED 530 nm, and LED 470 nm excitation light sources. Therefore it is weakly photo-degraded (its amount slightly decreases) with long-time exposure. Ret 405 and Ret 380 are thought to be deprotonated retinal Schiff base conformations. Their proper covalent binding to the opsin protein may have been lost. Ret 335 may be a deprotonated retinol bound to the opsin protein or released from it. The absorption spectrum of Ret 335 agrees with the absorption spectral shape of retinol [25]. The long-wavelength absorption tails of Ret 405 and Ret 380 overlap with the excitation spectrum of LED 470 nm and therefore Ret 405 and Ret 380 seem to be partly photo-degraded to Ret 335 in the case of long-time LED 470 nm exposure.
The quantum yield of photo-degradation φ d of CaRh in its light-adapted state (Rh-527) is determined by the absorption decrease of CaRh in its dark-adapted state (Rh-541) of Figure 11a  Ret520 absorbs in the spectral region of the LED 590 nm, LED 530 nm, and LED 470 nm excitation light sources. Therefore it is weakly photo-degraded (its amount slightly decreases) with long-time exposure. Ret405 and Ret380 are thought to be deprotonated retinal Schiff base conformations. Their proper covalent binding to the opsin protein may have been lost. Ret335 may be a deprotonated retinol bound to the opsin protein or released from it. The absorption spectrum of Ret335 agrees with the absorption spectral shape of retinol [25]. The long-wavelength absorption tails of Ret405 and Ret380 overlap with the excitation spectrum of LED 470 nm and therefore Ret405 and Ret380 seem to be partly photo-degraded to Ret335 in the case of long-time LED 470 nm exposure. The quantum yield of photo-degradation φd of CaRh in its light-adapted state (Rh-527) is determined by the absorption decrease of CaRh in its dark-adapted state (Rh-541) of Figure 11a at λ = 541 nm due to excitation photon absorptions around 596 nm (LED 590 nm), 520 nm (LED 530 nm) and 462 nm (LED 470 nm) of light-adapted CaRh in Figure 10. The calculation of φd from the experimental curves in Figures 11a and 10 Figure 12. φd decreased with exposure time (accumulated input excitation energy density). Some saturation is obsvered for very long-time sample exposure (wexc > 100 J cm −2 ). The efficiency of photo-degradation of light-adapted CaRh also depended on the excitation wavelength. It was highest for excitation with LED 590 nm (φd(wexc = 0.62 J cm −2 ) = 6.3 × 10 −4 , φd(wexc = 416 J cm −2 ) = 2.3 × 10 −5 ), in between for excitation with LED 470 nm (φd(wexc = 2.3 J cm −2 ) = 1.3 × 10 −4 , φd(wexc = 564 J cm −2 ) = 1.7 × 10 −5 ), and lowest for for excitation with LED 530 nm (φd(wexc = 2.7 J cm −2 ) = 9.6 × 10 −5 , φd(wexc = 910 J cm −2 ) = 5.3 × 10 −6 ). This excitation wavelength and excitation energy density dependence of CaRh photo-degradtion indicates an inhomogeneous nature of the CaRh protein concerning the excitation wavelength dependence (higher stability around wavelength position of maximum absorption) and the exposed excitation energy density (less stable protein fraction photo-degrades first). The obtained quantum yields of photo-degradation φ d of CaRh versus accumulated input excitation energy density w exc = I exc dt for sample excitation with LED 530 nm, LED 590 nm and LED 470 nm are displayed in Figure 12. φ d decreased with exposure time (accumulated input excitation energy density). Some saturation is obsvered for very long-time sample exposure (w exc > 100 J cm −2 ). The efficiency of photo-degradation of light-adapted CaRh also depended on the excitation wavelength. It was highest for excitation with LED 590 nm (φ d (w exc = 0.62 J cm −2 ) = 6.3 × 10 −4 , φ d (w exc = 416 J cm −2 ) = 2.3 × 10 −5 ), in between for excitation with LED 470 nm (φ d (w exc = 2.3 J cm −2 ) = 1.3 × 10 −4 , φ d (w exc = 564 J cm −2 ) = 1.7 × 10 −5 ), and lowest for for excitation with LED 530 nm (φ d (w exc = 2.7 J cm −2 ) = 9.6 × 10 −5 , φ d (w exc = 910 J cm −2 ) = 5.3 × 10 −6 ). This excitation wavelength and excitation energy density dependence of CaRh photo-degradtion indicates an inhomogeneous nature of the CaRh protein concerning the excitation wavelength dependence (higher stability around wavelength position of maximum absorption) and the exposed excitation energy density (less stable protein fraction photo-degrades first).

Discussion
The rhodopsin-guanylyl cyclase CaRhGC from the nematophagus fungus Catenaria anguillulae belongs to the class of enzymerhodopsins of microbial proteins which consist of a rhodopsin domain and an enzyme domain [8,9,[26][27][28]. Light excitation of the rhodopsin domain results in the activation of the guanylyl cyclase domain and causes the conversion of GTP (guanosine triphosphate) to cGMP (cyclic guanosine monophosphate).
The rhodopsin-guanylyl cyclase CaRhGC from Catenaria anguillulae was expressed recently and its light-activated guanylyl cyclase activity was demonstrated [4]. Previously, another rhodopsin-guanylyl cyclase BeRhGC from the aquatic fungus Blastocladiella emersonii was studied and applied as optogenetic tool in [4,29,30]. An absorption and emission spectroscopic characterization of the rhodopsin domain BeRh of BeRhGC was carried out in [6] (named Rh (BE) of RhGC in [6]). Here, an absorption and emission spectroscopic characterization of the rhodopsin part CaRh of CaRhGC was carried out and and its behavior is compared below with BeRh and with channelrhodopsin ChR2 from Chlamydomonas reinhardtii.

Spectral and Thermal Studies
The rhodopsin CaRh is thermally very stable. The apparent protein melting temperature of CaRh is ϑm = 62 ± 2 °C. For CaRh at 3.5 °C no attenuation coefficient rise in the transparency region (λ > 640 nm) was observered within the investigation period of 103 days. It occurred an attenuation coefficient spectrum reduction due to CaRh aggregate cluster compactization with storage time [24]. At room temperature (20.5 °C) some continuous attenuation coefficient rise in the transparency region was observed within the first 144 h due to protein aggregation, then the attenuation coefficient decreased because of protein aggregate sedimentation. The higher the apparent protein melting temperature ϑm the longer is the protein melting time tm at the temperature of experimental investigation of the protein (e.g., room temperature). The protein melting time or half-time tm is

Discussion
The rhodopsin-guanylyl cyclase CaRhGC from the nematophagus fungus Catenaria anguillulae belongs to the class of enzymerhodopsins of microbial proteins which consist of a rhodopsin domain and an enzyme domain [8,9,[26][27][28]. Light excitation of the rhodopsin domain results in the activation of the guanylyl cyclase domain and causes the conversion of GTP (guanosine triphosphate) to cGMP (cyclic guanosine monophosphate).
The rhodopsin-guanylyl cyclase CaRhGC from Catenaria anguillulae was expressed recently and its light-activated guanylyl cyclase activity was demonstrated [4]. Previously, another rhodopsin-guanylyl cyclase BeRhGC from the aquatic fungus Blastocladiella emersonii was studied and applied as optogenetic tool in [4,29,30]. An absorption and emission spectroscopic characterization of the rhodopsin domain BeRh of BeRhGC was carried out in [6] (named Rh (BE) of RhGC in [6]). Here, an absorption and emission spectroscopic characterization of the rhodopsin part CaRh of CaRhGC was carried out and and its behavior is compared below with BeRh and with channelrhodopsin ChR2 from Chlamydomonas reinhardtii.

Spectral and Thermal Studies
The rhodopsin CaRh is thermally very stable. The apparent protein melting temperature of CaRh is ϑ m = 62 ± 2 • C. For CaRh at 3.5 • C no attenuation coefficient rise in the transparency region (λ > 640 nm) was observered within the investigation period of 103 days. It occurred an attenuation coefficient spectrum reduction due to CaRh aggregate cluster compactization with storage time [24]. At room temperature (20.5 • C) some continuous attenuation coefficient rise in the transparency region was observed within the first 144 h due to protein aggregation, then the attenuation coefficient decreased because of protein aggregate sedimentation. The higher the apparent protein melting temperature ϑ m the longer is the protein melting time t m at the temperature of experimental investigation of the protein (e.g., room temperature). The protein melting time or half-time t m is defined as the time duration of unfolding of 50% of the protein [21]. The protein melting time of CaRh at ϑ = 3.5 • C was longer than the time of experimental observation of 103 days (during this time no measurable increase of light scattering, see Figure 5). Also at ϑ = 20.5 • C the melting time of CaRh was longer than the time of experimental observation of 312 h (scattering coefficient α s (750 nm, 312 h) ≈ 0.25 cm −1 in Figure 6 compared to α s (750 nm, 69.2 • C) ≈ 5 cm −1 in Figure 4a).
Fresh thawed CaRh exhibits a smooth inhomogeneous broadened S 0 -S 1 absorption band and shows the structure of less inhomogeneous broadened higher excitation bands (S 0 -S 2 and S 0 -S 3 transitions, see Figure 1). If the absorption coefficient spectrum of fresh CaRh in the wavelength range from 440 to 310 nm would belong to S 0 -S 1 transitions of different retinal isomers, then another photocycle behavior in this spectral range would be expected than observed in Figure 7. Inhomogeneous absorption line broadening means the presence of a distribution of species with shifted absorption spectra [31,32]. Here it indicates the presence of a distribution of retinal and opsin protein conformations with differing retinal-ospin interactions causing a distribution of ground-state and excited-state singlet potential energy surfaces.
The inhomogeneous nature of the S 0 -S 1 absorption band of CaRh shows up in the variation of fluorescence quantum distributions (Figure 2a,b) and the fluorescence quantum yield (Figure 3) with fluorescence excitation wavelength within the S 0 -S 1 absorption band (λ F,exc ≥ 440 nm). The isomerization path in the retinal S 1 potential energy surface depends on the excitation wavelength (locally excited state LE, see Figure S5 in Supplementary Material S8). S 1 -S 0 fluorescence emission along the S 1 potential energy surface relaxation path towards the S 1 state funnel Fu (conical intersection [14], position of S 1 -S 0 twisted internal conversion [15]) determines the excitation wavelength dependent fluorescence emission quantum distribution and fluorescence quantum yield. The small fluorescence quantum yield indicates a barrierless S 1 -state potential energy surface relaxation.
Higher excited state S 0 -S 2 and S 0 -S 3 transitions of PRSB in CaRh turned out to be less inhomogeneous broadened showing some vibronic structure. They follow higher excited state isomerization paths with activation barriers to funnel positions indicated by structured fluorescence emissions and higher fluorescence quantum yields.

Photocycle Studies
Generally photo-excitation of rhodopsins causes retinal spatial cis-trans isomerization [8]. The photo-isomerization of protonated retinal Schiff base PRSB in rhodopsins often leads to a deprotonated retinal Schiff base intermediate RSB in the photocycle process [33][34][35][36]. The absorption coefficient spectra development of CaRh during and after photo-excitation displayed in Figure  The experimental photocycle studies lead to the following interpretation: (i) the photo-excitation of dark-adapted CaRh da causes a primary all-trans-13-cis photo-isomerization cycle with protonated retinal Schiff base to deprotonated retinal Schiff base conversion (light-adapted CaRh la2 formation, Rh-365); (ii) it involves an all-trans back photo-isomerization and protein restructuring cycle changing CaRh da to a light-adapted ground-state conformation CaRh la1 (Rh-527); (iii) photo-excitation of CaRh la1 causes a secondary all-trans-13-cis photo-isomerization cycle without protonated retinal Schiff base deprotonation. (Structural formulae of PRSB all-trans , PRSB 13-cis , and RSB 13-cis are shown in Figure S2 of [6]).
The proposed photocycle schemes are displayed in Figure 13a (primary photocycle, and all-trans back-isomerization with protein restructuring cycle) and Figure 13b (secondary photocycle including photo-degradation). Schematic reaction coordinate diagrams for the primary photocycle including protein restructuring of initially dark-adapted CaRh and the secondary photocycle of light-adapted CaRh without deprotonation are shown in Figures S5 and S6 of Supplementary Material S8, respectively. In the following the primary all-trans-13-cis photocycle scheme of Figure 13a and Figure S5 is explained first, then the all-trans back-isomerization and protein restructuring cycle of Figure 13a and Figure S5 is described, and then follows a description of the secondary photocycle scheme of Figure 13b and Figure S6. After that relevant photocycle parameters are extracted from the experimental results (Tables 2 and 3).

Primary All-trans-13-cis Photocycle of Initially Dark-Adapted CaRh
The proposed all-trans-13-cis photocycle scheme of initially dark-adapted CaRh (named CaRh da ) is shown in Figure 13a (upper part) and Figure S5. The retinal in the CaRh da dark-adapted state (also named G da for dark-adapted ground-state and Rh-541 considering its first peak absorption wavelength position) is thought to be all-trans protonated retinal Schiff base PRSB all-trans [6,8]. Photo-excitation of PRSB all-trans to a locally excited electronic state PRSB all-trans * (LE) starts photo-isomerization by relaxation (twisting) along the excited state potential energy surface to a funnel position PRSB Fu (Fu, conical intersection position, twisted internal conversion position). The relaxation time constant from locally excited state LE to funnel Fu is experimentally given by the fluorescence lifetime τ F in the sub-picosecond region for barrier-less relaxation to the picosecond region for barrier-slowed down relaxation (see Table 1). It occurs internal conversion (IC) from the funnel position Fu to a transition state position TS 0 (PRSB TS 0 ) on the S 0 potential energy surface. This state is labled I. Relaxation out of the PRSB TS 0 labile transition state position I leads to a branching of relaxation along a cis isomerization paths (quantum yield of cis-isomerization φ cis ) and along the all-trans back-isomerization path (quantum yield of all-trans back-isomerization φ trans = 1 − φ cis ). The cis isomerization path leads to the formation of PRSB 13-cis (Rh-630, named K intermediate [5] following the bacteriorodospin photocycle nomencature [36]). PRSB 13-cis relaxes to PRSB 13-cis,cirp (Rh-460, L intermediate [5,36]) by counter ion repositioning. PRSB 13-cis,cirp relaxes to RSB 13-cis (Rh-365, M intermediate [5,36], light-adapted CaRh la2 ) by proton release. RSB 13-cis recovers back to PRSB all-trans (CaRh da , G da , Rh-541) by re-protonation and cis-trans isomerization with time-constant τ rec,la2 .

Photo-Induced all-trans Back-Isomerization and Opsin Restructuring Cycle
The photo-excitation of CaRh da (PRSB all-trans ) causes besides the all-trans-13-cis photo-isomerization cycle an all-trans back-isomerization and opsin protein restructuring cycle generating light-adapted ground-state CaRh la1 (also named G la1 , Rh-527, PRSB all-trans,la1 ). CaRh la1 recovers back to CaRh da by protein back-structuring with time-constant τ rec,la1 . The all-trans back-isomerization and protein restructuring photocycle is included in Figure 13a (lower part) and Figure S5.

Secondary Photocycle of Light-Adapted CaRh la1
The secondary photo-isomerization cycle of CaRh la1 is illustrated in Figure 13b and Figure S6. As in the primary photocycle, photo-excitation of PRSB all-trans,la1 leads to metastable transition state PRSB TS 0 ,la1 (I intermediate) formation from where all-trans back-isomerization and all-trans-13-cis isomerization occurs. The cis isomerization causes PRSB 13-cis,la1 (K, Rh-630) formation. Counter ion repositioning changes PRSB 13-cis,la1 to PRSB 13-cis,cirp,la1 (L, Rh-460). Contrary to the primary photo-isomerization cycle of CaRh da no reversible deprotonation of PRSB 13-cis,cirp,la1 takes place due to the protein restructuring. Instead PRSB 13-cis,cirp,la1 recovers back to PRSB all-trans,la1 by 13-cis-all-trans back-isomerization with time constant τ rec,cis-trans . The back-isomerization with time constant τ rec,cis-trans has to be short compared to the protein back-structuring time constant τ rec,la1 (i.e., τ rec,cis-trans << τ rec,la1 ) since only weak population accumulation of Rh-460 is observed (only rise of attenuation coefficient at 460 nm from 1.75 to 1.8 cm −1 due to 590 nm light exposure in Figure 9b). The photo-excitation of CaRh la1 causes some irreversible degradation to CaRh Prod photoproducts (Prod, quantum yield of photo-degradation φ d , for photoproduct characterization see above).

Photocycle Parameters
The photo-excitation of CaRh da causes only a partial conversion of CaRh da to CaRh la2 because of the parallel back-isomerization and protein restructuring photocycle of CaRh da to CaRh la1 conversion.
The limiting fraction k la1 of excited CaRh da * converted to CaRh la1 at high excitation intensity is obtained from the ratio of the absorption strength of the S 0 -S 1 transition of CaRh la1 at high excitation intensity (dashed curves in Figure 7 for t exc = 3 s) to the initial absorption strength of the S 0 -S 1 transition of CaRh da before excitation (solid curves in Figure 7). The analysis presented in the Supplementary Material S7 gives k la1 ≈ 0.73 (k la1 is included in Table 3). The limiting fraction k la2 of excited CaRh da * converted to Ca Rh la2 is k la2 = 1 − k la1 ≈ 0.27 (k la2 is included in Table 2).
The initial quantum yield of all-trans-13-cis photo-isomerization φ cis ( Figure S5) of CaRh da is deduced from the initial light induced absorption change at λ pr = 550 nm of middle part of Figure Table 2 for the CaRh primary retinal photocycle dynamics, and in Table 3 for the CaRh secondary retinal photo-isomerization and protein restructuring photocycle dynamics.
In Figure 9a the transient attenuation coefficient development at λ pr = 630 nm is shown where the K intermediate (PRSB 13-cis ) has its absorption peak. The sharp attenuation dip in Figure 9a at t = 0 is due to G da * → I relaxation. The time constant of G da * → I relaxation is expected to be on the sub-picosecond to picosecond time scale (τ G * da →I ≥ τ F ). This dip disappears within the time resolution step of 0.0125 s due to I → K cis isomerization (τ I→K < t res = 0.0125 s) and I → G la1 trans back-isomerization (τ I→G la1 < t res = 0.0125 s). The following slight attenuation decrease seen in the left inset of Figure 9a is due to conversion of K to L. The time constant is τ K→L = 0.048 ± 0.005 s. The further rise of α(630 nm) is due to conversion of G da to G la1 . Its build-up time is equal to the G la1 to G da recovery time. The obtained time constant is τ G la1 →G da = τ rec,la1 = 0.8 ± 0.1 s. At light switch-off CaRh recovers to the dark-adapted situation. The spike at the moment of light switch-off is due to I → G da conversion. The following absorption decrease is caused by K → L → M conversion. The final slow absorption rise is thought to be mainly due to M to G da recovery.  0.46 ± 0.05 Figure 8a and Figure S5, Equations (S11, S12, S13a, S13b) φ trans 0.54 ± 0.05 <0.0125 Figure S5 and   Figure 9b to be τ L→M = 0.123 ± 0.005 s. The following rise of absorption is caused by the conversion of G da to G la1 . It follows a slight decrease of α(460 nm) because of G la1 →Prod photoproduct formation. At light switch-off the absorption rise is caused by M → G da relaxation (time constant τ rec,la2 ). The following absorption decrease is thought to be due to G la1 → G da recovery. Table 3. Secondary photo-isomerization and protein restructuring photocycle of CaRh in pH 7.3 HEPES/MOPS buffer (G la1 → I → K → L → G la1 → G da ). Photo-excitation with LED 590 nm.
In Figure 9c, the transient attenuation coefficient development at λ pr = 370 nm is displayed where the M intermediate (RSB 13-cis , CaRh la2 ) has its absorption peak. The attenuation coefficient increase at the onset of light exposure has a slightly sigmoidal shape (delayed rise, see inset in Figure 9c) because of the delayed population of M in the I → K → L → M intermediate chain (τ I→K < 0.0125 s, τ K→L ≈ 0.048 s, τ L→M ≈ 0.12 s). The steepness of the attenuation coefficient rise depends on the excitation intensity I exc (increases with rising excitation intensity). The initially reached α(370 nm) attenuation peak decreases somewhat because of build-up of CaRh la1 (G da → G la1 conversion) whose photo-excitation cycle does not involve M intermediate formation (time constant of attenuation decrease is given by τ rec,la1 by equilibration between G da and G la1 ). The following slight rise of α(370 nm) is due to CaRh la1 → CaRh Prod photo-degradation. After light swich-off attenuation coefficient α decreases because of M → G da re-protonation and cis-trans isomerization (CaRh la2 recovery to CaRh da ) with dominant time constant τ rec,la2 = 0.35 ± 0.01 s, and slower relaxation of the other intermediates with attenuation contribution at λ pr = 370 nm to G da .
In Figure 9d, the transient attenuation coefficeint development at λ pr = 530 nm is displayed where the absorption is dominated by the initial CaRh da (G da ) and the formed CaRh la1 (G la1 ). The initial absorption decrease after light switch-on is caused by G da → I → K → L → M intermediate formation.
The steepness of the decrease is I exc dependent (sharper decrease for larger I exc ). It follows a slight absorption increase due to G la1 formation with its secondary photo-isomerization cycle. The following slight attenuation coefficient decrease is due to photo-degradation G la1 → Prod. After light switch-off the attenuation coefficient recovers mainly because of M → G da recovery.
In Figure 9e the transient attenuation development at λ pr = 350 nm is displayed where the absorption is dominated by M (RSB 13-cis ) absorption as in the case of Figure 9c. The transient attenuation behavior is the same as in Figure 9c, only at the moment of light switch-on an additional attenuation dip and at the moment of light switch-off an additional attenuation spike are present. The dip and the spike are thought to be present because of G da level depopulation (G da →I, dip) and G da level repopulation (I→G da , spike) with associated S o -S n absorption change.

Comparision of Behavior of CaRh from Catenaria anguillulae with Behavior of BeRh from Blastocladiella emersonii
The studied rhodopsin BeRh in [6] was thermally of low stability. The apparent protein melting temperature of BeRh was ϑ m = 48.8 ± 2 • C. BeRh protein melting times of t m (1.65 • C) = 8.1 ± 0.2 day and t m (21.9 • C) = 1.45 ± 0.15 h were determined from the onset of strong light-scattering due to aggregation of unfolding proteins. For optogenetic studies the stability of the photoreceptor is crucial. Due to the increased protein stability of CaRh compared to BeRh, the application of CaRh is beneficial, in particular for experiments, which require a prolonged functionality of the photoreceptor, e.g., when repetitive illumination protocols over extended time periods are used.
Fresh thawed BeRh was composed of a mixture of retinal-protein conformations showing up in the rhodopsin absorption spectrum. In the inhomogeneous broadened absorption spectrum of BeRh the presence of (at least) four retinal isomers Ret_1, Ret_2, Ret_3, Ret_4 could be resolved (see Figure  S3 of [6]). The retinal-protein conformation mixture also showed up in the fluorescence emission quantum distribution dependence on the fluorescence excitation wavelength (see Figure 2 in [6]). The retinal composition changed with storage time towards irreversible deprotonated (likely 13-cis) retinal Schiff base (Ret_4') (see Figures 4a,b, S5 and S6 of [6]).
The photo-excitation dynamics of BeRh in the case of protonated retinal Schiff base PRSB all-trans excitation (λ exc = 532 nm) resulted in all-trans-13-cis photo-isomerization with subsequent retinal intermediate formations (see experimental curves in Figures 5-8 and schemes of Figures 9 and 10b of [6]). The photodynamics studies in [6] were carried out only at one excitation wavelength (second harmonic of cw Nd:YAG laser, λ exc = 532 nm) with rather low excitation intensity (I exc ≈ 16 mW cm −2 for Figure 5 and Iexc ≈ 22 mW cm−2 for Figures 6-8). The experimental photocycle/photo-degradation behavior of BeRh was found to be quite similar to that CaRh: for short-time exposure (t exc = 0.1 s) a reversible photocycle behavior was observed (Figure 7a,b); the wavelength position of the first absorption maximum of Ret_1 shifted from λ a,max = 527 nm in the dark-adapted state to λ a,max = 518 nm in the light-adapted state (solid curve in Figure 5 for t exc = 0 and dotted curve in Figure  5 for t exc = 2.171 s); in the continued exposure over 990 s an attenuance plateau was reached within the first few seconds of excitation and then gradual irreversible photoproduct formation occurred ( Figure 6).
The light-adapted Ret_1 la1 (PRSB all-trans,la1 ) state formation with its photo-isomerization cycle without deprotonated retinal Schiff base RSB 13-cis formation was overlooked in [6] since no excitation intensity dependent photocycle experiments were carried out. With the new information on the excitation intensity dependent photocycle and photoproduct formation behavior for the thermally stable CaRh we think that the Ret_1 photocycle dynamics of BeRh is similar to the photocycle dynamics of CaRh. In the photo-isomerization scheme of Figure 9b in [6] the back-relaxation from TS 0 to Ret_1 (PRSB all-trans ) is thought to involve a meta-stable state Ret_1 la1 (PRSB all-trans,la1 ) with all-trans-13-cis photo-isomerization cycling without PRSB 13-cis (Ret_5) reversible deprotonation to RSB (no Ret_4 formation).

Comparision of Photocycle Behavior of CaRh from Catenaria anguillulae with Photocycle Behavior of Channelrhodopsin ChR2 from Chlamydomonas reinhardtii
Photocurrent response studies to light stimuli and time resolved spectroscopy on channelrhodopsin ChR2 revealed a coupled dark-adapted state (D) and light-adapted desensitized state (Des) two-photocycle model [37]. Initially dark-adapted (IDA) ChR2-C128T mutant recovered to two different dark-adapted states DAB and DAG after blue and green light exposure, respectively (DAB = blue-light dark-adapted state, DAG = green-light dark-adapted state, IDA = initially dark-adapted state). The photo-excitation of DAB and DAG led to two coupled photocycles [38]. Liquid and solid-state nuclear magnetic resonance spectroscopy and resonance Raman spectroscopy on ChR2 were carried out to understand the substantial reduction of photocurrents during illumination, a process named "light-adaptation" [39]. It was shown that longer light pulse excitation led to an apparent dark-adapted state with two isomer conformations: all-trans,15-anti (IDA initial dark adapted state, D480) and 13-cis,15-syn (light-induced dark-adapted state, D470'). Both isomers together were named apparent dark-adapted state (DA app ). The photo-excitation of both apparent dark-adapted state isomers caused two distinct photocycles [39].
The coupled photocycle occurrence of ChR2 has strong resemblance to the coupled dark-adapted-state G da and light-adapted state G la1 photocycle behavior of CaRh from Catenaria anguillulae studied in this paper.

Sample Preparation
CaRh was expressed and purified as described earlier [4]. Briefly, the Rh domain (1-396 aa) of the full-length CaRhGC was expressed in Pichia pastoris. All purification steps were performed in 50 mM HEPES/MOPS buffer pH 7.5, 100 mM NaCl, 0.1 mM PMSF at 4 • C. Fractions that contained the protein were pooled, concentrated (Amicon Ultra 100 kDa, Millipore) to yield 2.6 mg/mL and stored at −80 • C.
For fluorescence spectroscopic measurements a spectrofluorimeter (Cary Eclipse from Varian) was used (cell length in excitation direction 0.15 cm, cell width in detection direction 3 mm). Fluorescence quantum distributions E F (λ) were determined from fluorescence emission spectrum measurements at fixed excitation wavelengths [16,41,42]. The dye rhodamine 6G in methanol (fluorescence quantum yield φ F,ref = 0.94 [43]) was used as reference standard for fluorescence quantum distribution calibration. The fluorescence quantum yield is given by φ F = em E F (λ)dλ where the integration runs over the fluorescence emission wavelength region. The fluorescence spectra were deprived from scattering contributions by separate spectra measurements using a Ludox CL-X colloidal silica-water solution with particle size of 21 nm diameter and appropriate scattering contribution subtraction.
For absorption spectroscopic photocycle investigations, CaRh samples were excited with light emitting diodes LED 590 nm, LED 530 nm, and LED 470 nm from Thorlabs (spectral distributions included in Figure 7). The sample cell in the Cary 50 spectrophotometer was irradiated with the LEDs transverse to the transmission detection path (exposed area 3 × 5 mm 2 , sample thickness along excitation path 1.5 mm, transmission detection path length 3 mm). The excitation power P exc was measured with a power meter (model PD 300-UV-SH photodiode detector head with NOVA power monitor from Ophir). Photo-degradation studies were carried out by long-time sample exposure with LED 590 nm, LED 530 nm, and LED 470 nm.
The apparent protein melting temperature of CaRh was determined by stepwise sample heating up and then cooling down, whereby transmission spectra were measured and the rising light scattering with sample heating was analyzed [21]. The thermal protein stability at room temperature (20.5 • C) and refrigerator temperature (3.5 • C) was determined by storing CaRh samples at the selected temperatures in the dark and measuring transmission spectra at certain time intervals whereby the temporal light attenuation development was analyzed.

Conclusions
The rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase CaRhGC from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. Its photophysical behavior was compared with that of BeRh, the rhodopsin domain of the rhodopsin-guanylyl cyclase BeRhGC from the aquatic fungus Blastocladiella emersonii. Both rhodopsin-guanylyl cyclases belong to the class of emzymerhodopsins in microbial organisms. Both CaRhGC and BeRhGC found already application as tools in optogenetics [4,5,29,30].
CaRh in pH 7.3 HEPES/MOPS buffer has the advantage of high thermal stability compared to BeRh in pH 8.0 Tris buffer of low thermal stability. The low thermal stability of BeRh may be the reason of the presence of a mixture of retinal-protein conformations in fresh thawed samples and conformational changes within the time of some hours at room temperature which show up in the UV-Vis absorption spectral shape of fresh thawed samples and the spectral development with time. On the other side the CaRh absorption spectrum of a fresh thawed sample exhibits inhomogeneous broadened singlet ground-state to first, second, and third singlet excited-state excitations with aggregation dependent changes on a several day timescale. The inhomogeneous absorption line broadening indicates a variation of the retinal structural shape and a variation of the arrangement and charge distribution of the surrounding amino acid residues of the opsin protein.
The photocycle dynamics of BeRh and CaRh by PRSB all-trans excitation were found to behave quite similar. Sample exposure of dark-adapted BeRh and CaRh in the S 0 -S 1 absorption band region of PRSB all-trans caused (i) all-trans-13-cis photo-isomerization to PRSB 13-cis , counter ion repositioning to PRSB 13-cis,cirp , proton release to RSB 13-cis and recovery to PRSB all-trans by re-protonation and cis-trans isomerization (primary photocycle) and (ii) it involved all-trans back-isomerization with protein restructuring to light-adapted PRSB all-trans,la1 with recovery in the dark to PRSB all-trans . Continued light exposure caused PRSB all-trans,la1 all-trans-13-cis photo-isomerization to PRSB 13-cis,la1 , counter ion repositioning to PRSB 13-cis,cirp,la1 and cis-trans back-isomerization to PRSB all-trans,la1 without the involvement of proton release and re-protonation (secondary photocycle). The prolonged photo-excitation caused some low-efficient photo-degradation of the protonated retinal Schiff base.