## 3 Results
### Spectroscopic Properties of the Dark State of the T127 Variants of CrChR2  
As T127 is part of the retinal binding pocket, we first examined the potential influence of the amino acid exchange on the electronic properties of the retinal. It was evident from comparison of the UV/Vis spectra (Figure 3, red and blue spectra) that both T-A and T-S amino acid exchanges shifted the visible absorption spectrum only slightly to the blue as compared to WT (black spectrum in Figure 3). The second derivative of the absorption spectra (bottom panel) resolved a blue shift of about 2 nm in the T127A variant as compared to the WT (similar to T127S, not shown). The vibronic fine structure of the electronic absorption spectrum was retained in the T127A and the T127S variants. The latter observation was different to the C128T variant, where a 20 nm redshift of the retinal absorption was accompanied by a loss in vibronic fine structure [27]. We also computed the excitation energies for the WT protein and the two variants. The results displayed in Table 2 showed virtually no alteration of the excitation energies, which agreed well with the experimental results (Figure 3). These results present an additional validation of our QM/MM model. \begin{table}
\begin{tabular}{l c c c} \hline \hline
**RSBH\({}^{+}\) Atom Pairs** & & & \\ (**Distances in Å**) & & & \\ \hline C=N & 1.310 & 1.308 & 1.309 \\ \hline N–H & 1.076 & 1.072 & 1.092 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Bond length in selected RSBH\({}^{+}\) atom pairs, based on the hybrid quantum mechanics/molecular mechanics (QM/MM)-optimized geometries. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Model** & **WT** & **T127S** & **T127A** \\ \hline Excitation energy nm (eV) & 389 (3.18) & 392 (3.16) & 388 (3.19) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Computed excitation energies at the sTD-DFT CAM-B3 LYP/cc-pVDZ level of theory. Figure 2: Optimized geometry of (**A**) CrChR2-WT, (**B**) T127S, and (**C**) T127A variants. The retinal Schiff base and side chains of the counterion complex are shown as grey and yellow sticks, respectively. Dotted lines correspond to the hydrogen-bonding network. As the wavelength of the electronic absorption in the visible correlated with the C=C stretching vibration [28], the strongest band in the resonance Raman spectrum of the T127A variant (Figure 4, green trace) was 1557 cm\({}^{-1}\), slightly blue-shifted by 6 cm\({}^{-1}\) with respect to the WT (Figure 5A, [29]). All of the other Raman bands were essentially the same as in the WT, except for the C=N-H vibration of the retinal Schiff base (Figure 5B). The frequency of the latter was observed at 1674 cm\({}^{-1}\) for T127A, which is, to our knowledge, the highest frequency so far observed for any rhodopsin. It even outperformed the frequency of the Schiff base vibration in rhodopsin (1660 cm\({}^{-1}\), [30]). The C=N-H vibrational band was downshifted by 42 cm\({}^{-1}\) upon H/D exchange, which was the largest isotope effect reported (Figure 4). In comparison, the C=N-H stretch in WT resonated with a frequency of 1657 cm\({}^{-1}\) and a \(\Delta\)\(\upmu\)\({}_{\text{H/D}}\) = 28 cm\({}^{-1}\) was reported [29]. As the hydrogen bond between T127 and E123 was removed in the T127A variant (Figure 2C), the carboxylic side chain of E123 was able to form a stronger salt bridge with the retinal Schiff base, resulting in a 0.016 A elongated N-H bond (Table 1). Figure 4: Resonance Raman spectra of the T127A variant in H\({}_{2}\)O (green trace) and in D\({}_{2}\)O (red trace). The indicated vibrational bands are discussed in the text. Figure 3: UV/Vis absorption spectra for the CrChR2-WT, T127A, and T127S variants. The second derivatives of the absorption spectra are displayed in the bottom panel; the second derivative of the T127S spectrum has been omitted for clarity as it overlaps with the T127A trace.