## 4 Discussion
This result is particularly interesting in light of the exceptionally high vibrational frequency recorded for the C=N-H vibration of the retinal Schiff base in T127A, as the hydrogen-bonding of the retinal Schiff base has been proposed to be a molecular determinant of the opsin shift [40]. QM/MM calculations showed nearly identical excitation energies for WT, T127S, and T127A variants. The same trend was also observed in the experimental absorption maxima of the retinal chromophore of _Cr_ChR2. However, a large shift of 19 cm\({}^{-1}\) was calculated for the C=N-H vibration in the alanine variant, but no shift for the serine variant, again confirming the resonance Raman spectroscopic results. Since our QM/MM models were able to reproduce the relative trends in the UV/Vis and vibrational spectra of the variants, we considered these reliable for our analysis of the molecular changes. In the T127A variant, the hydrogen bond between the threonine and E123 was missing, affecting the hydrogen-bonding network around the Schiff base. To compensate for the missing hydrogen bond, the interaction between E123 and the Schiff base (N(RSBH\({}^{+}\))\({}^{\cdots}\) O(E123)) increased, resulting in a longer N-H bond which manifested in the upshift of the C=N-H vibrational frequency. The elongation of the Schiff base N-H bond was 0.016 A, and possibly accelerates the deprotonation of the Schiff base in this variant. Hence, the stronger interaction between the Schiff base and E123 provided an explanation for the faster rise time of the P\({}_{2}\)[380] intermediate in the T127A variant, in a similar way to the D85E variant of bacteriorhodopsin [41]. Detailed analysis of the Schiff base vibration (coupled mode of the C=N stretching and the N-H bending vibration) by resonance Raman spectroscopy revealed the presence of two overlapping bands. The frequency shifts of these vibrational bands in the different variants were compared to the WT and their presence can be rationalized on the basis of the molecular model proposed in Reference [31]. In this model, the E123 side chain had one rotamer pointing towards the Schiff base and another rotamer pointing to a different hydrogen-bonding network that gave rise to two different vibrational bands of the C=N-H mode. This interpretation was supported by the band shape of the Schiff base vibration in the E123D variant, which was missing the high-frequency component. We can therefore lend support to the model of two rotamers of E123 in _Cr_ChR2, where a direct hydrogen bond with the Schiff base was formed in only one of the two configurations. The presence of two rotamer configurations may be essential for a voltage-sensing mechanism involving E123 [42]. While this seems to be a plausible scenario, further experimental evidence needs to be collected to support this model. Conceptualization, R.S. and J.H. ; Funding acquisition, R.S. and J.H. ; Investigation, D.E., N.K., M.S., C.B., R.K.K., K.H., D.H. and IS. ; Writing--original draft, R.S. and N.K. ; Writing--review & editing, J.H. and M.S. The research was funded by the German Research Foundation via the SFB-1078 projects B3 (to J.H.) and B4 (to R.S.). I.S. thanks the SFB 1078 for support within the Mercator program. R.K.K. acknowledges support from the Lady Davis Trust for Shunbrun postdoctoral fellowship. I.S. gratefully acknowledges funding by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 678169 \"PhotoMutant\"). The publication of this article was funded by Freie Universitat Berlin. The authors declare no conflict of interest.