## 4 Discussion  
T127 of CrChR2 is localized in close vicinity to the retinal Schiff base and is hydrogen-bonded to E123 which, in turn, is the hydrogen bond acceptor of the Schiff base. The position of the threonine is also strategic for Schiff base reproduction, as its hydroxylic group is supposed to be part of the pathway between the proton donor D156 and the Schiff base. There is no direct hydrogen-bonding network in the ground state that connects the RSBH\({}^{+}\) and D156, but it is only with the rise of the photocycle intermediate P\({}_{3}\)[530] that this connection is formed [13]. T127A substitution removed the hydroxylic group of the threonine, which affected the photocycle kinetics. The P\({}_{2}\)[380] intermediate, during the lifetime of which the Schiff base is deprotonated, had a \(\sim\)10 times faster risetime, meaning that the deprotonation of the Schiff base was facilitated in this variant. The acceleration of the rise of the P\({}_{2}\)[380] intermediate may have been due to the stronger hydrogen-bonding interaction between the Schiff base and E123 that we reported in the present work. The lifetime of the P\({}_{2}\)[380] state was longer in the T127A variant and, as a consequence, the accumulation of the subsequent intermediate P\({}_{3}\)[530] was very low. A longer lifetime of the state with a deprotonated Schiff base is a sign that the reprotonation pathway was blocked in T127A. The T127S variant conserved the hydroxyl group of the side chain, and we showed here that the photocycle kinetics were almost indistinguishable from the WT. We concluded, therefore, that the hydrogen-bonded network between D156 and the Schiff base, which is necessary for the reprotonation of the latter, involves the hydroxylic group of T127. The formation of this chain of hydrogen bonds is necessary for proton translocation and marks the transition between the P\({}_{2}\)[380] and P\({}_{3}\)[530] states. T127 is important for the opening of the ion channel after light activation, since we have shown here that the T127A, as well as the more conservative T127S variants, showed less than 10% of the WT current in electrophysiology experiments. We note, though, that the reduced conductance may also have been related to lower expression yields of the variants in the different hosts (_Xenopus oocytes_ and HEK cells). It has previously been shown that the channel closes with the decay of the P\({}_{3}\)[530] intermediate [36, 37], but the channel opening is an optically silent transition in the visible range [38]. Figure 8: Time-resolved FTIR difference spectra of ChR2-WT (black spectra, taken from Reference [13]), T127A (red spectrum), and T127S variants (blue spectrum). The ChR2-WT spectrum at 300 \(\upmu\)s and 6.7 ms was dominated by the P\({}_{2}\)[380] and P\({}_{3}\)[530] states, respectively. The spectra of the T127 variants were recorded at 8.4 ms, at which the P\({}_{2}\)[380] with smaller contributions of P\({}_{3}\)[530] was predominant. We confirmed from the present work that channel opening in _Cr_ChR2 is not related to changes in the photocycle kinetics as recorded by time-resolved UV/Vis spectroscopy. The T127S variant, in fact, showed WT-like photocyle kinetics, whereas a long-lived P\({}_{2}\)[380] intermediate occurred in the T127A variant with both variants exhibiting low channel conductance. A threonine residue at this position was conserved for most of the cation- and anion-conducting channelrhodopsins. It is also present in BR [12; 39], where exchanges to valine or alanine resulted in blue shifts in the absorption maxima by 146 nm and 28 nm, respectively, with minor effects on pumping activity [3]. By contrast, the exchange of threonine to serine and alanine in _Cr_ChR2 had only negligible effects on the electronic properties of the retinal chromophore, as registered by its visible absorption.