Channelrhodopsin-2 is a photoactive membrane protein serving as an ion channel, gathering significant interest for its applications in optogenetics. Despite extensive investigation, several aspects of its photocycle remain elusive and continue to be subjects of ongoing debate. Of particular interest are the localization of the P480 intermediate within the photocycle and the timing of the deprotonation of glutamic acid E90, a critical residue for ChR2 functioning. In this study, we explore the possibility of an early-P480 state, formed directly upon photoillumination of the dark-adapted state, where E90 is deprotonated, as hypothesized in a previous work [Kuhne et al. Proc. Natl. Acad. Sci. 116.19 (2019): 9380]. Employing extended molecular dynamics simulations, deprotonation free energy calculations, and the computation of the infrared band associated with E90, we provide support to the photocycle model proposed by Kuhne et al. Furthermore, our findings show that E90 protonation state is influenced by diverse interconnected variables and provide molecular detail insights that connect E90 interaction pattern with its deprotonation propensity. Our data demonstrate in fact that both protonated and deprotonated E90 are possible in P480 depending on E90 hydrogen bonding pattern and explaining the molecular mechanism at the basis of P480 accumulation under continuous illumination.