[en] Highlights: • Photocycle of E46Q-PYP was investigated by TG spectroscopy. • Kinetics and diffusion coefficients were compared between E46Q-PYP and wt-PYP. • The blue-shifted intermediate is formed more slowly in E46Q-PYP. • The smaller ΔD of E46Q-PYP is mostly contributed by the smaller D_pG than that of wt-PYP. To elucidate the role of internal proton transfer in the photodynamics of photoactive yellow protein (PYP), the photocycle of the Glu46Gln mutant of PYP (E46Q-PYP) is investigated by transient grating (TG) spectroscopy. Compared with wild-type PYP (wt-PYP), the first spectrally blue-shifted intermediate of E46Q-PYP is formed more slowly, which is consistent with the absence of direct protonation from Glu46 residue, if the parallel kinetic model for wt-PYP is invoked. The smaller conformational change in E46Q-PYP, as manifested by the smaller change in the diffusion coefficient, mainly arises from the relatively larger volume of the ground state E46Q-PYP than wt-PYP rather than from the smaller volume of the pB state.