The perturbations on conversion from the dark state to the signaling state in photoactive yellow protein have been determined by solution-phase hydrogen/deuterium exchange and mass spectrometry. Both the wild type and M100A mutant are used in this study, with the mutant providing over 90% conversion to the bleached state under steady-state illumination. We found perturbations in both the wild type and the mutant on illumination, consistent with a more flexible structure in the long-lived signaling (I2') state. In the case of the wild type, the conformational changes detected are mainly around the chromophore region. With the M100A mutant, differences in H/D exchange between the light and dark are more extensive as compared to wild type; not only are the chromophore surroundings affected, but significant increases in deuterium uptake in the N-terminus and central beta-sheet are observed as well. On the basis of the data obtained from this study and previous findings, a sequence of events that leads to the perturbation of PYP following chromophore photoisomerization is proposed.