The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatio-temporal control of cell signalling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds timescale, which then modulate the activity of output domains responsible for biological signalling. Using time resolved vibrational spectroscopy coupled with isotope labelling we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 femtoseconds and one millisecond after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labelled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a sub-picosecond perturbation of the protein matrix occurs. In this perturbed environment the previously characterised reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the -sheet then -helix regions of the AsLOV2 domain, which ultimately gives rise to J-helix unfolding, yielding the signalling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.