Protochlorophyllide (Pchlide), an intermediate in the biosynthesis of chlorophyll, is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase (POR). Pchlide has excited state properties that allow it to initiate photochemistry in the enzyme active site that involves reduction of Pchlide by sequential hydride and proton transfer. The basis of this photochemical behavior is investigated here using a combination of time-resolved spectroscopies and DFT calculations of a number of Pchlide analogues with modifications to various substituent groups. A keto group on ring E is essential for excited state charge separation in the molecule, which is the driving force for the photoreactivity of the pigment. Vibrational “fingerprints” of specific regions of the Pchlide chromophore have been assigned, allowing identification of the modes that are crucial for excited state chemistry in the enzyme. This work provides an understanding of the structural determinants of Pchlide that are so important for harnessing light energy.