In proteins and enzymes, the local environment of an active cofactor plays an important role in controlling the outcome of a functional reaction. In photoactive yellow protein (PYP), it ensures photoisomerization of the chromophore, a prerequisite for formation of a signaling state. PYP is the prototype of a PAS domain, and the preferred model system for the studies of molecular mechanisms of biological light sensing. We investigated the effect of replacing proline-68, positioned near but not in direct contact with the chromophore, with other neutral amino acids (alanine, glycine, and valine), using ultrafast spectroscopy probing the visible and the mid-IR spectral regions, and molecular simulation to understand the interactions tuning the efficiency of light signaling. Transient absorption measurements indicate that the quantum yield of isomerization in the mutants is lower than the yield observed for the wild type. Subpicosecond mid-IR spectra and molecular dynamics simulations of the four proteins reveal that the hydrogen bond interactions around the chromophore and the access of water molecules in the active site of the protein determine the efficiency of photoisomerization. The mutants provide additional hydrogen bonds to the chromophore, directly and by allowing more water molecules access to its binding pocket. We conclude that proline-68 in the wild type protein optimizes the yield of photochemistry by maintaining at weak hydrogen bond with the chromophore, at the same time restraining the entrance of water molecules close to the allcylic part of pCa. This study provides a molecular basis for the structural optimization of biological light sensing.