Phycocyanobilin:ferredoxinoxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction of biliverdin IXα's two vinyl groups to produce phycocyanobilin, an essential chromophore for phytochromes, cyanobacteriochromes and phycobiliproteins.Previous site directed mutagenesis studies indicated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits proton transfer. Here, we exploit X-ray crystallography, enzymology, and molecular dynamics simulations to understand the functional role of this invariant histidine. The structures of the H74A, H74E, and H74Q variants of PcyA reveal that a "conserved" buried water molecule that bridges His74 and catalytically essential His88is not required for activity. Despite distinct conformations of Glu74 and Gln74 in theH74E and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggestingsmaller residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular dynamic simulationsfurtherreveal that the crucial active site residue Asp105 is more dynamic in H74A compared towild-typePcyA and the two other His74 variants, supporting the conclusion that theAla74 mutation has increased the flexibility of the active site.