In P450cin, Tyr81, Asp241, Asn242, two water molecules, and the substrate participate in a complex H-bonded network. The role of this H-bonded network in substrate binding and catalysis has been probed by crystallography, spectroscopy, kinetics, isothermal titration calorimetry (ITC), and molecular dynamics. For the Y81F mutant, the substrate binds about 20-fold more weakly and Vmax decreases by about 30% in comparison to WT. The enhanced susceptibility of the heme to H₂O₂-mediated destruction in Y81F suggests that this mutant favors the open, low-spin conformational state. Asn242 H-bonds directly with the substrate, and replacing this residue with Ala results in water taking the place of the missing Asn side chain. This mutant exhibits a 70% decrease in activity. Crystal structures and molecular dynamics simulations of substrate-bound complexes show that the solvent has more ready access to the active site, especially for the N242A mutant. This accounts for about a 64% uncoupling of electron transfer from substrate hydroxylation. These data indicate the importance of the interconnected water network on substrate binding and on the open/closed conformational equilibrium, which are both critically important for maintaining high-coupling efficiency.