Δ5-3-Ketosteroid isomerase (EC 18.104.22.168) from Pseudomonas testosteroni catalyzes the highly efficient conversion of Δ5-3-ketosteroids to Δ4-3-ketosteroids by a stereoselective and intramolecular transfer of the 4β-proton to the 6β-position. Tyr-14 is the critical general acid and Asp-38 is the general base involved in catalysis. The UV absorption bandwidths of Tyr-14 were much narrower than those of the other two tyrosines (Tyr-55 and Tyr-88) of isomerase or of the N-acetyltyrosine ethyl ester in aqueous solution, suggesting that Tyr-14 is restricted in its mobility. Further immobilization of this residue occurs upon steroid binding. Thus, 5α-estrane-3, 17-dione, an A-ring saturated steroid, induces significant narrowing of the tyrosine absorption bands (π → π) of the main peak (279.5 nm) and the shoulder (285.5 nm) of Tyr-14, with no significant changes in Amax. No effects of steroid binding were found on the absorption bandwidths of Tyr-55, Tyr-88, or the phenylalanine residues. The ratio of the absorbance (Amax) at the absorption maximum (Amax) to that at γmaxplus 4 nm (Amax+4) was used as a measure of peak sharpness. Specifically, the ratios of A279.5/A283.5(main peak) and A285.5/A289.5(shoulder) of Tyr-14 of the free enzyme at 25 °C were 1.25 and 1.89, respectively, and they increased to 1.41 and 2.70, respectively, in the complex. A more precise measurement of the band narrowing from 4.2 to 3.1 nm between the inflection points was obtained from the derivative spectra. The absorption bands of free and steroid-bound isomerase were narrowed significantly by lowering the temperature and were broadened by denaturation, suggesting that the unusual peak-sharpening effects induced by steroid binding arise from the restricted motion of Tyr-14, as well as from more directional hydrogen bonding resulting from the displacement of water molecules from the active site and decreased flexibility of the protein. Larger enthalpy of the sharpening effects was observed for the steroid-bound enzyme (-0.527 ± 0.016 kcal/mol) than for the free enzyme (-0.250 ± 0.018 kcal/mol) by lowering the temperature, indicating that interactions of Tyr-14 with its environment, which restrain its motion, are stronger in the steroid-bound enzyme than in the free enzyme. Hydrogen-bonding modes of Tyr-14, mobility of the active site, and protein flexibility are the environmental factors determining the absorption bandwidths of the critical Tyr-14 residue.