where n indexes ionization state of 4F d 2 (1 = neutral; 2 = anionic), I n KSI is the integrated intensity of a C-F peak in the protein ( Figure 2F ), and I n ref is the integrated intensity of the basis spectrum of neutral or ionized 4F d 2 ( Figure 2E ). We note that equation (2) assumes that the intensity of a transition is unperturbed by the active site environment. This assumption appears valid, as the sum of the two referenced intensities in the KSI D40N -bound spectrum ( Figure 2F , Table 2 ) is , quite close to unity. The doubly-peaked spectra of KSI D40N •4F d 2 ( Figure 4 ) were fit to two Gaussians using the OPUS software package with the Levenberg-Maquardt algorithm ( Figure 2F , red and blue traces). The Gaussian fits were integrated to give the integrated intensities, I n KSI . The integrated intensities were referenced (according to equation (2) ) to give relative populations of the ionization states, p n (1 = neutral; 2 = anionic). A simple ratio of the populations
Ketosteroid isomerase has become a test enzyme in the debate over the existence of low barrier hydrogen bonds (LBHBs) in accordance with the hydrogen bonding discussed above. Nuclear magnetic resonance (NMR) studies of KSI in complex with transition state analogs have revealed the presence of a highly shielded proton characteristic of the formation of a LBHB between Tyr 14 and the O-3 atom of the analogs and suggestive of the formation of a bridging hydrogen with short bonds.  ,  ,  Additionally, NMR fractionation studies with deuterium substitution are strongly suggestive of the high strength of this bond as deuterium is retained preferentially in this position.  The energy of this bond along with that of the normal hydrogen bond from Asp 99 most likely contribute to the energy needed to support proton abstraction from the C-4 position.