A Predictive Model of Strong Hydrogen Bonding in Proteins: The N δ1 -H-O δ1 Hydrogen Bond in Low-pH r-Chymotrypsin and r-Lytic Protease Pablo A. Molina and Jan H. Jensen* Department of Chemistry, UniVersity of Iowa, Iowa City, Iowa 52242 ReceiVed: January 10, 2003; In Final Form: April 10, 2003 A computational QM/MM methodology for the systematic study of various structural and spectroscopic properties of strong hydrogen bonds in enzymes is presented. The theoretical model is applied to the N δ1 - H-O δ1 hydrogen bond between His57 and Asp102 in the active sites of low-pH R-chymotrypsin and R-lytic protease. The minimum energy structures of both enzymes reproduce the experimental N δ1 -O δ1 distance and are used to obtain computational values for the H-D fractionation factor (φ), the proton chemical shift (δ H ) of the H δ1 , and changes in δ H upon isotope substitution (Δδ H-D and Δδ H-T ). For both enzymes, calculated parameters are in good agreement with available experimental data. Predictions are made for other properties for which experimental values are not available. I. Introduction The hydrogen bond (HB) is central to biomolecular structure and function. The spectroscopic characterization of HBs is a vital component of biochemistry, and an increasing amount of detailed information about HBs in biomolecules is becoming available. A good example is the N δ1 -H-O δ1 HB in the catalytic triad of serine proteases and esterases for which X-ray structures, proton chemical shifts (δ H ), changes in δ H upon deuteration (Δδ H-D ) δ H - δ D ), and H-D fractionation factors (φ) have been measured. 1,2 Recently, the first accurate measure- ments of Δδ H-T in a protein were reported for the serine protease R-chymotrypsin (Cht) complexed with three transition state analogue inhibitors. 3 The interest in the N δ1 -H-O δ1 HB was initially sparked by the unusually low-field δ H of 18 ppm first observed by Robillard and Shulman 4 in the low-pH form of Cht. On the basis of detailed studies of HBs in small organic molecules, Hibbert and Emsley 5 have suggested that a low-field δ H is consistent with a strong HB and is accompanied by a positive Δδ H-D ,a φ < 1, and 1.0 e ν H st /ν D st e 1.4 (where ν st is the vibrational stretch frequency of the hydrogen donor). Subsequently, Δδ H-D ) 1.0 ( 0.4 ppm and φ ) 0.4 were first measured for low-pH Cht and chymotrypsinogen, respectively, by Frey et al. 6 and Markley and Westler. 7 A Δδ H-D of 1.1 ( 0.5 ppm has also been measured for the Cht-N-Ac-Leu-Phe-CF 3 complex, 8 while low- field δ H and low φ’s have been measured for many other serine proteases. Due to the difficulty of assigning vibrational spectra of proteins, experimental measurements of ν H st /ν D st for serine proteases have not appeared in the literature. These measurements provide valuable benchmarks for theo- retical models of strong HBs, which in turn would provide valuable tools for the interpretation of these properties. However, the accurate ab initio predictions of these spectroscopic proper- ties for strong HBs present a significant challenge, even for very simple systems. For example, Barich, Nicholas, and Haw 9 have shown that the effect of electron correlation (at the MP2 level) on proton chemical shifts is unusually large for strong HBs. At the highest level of theory considered by Barich et al., the predicted proton chemical shift of hydrogen maleate was still 1.3 ppm larger than the experimental value. Interestingly, Garcia-Viloca et al. 10 have shown that vibrational averaging of the proton-transfer coordinate reduces the proton chemical shift by 1.6 ppm for hydrogen maleate at the RHF level of theory. Because of their use of RHF, δ H was still significantly overestimated, though the predicted Δδ H-D was in excellent agreement with experiment. Very recently, Westler, Weinhold, and Markley 11 were finally able to reproduce the proton chemical shift of hydrogen maleate (as well as FHF - , hydrogen 2,2-dimethylmalonate, and hydrogen phthalate) to within a few tenths of a ppm by including both vibrational averaging and electron correlation effects. Here, the electron correlation was treated at the B3LYP level, with an empirical scaling correction. The same methodology was applied to compute a δ H value of 18.3 ppm for an 78-atom ab initio model of complexed Cht. Though vibrational averaging could not be performed for this model due to computational expense, the value agrees well with the experimental range of 18.6- 18.9 ppm measured for various closely related inhibitors complexed to Cht. The use of a smaller (33-atom) model that allowed for vibrational averaging gave less satisfactory results. The small model of Westler et al. 11 was also used to calculate Δδ H-T and φ values at several constrained N δ1 -O δ1 distances. At the N δ1 -O δ1 distance (2.6 Å) most closely resembling the measured values (2.57-2.62 Å), the predicted values of Δδ H-T (0.39 ppm) and φ (0.50) were in good agreement with experimental values (0.63-0.68 ppm and 0.32-0.43, respec- tively), although the vibrationally averaged δ H was significantly overestimated (20.91 vs 18.61-18.95 ppm). The good agree- ment for φ is especially remarkable as only the change in zero- point energy of the stretch frequency was considered (a limitation imposed by the constraint). A previous study of Edison, Weinhold, and Markley 12 has shown that other vibra- tional modes associated with the HB, such as bends, can contribute significantly to φ in model compounds. In this paper, we use a hybrid QM/MM model (the effective fragment potential method) 13 to construct a predictive model of the N δ1 -H-O δ1 HB in the low-pH forms of Cht and R-lytic protease (Alp). The use of a hybrid approach allows for a (static) treatment of a significant portion of the protein (ca. 600 atoms) close to the active site, while retaining an (flexible) ab initio description of the active site. The low-pH form is the simplest * To whom correspondence should be addressed. E-mail: Jan- Jensen@uiowa.edu. 6226 J. Phys. Chem. B 2003, 107, 6226-6233 10.1021/jp0340663 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/29/2003