The design, synthesis and evaluation of high affinity macrocyclic carbohydrate inhibitors

Carbohydrate-protein interactions have been investigated for a model system of a monoclonal antibody, SYA/J6, which binds a trisaccharide epitope of the O-polysaccharide of the Shigella flexneri variant Y lipopolysaccharide. The thermodynamics of binding for the methyl glycoside of the native trisaccharide epitope, Rha-Rha-GlcNAc () to SYA/J6 over a range of temperatures exhibits strong, linear enthalpy-entropy compensation and a negative heat capacity change (DeltaC(p)=-152 cal mol(-1) degree(-1)). At 293 K the free energy of association is the sum of favourable enthalpy and entropy contributions (DeltaH=-3.9 kcal mol(-1) and -TDeltaS=-2.9 kcal mol(-1)). Crystal structures for SYA/J6 Fab detailed the position of the native trisaccharide epitope, Rha-Rha-GlcNAc, and facilitated a strategy to design a tighter binding, low molecular weight ligand. This involved pre-organization of the native trisaccharide in its bound conformation by addition of intramolecular constraints (a beta-alanyl or glycinyl tether). ELISA measurements indicated that the glycinyl tethered trisaccharide was not an optimal candidate for further analysis, while microcalorimetry provided data showing that the beta-alanyl tethered trisaccharide displayed a 15-fold increase in affinity for SYA/J6. Tethering resulted in a favourable entropic contribution to binding, relative to the native trisaccharide (-TDeltaDeltaS=-1.2 kcal mol(-1)). Potential energy and dynamics calculations using the AMBER Plus force fields indicated that trisaccharide adopted a rigid conformation similar to that of the bound conformation of the native trisaccharide epitope. While this strategy resulted in modest free energy gains by minimizing losses due to conformational entropy, thermodynamic data are consistent with significant contributions from solvent reorganization.     

Thermodynamics of buried water clusters at a protein-ligand binding interface

The structure of the complex of cyclophilin A (CypA) with cyclosporin A (CsA, 1) shows a cluster of four water molecules buried at the binding interface, which is rearranged when CsA is replaced by (5-hydroxynorvaline)-2-cyclosporin (2). The thermodynamic contributions of each bound water molecule in the two complexes are explored with the inhomogeneous fluid solvation theory and molecular dynamics simulations. Water (WTR) 133 in complex 1 contributes little to the binding affinity, while WTR6 and 7 in complex 2 play an essential role in mediating protein-ligand binding with a hydrogen bond network. The calculations reveal that the rearrangement of the water molecules contributes favorably to the binding affinity, even though one of them is displaced going from ligand 1 to 2. Another favorable contribution comes from the larger protein-ligand interactions of ligand 2. However, these favorable contributions are not sufficient to overcome the unfavorable desolvation free energy change and the conformational entropy of the hydroxylpropyl group of ligand 2 in the complex, leading to a lower binding affinity of ligand 2. These physical insights may be useful in the development of improved scoring functions for binding affinity prediction.     

Involvement of water in carbohydrate-protein binding.

The interactions of trimannosides 1 and 2 with Con A were studied to reveal the effects of displacement of well-ordered water molecules on the thermodynamic parameters of protein-ligand complexation. Trisaccharide 2 is a derivative of 1, in which the hydroxyl at C-2 of the central mannose unit is replaced by a hydroxyethyl moiety. Upon binding, this moiety displaces a conserved water molecule present in the Con A binding site. Structural studies by NMR spectroscopy and MD simulations showed that the two compounds have very similar solution conformational properties. MD simulations of the complexes of Con A with 1 and 2 demonstrated that the hydroxyethyl side chain of 2 can establish the same hydrogen bonds in a low energy conformation with the protein binding site as those mediated by the water molecule in the complex of 1 with Con A. Isothermal titration microcalorimetry (ITC) measurements showed that 2 has a more favorable entropy of binding compared to 1. This term, which was expected, arises from the return of the highly ordered water molecule to bulk solution. The favorable entropy term was, however, offset by a relatively large unfavorable enthalpy term. This observation was rationalized by comparing the extent of hydrogen bond and solvation changes during binding. It is proposed that an indirect interaction through a water molecule will provide a larger number of hydrogen bonds in the complex that have higher occupancies than in bulk solution, thereby stabilizing the complex.     

Role of structural water molecule in HIV protease-inhibitor complexes: a QM/MM study

Structural water molecule 301 found at the interface of HIV protease-inhibitor complexes function as a hydrogen bond (H-bond) donor to carbonyl groups of the inhibitor as well as H-bond acceptor to amide/amine groups of the flap region of the protease. In this study, six systems of HIV protease-inhibitor complexes were analyzed, which have the presence of this "conserved" structural water molecule using a two-layer QM/MM ONIOM method. The combination of QM/MM and QM method enabled the calculation of strain energies of the bound ligands as well as the determination of their binding energies in the ligand-water and ligand-water-protease complexes. Although the ligand experiences considerable strain in the protein bound structure, the H-bond interactions through the structural water overcomes this strain effect to give a net stability in the range of 16-24 kcal/mol. For instance, in 1HIV system, the strain energy of the ligand was 12.2 kcal/mol, whereas the binding energy associated with the structural water molecule was 20.8 kcal/mol. In most of the cases, the calculated binding energy of structural water molecule showed the same trend as that of the experimental binding free energy values. Further, the classical MD simulations carried out on 1HVL system with and without structural water 301 showed that this conserved water molecule enhances the H-bond dynamics occurring at the Asp-bound active site region of the protease-inhibitor system, and therefore it will have a direct influence on the mechanism of drug action.     

Structural properties of water: comparison of the SPC, SPCE, TIP4P, and TIP5P models of water

Molecular-dynamics simulations were carried out for the SPC, SPCE, TIP4P, and TIP5P models of water at 298 K. From these results we determine the following quantities: the absolute entropy using the two-particle approximation, the mean lifetime of the hydrogen bond, the mean number of hydrogen bonds per molecule, and the mean energy of the hydrogen bond. From the entropy calculations we find that nearly all contributions to the total entropy originates from the orientation effects. Moreover, we determine the contributions to the total entropy which originate from the first, second, and higher solvation shells. It is interesting that the limits between solvation shells are clearly visible. The first solvation shell (0.22 < r < 0.36 nm) contributes approximately 43 J mol K to the total entropy; the second solvation shell (0.36 < r < 0.60 nm) contributes approximately 12 J mol K, while contributions from the third and other solvation shells are very small, approximately 2 J mol K in summary. This indicates that water molecules are strongly ordered up to 0.55-0.6 nm around the central water molecule, and beyond this limit the ordering diminishes. The results of calculations (entropy and hydrogen bonds) are compared with the experimental data for the choosing of the best water model. We find that the SPC and TIP4P models reproduce the best experimental values, and we recommend these models for computer simulations of the aqueous solution of biomolecules.     

WATsite: Hydration site prediction program with PyMOL interface

Water molecules that mediate protein–ligand interactions or are released from the binding site on ligand binding can contribute both enthalpically and entropically to the free energy of ligand binding. To elucidate the thermodynamic profile of individual water molecules and their potential contribution to ligand binding, a hydration site analysis program WATsite was developed together with an easy‐to‐use graphical user interface based on PyMOL. WATsite identifies hydration sites from a molecular dynamics simulation trajectory with explicit water molecules. The free energy profile of each hydration site is estimated by computing the enthalpy and entropy of the water molecule occupying a hydration site throughout the simulation. The results of the hydration site analysis can be displayed in PyMOL. A key feature of WATsite is that it is able to estimate the protein desolvation free energy for any user specified ligand. The WATsite program and its PyMOL plugin are available free of charge from http://people.pnhs.purdue.edu/~mlill/software . © 2014 Wiley Periodicals, Inc.     

Molecular recognition of amines and amino esters by zinc porphyrin receptors: binding mechanisms and solvent effects

Zinc porphyrin receptors bearing 12 ester groups in the meso phenyl groups (1-3) were prepared, and binding of amines and alpha-amino esters was studied with emphasis on the binding mechanisms. The X-ray crystallographic analysis of 5,10,15,20-tetrakis(2, 6-bis(carbomethoxymethoxy)-4-carbomethoxyphenyl)porphyrin (free base of 1) showed that the receptor has a binding pocket above the porphyrin plane. UV-visible titration experiments revealed that the zinc porphyrin receptors bound amines and alpha-amino esters with binding constants (K(a)) ranging from 0.5 to 52 700 M(-1) in CH(2)Cl(2) at 25 degrees C. The ester functional groups of 1 assisted the binding of aromatic alpha-amino esters (K(a) = 8 000-23 000 M(-1) in CH(2)Cl(2) at 25 degrees C) and inhibited the binding of bulky aliphatic alpha-amino esters (K(a) = 460 M(-1) for Leu-OMe in CH(2)Cl(2) at 25 degrees C), indicating that CH-pi type interactions and steric repulsions control the selectivity. The binding of amines and alpha-amino esters was tight both in a nonpolar solvent (CH(2)Cl(2)) and in a polar solvent (water) but loose in a solvent of intermediate polarity (H(2)O-MeOH (1:1)), demonstrating that two competitive driving forces are operating: (1) attractive electrostatic forces between host and guest such as coordination of the amino group to the zinc atom, and (2) entropic forces stemming from desolvation as well as enthalpic forces due to the host-guest dispersion forces. The former forces drive the binding in CH(2)Cl(2) while the latter forces drive the binding in water. The enthalpy changes in the binding in CH(2)Cl(2) and those in water range from -50 to -30 kJ mol(-1) and from -35 to 0 kJ mol(-1), respectively. The entropy changes in CH(2)Cl(2) and those in water range from -120 to -60 J K(-1) mol(-1) and from -50 to +60 J K(-1) mol(-1), respectively. Thus the binding in CH(2)Cl(2) is characterized by large negative enthalpy changes, while that in water by less negative entropy changes. These thermodynamic parameters also indicate that host-guest polar interactions (enthalpic forces) drive the binding in CH(2)Cl(2) while both host-guest dispersion interactions (an enthalpic force) and desolvation (an entropic force) drive the binding in water. Enthalpy-entropy compensation observed for the binding in water indicates that the binding of amines and amino esters in water by zinc porphyrins is associated with conformational changes as well as a high degree of dehydration. In CH(2)Cl(2), no clear compensation was observed, consistent with the mechanism that neither desolvation processes nor conformational changes contribute significantly to the binding energetics.     

Quantifying the role of water in protein-carbohydrate interactions

Water-mediated interactions play a key role in carbohydrate-lectin binding, where the interactions involve a conserved water that is separated from the bulk solvent and present a bridge between the side chains of the protein and the carbohydrate ligand. To apply quantum mechanical methods to examine the role of conserved waters, we present an analysis in which the relevant carbohydrate atoms are modeled by methanol, and in which the protein is replaced by a limited number of amino acid side chains. Clusters containing a conserved water and a representative amino acid fragment were also examined to determine the influence of amino acid side chains on interaction energies. To quantify the differential binding energies of methanol versus water, quantum mechanical calculations were performed at the B3LYP/6-311++G(3df,3pd)//B3LYP/6-31+G(d) level in which either a methanol molecule was bound to the conserved water (liganded state) or in which a water molecule replaces the methanol (unliganded state). Not surprisingly, the binding of a water to clusters containing charged amino acid side chains was more favorable by 1.55 to 7.23 kcal/mol than that for the binding of a water to the corresponding pure water clusters. In contrast, the binding energy of water to clusters containing polar-uncharged amino acid side chains ranged from 4.35 kcal/mol less favorable to 4.72 kcal/mol more favorable than for binding to the analogous pure water clusters. The overall trend for the binding of methanol versus water, in any of the clusters, favored methanol by an average value of 1.05 kcal/mol. To extend these studies to a complex between a protein (Concanavalin A) and its carbohydrate ligand, a cluster was examined that contained the side chains of three key amino acids, namely asparagine, aspartate, and arginine, as well as a key water molecule, arranged as in the X-ray diffraction structure of Con A. Again, using methanol as a model for the endogenous carbohydrate ligand, energies of -5.94 kcal/mol and -5.70 kcal/mol were obtained for the binding of methanol and water, respectively, to the Con A-water cluster. The extent to which cooperativity enhanced the binding energies has been quantified in terms of nonadditive three-body contributions. In general, the binding of water or methanol to neutral dimers formed cooperative clusters; in contrast, the cooperativity in charged clusters depended on the overall geometry as well as the charge.     

Computation of relative binding free energy for an inhibitor and its analogs binding with Erk kinase using thermodynamic integration MD simulation

In the present study, we carried out thermodynamic integration molecular dynamics simulation for a pair of analogous inhibitors binding with Erk kinase to investigate how computation performs in reproducing the relative binding free energy. The computation with BCC-AM1 charges for ligands gave ?1.1?kcal/mol, deviated from experimental value of ?2.3?kcal/mol by 1.2?kcal/mol, in good agreement with experimental result. The error of computed value was estimated to be 0.5?kcal/mol. To obtain convergence, switching vdw interaction on and off required approximately 10 times more CPU time than switching charges. Residue-based contributions and hydrogen bonding were analyzed and discussed. Furthermore, subsequent simulation using RESP charge for ligand gave ΔΔG of ?1.6?kcal/mol. The computed results are better than the result of ?5.6?kcal/mol estimated using PBSA method in a previous study. Based on these results, we further carried out computations to predict ΔΔG for five new analogs, focusing on placing polar and nonpolar functional groups at the meta site of benzene ring shown in the Fig.?1, to see if these ligands have better binding affinity than the above ligands. The computations resulted that a ligand with polar –OH group has better binding affinity than the previous examined ligand by ~2.0?kcal/mol and two other ligands have better affinity by ~1.0?kcal/mol. The predicted better inhibitors of this kind should be of interest to experimentalist for future experimental enzyme and/or cell assays.     

Implicit solvent simulations of DPC micelle formation

The formation of micelles by dodecylphosphocholine (DPC) is modeled by treating the surfactants in atomic detail and the solvent implicitly, in the spirit of the EEF1 solvation model for proteins. The solvation parameters of the DPC atoms are carried over from those of similar atoms in proteins. A slight adjustment of the parameters for the headgroup was found necessary for obtaining an aggregation number consistent with experiment. Molecular dynamics simulations of 960 DPC molecules at different concentrations are used to obtain the aggregation number, the micelle size distribution, and the CMC. At 20 mM concentration we obtain an aggregation number of 53-56 and a CMC of 1.25 mM, values close to the experimental ones. At 100 mM the aggregation number increases to 90. Simulations of individual micelles of varying size show that the effective energy per surfactant molecule is initially a decreasing function of aggregation number but stabilizes at about 60 molecules. The van der Waals term and the desolvation of nonpolar groups contribute to micellization, whereas the desolvation of polar groups opposes it. From the difference between the effective energy and the free energy (calculated from the CMC), the translational and rotational entropy contributions to the free energy are estimated at about 7 kcal/mol per monomer. The micelles obtained here are more irregular than those obtained in explicit water simulations. This modeling approach allows the study of larger surfactant aggregates for longer times and the extraction of thermodynamic in addition to structural information.     

Calculations of protein-ligand binding entropy of relative and overall molecular motions

In the context of virtual database screening, calculations of protein-ligand binding entropy of relative and overall molecular motions are challenging, owing to the inherent structural complexity of the ligand binding well in the energy landscape of protein-ligand interactions and computing time limitations. We describe a fast statistical thermodynamic method for estimation the binding entropy to address the challenges. The method is based on the integration of the configurational integral over clusters obtained from multiple docked positions. We apply the method in conjunction with 11 popular scoring functions (AutoDock, ChemScore, DrugScore, D-Score, F-Score, G-Score, LigScore, LUDI, PLP, PMF, X-Score) to evaluate the binding entropy of 100 protein-ligand complexes. The averaged values of binding entropy contribution vary from 6.2 to 9.1 kcal/mol, showing good agreement with literature. We calculate positional sizes and the angular volume of the native ligand wells. The averaged geometric mean of positional sizes in principal directions varies from 0.8 to 1.4 A. The calculated range of angular volumes is 3.3-11.8 rad(2). Then we demonstrate that the averaged six-dimensional volume of the native well is larger than the volume of the most populated non-native well in energy landscapes described by all of 11 scoring functions.     

An Artificial Receptor for Dimethyl Aspartate

[trans-5,15-Bis(2,7-dihydroxy-1-naphthyl)-2,3,7,8,12,13,17,18-octaethylporphyrinato]zinc(II) (1), a trifunctionalized porphyrin host, was prepared as a receptor for amino acid derivatives, particularly those having a hydrogen-bonding site in the side chain. The free energy changes for the binding of Leu-OMe, Asp-OMe, and Glu-OMe to 1 were -5.8 kcal/mol, -6.6 kcal/mol, and -5.9 kcal/mol, showing a selectivity for Asp-OMe. (1)H NMR titration experiments indicated that three simultaneous attractive interactions, one coordination interaction, and two hydrogen-bonding interactions, are operating in the host-guest complex. The preference for Asp-OMe over Glu-OMe was found to originate from the favorable enthalpy term for Asp-OMe. The free energy change, the enthalpy change, and the entropy change were determined and split into contributions arising from coordination interaction and from hydrogen-bonding interactions by use of reference hosts. Comparison of enthalpy and entropy changes suggests that the host-guest complex becomes more ordered as the number of recognition pairs increases.     

Thermodynamic contributions of the ordered water molecule in HIV-1 protease

Binding between biomolecules is usually accompanied by the formation of direct interactions with displacement of water from the binding sites. In some cases, however, the interactions are mediated by ordered water molecules, whose effect on binding affinity and the other thermodynamic functions is unclear. In this work, we compute the contribution of one such water molecule, the strongly bound water molecule at the binding site of HIV-1 protease, to the thermodynamic properties using statistical mechanical formulas for the energy and entropy. The requisite correlation functions are obtained by molecular dynamics simulations. We find that the entropic penalty of ordering is large but is outweighed by the favorable water-protein interactions. We also find a large negative contribution from this water molecule to the heat capacity. This approach could be useful in rational drug design by estimating which bound water molecules would be most favorable to displace.     

Binding free energy, energy and entropy calculations using simple model systems

Free energy differences are calculated for a set of two model host molecules, binding acetone and methanol. Two active sites of different characteristics were constructed based on an artificially extended C60 fullerene molecule, possibly functionalised to include polar interactions in an otherwise apolar, spherical cavity. The model host systems minimise the necessary sampling of conformational space while still capturing key aspects of ligand binding. The estimates of the free energies are split up into energetic and entropic contributions, using three different approaches investigating the convergence behaviour. For these systems, a direct calculation of the total energy and entropy is more efficient than calculating the entropy from the temperature dependence of the free energy or from a direct thermodynamic integration formulation. Furthermore, the compensating surrounding–surrounding energies and entropies are split off by calculating reduced ligand-surrounding energies and entropies. These converge much more readily and lead to properties that are more straightforwardly interpreted in terms of molecular interactions and configurations. Even though not experimentally accessible, the reduced thermodynamic properties may prove highly relevant for computational drug design, as they may give direct insights into possibilities to further optimise ligand binding while optimisation in the surrounding–surrounding energy or entropy will exactly cancel and not lead to improved affinity.     

Thermodynamic profiles for CO photodissociation from heme model compounds: effect of proximal ligands

Here we present a comprehensive study of the thermodynamic parameters (enthalpy, entropy, and volume changes) associated with carbon monoxide photodissociation and rebinding to Fe(II) microperoxidase-11 (Fe(II)MP11) and Fe(ll) tetrakis(4-sulfonatophenyl)porphine complex (FeII4SP) with water and 2-methylimidazole as proximal ligands. CO photodissociation from FeII4SP complexes is accompanied by a positive volume change of approximately 17 mL mol(-1). A smaller volume change of approximately 12 mL mol(-1) was observed for CO dissociation from Fe(II)MP-11. We attribute the positive volume change to cleavage of the Fe-CO covalent bond and to solvent reorganization due to the low-spin to high-spin transition. CO binding is an exothermic reaction with an enthalpy change of -17 kcal mol(-1) for the CO-FeII4SP complexes and -13 kcal mol(-1) for the CO-Fe(II)MP11 complex. In all cases, the ligand recombination occurs as a single-exponential process indicating that CO dissociation is followed by direct CO rebinding to a high-spin five-coordinate complex without concomitant dissociation of the proximal base. In addition, observed negative activation entropies and volumes for ligand binding to (2-Melm)FeII4SP and MP-11, respectively, suggest that CO rebinding can be described by an associative mechanism with bond formation being the rate-limiting step.     

Entropic control of the relative stability of triple-helical collagen peptide models

Herein, we show that current methodologies in atomistic simulations can yield reliable standard free energy values in aqueous solution for the transition from the dissociated monomeric form to the triple-helix state of collagen model peptides. The calculations are performed on a prototypical highly stable triple-helical peptide, [(Pro-Hyp-Gly)10]3 (POG10), and on the so-called T3-785 triple-helix mimicking a fragment from the type III human collagen, which is more thermally labile. On the basis of extensive MD simulations in explicit solvent followed by molecular-mechanical and electrostatic Poisson-Boltzmann calculations complemented with an accurate estimation of the nonpolar contributions to solvation, the computed free energy change for the aggregation processes of the POG10 and T3-785 peptides leading to their triple-helices is -6.6 and -6.1 kcal/mol, respectively. For POG10, this value is in agreement with differential scanning calorimetric data. However, it is shown that conformational entropy, which is estimated by means of an expansion of mutual information functions, preferentially destabilizes the triple-helical state of T3-785 by around 4.6 kcal/mol, thus explaining its lower thermal stability. Altogether, our computational results allow us to ascertain, for the first time, the actual thermodynamic forces controlling the absolute and relative stability of collagen model peptides.     

Hydrogen bonding of the nucleobase mimic 2-pyridone to fluorobenzenes: an ab initio investigation

The hydrogen-bonded complexes of the nucleobase mimic 2-pyridone (2PY) with seven different fluorinated benzenes (1-, 1,2-, 1,4-, 1,2,3-, 1,3,5-, 1,2,3,4-, and 1,2,4,5-fluorobenzene) are important model systems for investigating the relative importance of hydrogen bonding versus pi-stacking interactions in DNA. We have shown by supersonic-jet spectroscopy that these dimers are hydrogen bonded and not pi-stacked at low temperature (Leist, R.; Frey, J. A.; Leutwyler, S. J. Phys. Chem. A 2006, 110, 4180). Their geometries and binding energies D(e) were calculated using the resolution of identity (RI) M?ller-Plesset second-order perturbation theory method (RIMP2). The most stable dimers are bound by antiparallel N-H...F-C and C-H...O=C hydrogen bonds. The binding energies are extrapolated to the complete basis set (CBS) limit, , using the aug-cc-pVXZ basis set series. The CBS binding energies range from -D(e,CBS) = 6.4-6.9 kcal/mol and the respective dissociation energies from -D(0,CBS) = 5.9-6.3 kcal/mol. In combination with experiment, the latter represent upper limits to the dissociation energies of the pi-stacked isomers (which are not observed experimentally). The individual C-H...O=C and N-H...F-C contributions to D(e) can be approximately separated. They are nearly equal for 2PY.fluorobenzene; each additional F atom strengthens the C-H...O=C hydrogen bond by approximately 0.5 kcal/mol and weakens the C-F...H-N hydrogen bond by approximately 0.3 kcal/mol. The single H-bond strengths and lengths correlate with the gas-phase acid-base properties of the C-H and C-F groups of the fluorobenzenes.     

Absolute hydration free energy scale for alkali and halide ions established from simulations with a polarizable force field

A polarizable potential function for the hydration of alkali and halide ions is developed on the basis of the recent SWM4-DP water model [Lamoureux, G.; MacKerell, A. D., Jr.; Roux, B. J. Chem. Phys. 2003, 119, 5185]. Induced polarization is incorporated using classical Drude oscillators that are treated as auxiliary dynamical degrees of freedom. The ions are represented as polarizable Lennard-Jones centers, whose parameters are optimized to reproduce the binding energies of gas-phase monohydrates and the hydration free energies in the bulk liquid. Systematic exploration of the parameters shows that the monohydrate binding energies can be consistent with a unique hydration free energy scale if the computed hydration free energies incorporate the contribution from the air/water interfacial electrostatic potential (-540 mV for SWM4-DP). The final model, which can satisfyingly reproduce both gas and bulk-phase properties, corresponds to an absolute scale in which the intrinsic hydration free energy of the proton is -247 kcal/mol.     

The effect of heme environment on the hydrogen abstraction reaction of camphor in P450cam catalysis: a QM/MM study

The discrepancies between the published QM/MM studies (Sch?neboom, J. C.; Cohen, S.; Lin, H.; Shaik, S.; Thiel, W. J. Am. Chem. Soc. 2004, 126, 4017; Guallar, V.; Friesner, R. A. J. Am. Chem. Soc. 2004, 126, 8501) on H-abstraction of camphor in P450cam have largely been resolved. The crystallographic water molecule 903 situated near the oxo atom of Compound I acts as a catalyst for H-abstraction, lowering the barrier by about 4 kcal/mol. Spin density at the A-propionate side chain of heme can occur in the case of incomplete screening but has no major effect on the computed barrier.