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The evaluation of ab inito QM/MM free-energy surfaces and activation free energies of reactions in enzymes and in solutions is carried out by using the empirical valence bond (EVB) method as a reference potential and then using linear response approximation (LRA). This new method evaluates the free energy associated with both the substrate and the solvent and helps in obtaining a free-energy barrier that reflects the solute entropy properly.

Microscopic calculations of free energy profiles for ion transport through biological ion channels present a very serious challenge to modern simulation approaches. The main problem is due to the major convergence problems associated with the heterogeneous landscape of the electrostatic environment in ion channels and with the need to evaluate the profile associated with the transfer of the ion from bulk water to the channel environment.

The molecular basis of proton conductance the gA channel is examined by using a full empirical valence bond (EVB) model to obtain the free energy profile and then using a simplified EVB model to obtain the time dependence of the proton transduction (PTR) process. It concludes that the barrier associated with the electrostatic energy of the transferred proton determines the rate of the PTR process.

Several strategies that allow one to perform practical ab initio QM/MM calculations of free energy profiles in solutions and proteins are presented. The performance of both approaches is examined by calculating by calculating the potential of mean force for the autodissociation reaction of water in solution.

The relative stability of Watson-Crick and mismatched dNTP template base pairs in the active site of T7 DNA polymerase (pol T7) was examined using linear-response molecular dynamics simulations. It was found that pol T7 provides a larger binding contribution to the replication fidelity than pol beta by discriminating more effectively against mismatches that are most probable to occur in the neutral anti-anti configurations.

A theoretical study of the selection of right/wrong deoxy-ribonucleotide triphosphates substrates by DNA polymerases at the initial binding step, a major component of the DNA replication fidelity is presented. Calculations are performed to predict the effects of amino acid substitutions on the fidelity for mutant forms of pol beta, thus providing a deeper understanding of the role of the polymerase active site in ensuring replication accuracy.

Research is presented using a computational approach within a Langevin dipoles solution model to determine the entropies of transfer of molecules from the gas phase to aqueous solution.

Research was conducted to examine the potentials of mean force for the association of adenine, guanine, uracil, purine, thymine and cytosine in aqueous solution using Langevin dipoles solvation model and ab initio MP2/6-31G measurements. Using the linear relationship between entropies and binding enthalpies, the entropy contributions to the free energies for hydrogen bonding and stacking were calculated. Results indicate that pure bases are more stable than their pyrimidine-containing counterparts.

Wave function propagation approaches are used to simulate isotope effects in enzymes with focus on the large H/D kinetic isotope effect of soybean lipoxygenase-1. The H/D kinetic isotope effect is evaluated as the ratio of the rate constants for hydrogen and deuterium transfer.

Several quantum mechanical/ molecular mechanics (QM/MM) free energy calculations are conducted to study the solvent effects on surroundings by an updated mean charge distribution. The approach accurately evaluates the solvation free energies and can be hence used for several other cases.

The frozen density functional (FDFT) approach is used for generating diabatic surfaces, which are used for generating a mapping potential that has taken the system from the reactant to the product state. The results have shown that the present implementation of the FDFT approach has provided a very good approach for evaluating ab initio quantum mechanical/ molecular mechanics (QM(ai)/MM ) free energy surfaces.

An investigation was carried out to explore the long-standing question of the validity of the empirical valence bond's (EVB's) key assumption that the off diagonal mixing terms are transferable between different environments. The findings reveal that at least for the test case of [S.sub.N]2 reactions, the off diagonal element does not change significantly upon moving from the gas phase to solutions and thus the EVB assumption is valid and extremely useful.

Charge induced structural changes are studied by a new approach by observing the [pK.sub.a] of the Val66Glu mutant of staphylococcal nuclease. It is shown that standard free energy perturbation (FEP) approaches give extremely disappointing results while the new approach gives realistic results and the reduced [pK.sub.a] reflects the protein structural change and water penetration process and the barrier for partial unfolding cannot be surmounted by the regular FEP approach.

Reliable studies of enzymatic reactions by combined quantum mechanics/molecular mechanics (QM/MM) approaches, with an ab initio description of the quantum region, presents a major challenge to computational chemists. The hypothetical reaction of a metaphosphate ion with water in the Ras. GAP complex was chosen, to examine the problems with energy minimization QM/MM approaches.

A multidimensional density-matrix model to explore the temperature dependence of electron transfers from the excited singlet state of the primary electron donor (P*) to the neighboring bacteriochlorophyll in photosynthetic bacterial reaction centers is presented. It is examined how the rate of electron transfer in the density matrix model depends on temperature under several different assumptions about temperature, the time constant for vibrational equilibration of the two lowest levels of a vibrational mode.

The search of the origin of the catalytic power of enzymes must involve dissection of partial energy contribution, which is accomplished by computer simulation approaches is emphasized. The catalytic proposals are examined by two examples: the popular proposal that enzymes catalyze reaction by special dynamic effects is considered and the effectiveness of simulation approaches is demonstrated.

A study is conducted to understand the quantitative relevant energetics of the catalytic reaction of cysteine proteases and the free energy surface for the corresponding reference solution reaction is determined. The calculations involve self-consistent evaluation of the solute charges in solution as \well as mapping the solution free energy surface.

A study is conducted to evaluate the solvation free energy cost associated with thymine (T) -> difluorotoluene (F), T -> cytosine (C) and C -> T transformation in aqueous solution and in a DNA duplex. The dependence of the calculated free energies on the DANA sequence, the simulation length, size of the water droplet and phosphate charges are examined.

The same Boltzmann probability law governs enzyme catalysis as other reactions in condensed phases. Simulations that actually examined the activation of enzyme catalysis indicate that the enzyme reduces the activation barrier by providing a preorganized polar environment that stabilizes the transition rate.

The structures of transport proteins have been steadily revealed in the last few decades, and yet the conversion of this information into molecular-level understanding of their function is still lagging behind. In this study, we try to elucidate how the action of the archaeal sodium/proton antiporter MjNhaP1 depends on its structure-energy relationship. To this end, we calculate the binding energies of its substrates and evaluate the conformational change barrier, focusing on the rotation of the catalytic residue D161. We find that sodium ions and protons compete against a common binding site and that the accessibility of this binding site is restricted to either the inside or outside of the cell. We suggest that the rotation of D161 χ1 angle correlates with the conformational change and is energetically unfavorable when D161 does not bind any substrate. This restriction ensures coupling between the sodium ions and the protons, allowing MjNhaP1 and probably other similar transporters to exchange substrates with minimal leak. Using Monte Carlo simulations we demonstrate the feasibility of our model. Overall we present a complete picture that reproduces the electroneutral (at 1:1 substrate ratio) and coupled transport activity of MjNhaP1 including the energetic basis for the criteria provided by Jardetzky half a century ago. [ABSTRACT FROM AUTHOR]

Although QM/MM calculations are the primary current tool for modeling enzymatic reactions, the reliability of such calculations can be limited by the size of the QM region. Thus, we examine in this work the dependence of QM/MM calculations on the size of the QM region, using the reaction of catechol-O-methyl transferase (COMT) as a test case. Our study focuses on the effect of adding residues to the QM region on the activation free energy, obtained with extensive QM/MM sampling. It is found that the sensitivity of the activation barrier to the size of the QM is rather limited, while the dependence of the reaction free energy is somewhat larger. Of course, the results depend on the inclusion of the first solvation shell in the QM regions. For example, the inclusion of the Mg2+ ion can change the activation barrier due to charge transfer effects. However, such effects can easily be included in semiempirical approaches by proper parametrization. Overall, we establish that QM/MM calculations of activation barriers of enzymatic reactions are not highly sensitive to the size of the QM region, beyond the immediate region that describes the reacting atoms. [ABSTRACT FROM AUTHOR]

The voltage sensitivity of membrane proteins is reflected in the response of the voltage sensing domains (VSDs) to the changes in membrane potential. This response is implicated in the displacement of positively charged residues, associated with the conformational changes of VSDs. The displaced charges generate nonlinear (i.e., voltage-dependent) capacitance current called the gating current (and its corresponding gating charge), which is a key experimental quantity that characterizes voltage activation in VSMP. However, the relevant theoretical/computational approaches, aimed to correlate the structural information on VSMP to electrophysiological measurements, have been rather limited, posing a broad challenge in computer simulations of VSMP. Concomitant with the development of our coarse-graining (CG) model of voltage coupling, we apply our theoretical framework for the treatments of voltage effects in membrane proteins to modeling the VSMP activation, taking the VSDs (Ci-VSD) derived from the Ciona intestinalis voltage sensitive phosphatase (Ci-VSP) as a model system. Our CG model reproduces the observed gating charge of Ci-VSD activation in several different perspectives. In particular, a new closed-form expression of the gating charge is evaluated in both nonequilibrium and equilibrium ways, while considering the fluctuation-dissipation relation that connects a nonequilibrium measurement of the gating charge to an equilibrium measurement of charge fluctuations (i.e., the voltage-independent linear component of membrane capacitance). In turn, the expression uncovers a novel link that connects an equilibrium measurement of the voltage-independent linear capacitance to a nonequilibrium measurement of the voltage-dependent nonlinear capacitance (whose integral over voltage is equal to the gating charge). In addition, our CG model yields capacitor-like voltage dependent free energy parabolas, resulting in the free energy difference and the free energy barrier for the Ci-VSD activation at "zero" (depolarization) membrane potential. Significantly, the resultant voltage dependent energetics enables a direct evaluation of capacitance-voltage relationship (C-V curve) as well as charge-voltage relationship (Q-V curve) that is in a good agreement with the observed measurement of Ci-VSD voltage activation. Importantly, an extension of our kinetic/thermodynamic model of voltage dependent activation in VSMP allows for novel derivations of voltage-dependent rate constants, whose parameters are expressed by the intrinsic properties of VSMP. These novel closed-form expressions offer a physicochemical foundation for the semiempirical Eyring-type voltage dependent rate equations that have been the cornerstone for the phenomenological (kinetic) descriptions of gating and membrane currents in the mechanistic study of ion channels and transporters. Our extended theoretical framework developed in the present study has potential implications on the roles played by gating charge fluctuations for the spike generations in nerve cells within the framework of the Hodgkin-Huxley-type model. [ABSTRACT FROM AUTHOR]

Research was conducted to examine the effect of solvent discreteness on solvation. The analysis consisted of two main components. The first used different dipole lattices as solvents while the second involved a mapping of continuum polarization onto the radial distribution function of a solute-solvent system. Results suggest that the direct effect of discreteness on solvation energy can easily exceed 20%.

The first systematic study of the free energy surfaces for the nonenzymatic hydrolysis of monomethyl phosphate in aqueous solution was conducted. In addition, this study examined the energetics of different reaction mechanisms and the structures and charge distributions of analogous reaction intermediates and transition states. Findings revealed that the associative and dissociative reaction pathways possess similar activation barriers for the hydrolysis of the mono- and dianionic methyl phosphates.

Thenature of the biological free energy scale (ΔGapp), obtained from translocon mediated insertionstudies, has been a major puzzle and the subject of major controversies.Part of the problem has been the complexity of the insertion processthat discouraged workers from considering the feasible kinetics schemesand left the possible impression that ΔGapppresents some simple partition. Here we extend and clarifyour recent analysis of the insertion problem using well-defined kineticsschemes and a free energy profile. We point out that although therate constants of some steps are far from being obvious, it is essentialto consider explicitly such schemes in order to advance in analyzingthe meaning of ΔGapp. It is thenshown that under some equilibrium conditions the kinetics scheme leadsto a simple formula that allows one to relate ΔGappto the actual free energy of partitioning betweenthe water, the membrane, and the translocon. Other options are alsoconsidered (including limits with irreversible transitions that canbe described by linear free energy relationships (LFERs)). It is concludedthat it is unlikely that a kinetics plus thermodynamic based analysiscan lead to a result that identifies ΔGappwith the partition between the membrane and the translocon.Thus, we argue that unless such analysis is presented, it is unjustifiedto assume that ΔGappcorrespondsto the membrane translocon equilibrium or to some other arbitrarydefinition. Furthermore, we point out that the presumption that itis sufficient to just calculate the PMF for going from the translocon(TR) to the membrane and then to assume irreversible diffusive motionto water and for further entrance to the membrane is not a valid analysis.Overall, we point out that it is important to try to relate ΔGappto a well-defined kinetics scheme (regardlessof the complication of the system) in order to determine whether theenergies of inserting positively charged residues to the membraneare related to the corresponding ΔGapp. It is also suggested that deviations from our simple formula forequilibrium conditions can help in identifying and analyzing kineticsbarriers. [ABSTRACT FROM AUTHOR]

The performance of the paradynamics (PD) reference potentialapproachin QM/MM calculations is examined. It is also clarified that, in contrastto some possible misunderstandings, this approach provides a rigorousstrategy for QM/MM free energy calculations. In particular, the PDapproach provides a gradual and controlled way of improving the evaluationof the free energy perturbation associated with moving from the EVBreference potential to the target QM/MM surface. This is achievedby moving from the linear response approximation to the full freeenergy perturbation approach in evaluating the free energy changes.We also present a systematic way of improving the reference potentialby using Gaussian-based correction potentials along a reaction coordinate.In parallel, we review other recent adaptations of the reference potentialapproach, emphasizing and demonstrating the advantage of using theEVB potential as a reference potential, relative to semiempiricalQM/MM molecular orbital potentials. We also compare the PD resultsto those obtained by direct calculations of the potentials of themean force (PMF). Additionally, we propose a way of accelerating thePMF calculations by using Gaussian-based negative potentials alongthe reaction coordinate (which are also used in the PD refinement).Finally, we discuss performance of the PD and the metadynamics approachesin ab initio QM/MM calculations and emphasize the advantage of usingthe PD approach. [ABSTRACT FROM AUTHOR]

Cysteine proteases play a major role in many life processes and are the target of key drugs. The reaction mechanism of these enzymes is a complex process, which involves several steps that are divided into two main groups: acylation and deacylation. In this work, we studied the energy profile for the acylation and a part of the deacylation reaction of three different enzymes, cruzain, papain, and the Q19A-mutated papain with the benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide (CBZ-FR-AMC) substrate. The calculations were performed using the EVB and PDLD/S-LRA methods. The overall agreement between the calculated and observed results is encouraging and indicates that we captured the correct reaction mechanism. Finally, our finding indicates that the minimum of the reaction profile, between the acylation and deacylation steps, should provide an excellent state for the binding of covalent inhibitors.

The COVID-19 caused by SARS-CoV-2 has spread globally and caused tremendous loss of lives and properties, and it is of utmost urgency to understand its propagation process and to find ways to slow down the epidemic. In this work, we used a coarse-grained model to calculate the binding free energy of SARS-CoV-2 or SARS-CoV to their human receptor ACE2. The investigation of the free energy contribution of the interacting residues indicates that the residues located outside the receptor binding domain are the source of the stronger binding of the novel virus. Thus, the current results suggest that the essential evolution of SARS-CoV-2 happens remotely from the binding domain at the spike protein trimeric body. Such evolution may facilitate the conformational change and the infection process that occurs after the virus is bound to ACE2. By studying the binding pattern between SARS-CoV antibody m396 and SARS-CoV-2, it is found that the remote energetic contribution is missing, which might explain the absence of cross-reactivity of such antibodies.

The proteasome is a key protease in the eukaryotic cells which is responsible for various important cellular processes such as the control of the cell cycle, immune responses, protein homeostasis, inflammation, apoptosis, and the response to proteotoxic stress. Acting as a major molecular machine for protein degradation, proteasome first identifies damaged or obsolete regulatory proteins by attaching ubiquitin chains and subsequently utilizes conserved pore loops of the heterohexameric ring of AAA+ (ATPases associated with diverse cellular activities) to pull and mechanically unfold and translocate the misfolded protein to the active site for proteolysis. A detailed knowledge of the reaction mechanism for this proteasomal proteolysis is of central importance, both for fundamental understanding and for drug discovery. The present study investigates the mechanism of the proteolysis by the proteasome with full consideration of the protein's flexibility and its impact on the reaction free energy. Major attention is paid to the role of the protein electrostatics in determining the activation barriers. The reaction mechanism is studied by considering a small artificial fluorogenic peptide substrate (Suc-LLVY-AMC) and evaluating the activation barriers and reaction free energies for the acylation and deacylation steps, by using the empirical valence bond method. Our results shed light on the proteolysis mechanism and thus should be important for further studies of the proteasome action.

Simulating the nature of voltage-activated systems is a problem of major current interest, ranging from the action of voltage-gated ion channels to energy storage batteries. However, fully microscopic converging molecular simulations of external voltage effects present a major challenge, and macroscopic models are associated with major uncertainties about the dielectric treatment and the underlying physical basis. Recently we developed a coarse-grained (CG) model that represents explicitly the electrodes, the electrolytes, and the membrane/protein system. The CG model provides a semimacroscopic way of capturing the microscopic physics of voltage-activated systems. Our method was originally validated by reproducing macroscopic and analytical results for key test cases and then used in modeling voltage-activated ion channels and related problems. In this work, we further establish the reliability of the CG voltage model by comparing it to the results of Monte Carlo (MC) simulations with a microscopic electrolyte model. The comparison explores different aspects of membrane, electrolyte, and electrode systems ranging from the Gouy-Chapman model to the determination of the electrolyte charge distribution in the solution between two electrodes (without and with a separating membrane), as well as the evaluation of gating charges. Overall the agreement is very impressive. This provides confidence in the CG model and also shows that the MC model can be used in realistic simulation of voltage activation of membrane proteins with sufficient computer time.

The ability to design effective enzymes presents a fundamental challenge in biotechnology and also in biochemistry. Unfortunately, most of the progress on this field has been accomplished by bringing the reactants to a reasonable orientation relative to each other, rather than by rational optimization of the polar preorganization of the environment, which is the most important catalytic factor. True computer based enzyme design would require the ability to evaluate the catalytic power of designed active sites. This work considers the evolution from a phosphotriesterase (with the paraoxon substrate) to arylesterase (with the 2-naphthylhexanoate (2NH) substrate) catalysis. Both the original and the evolved enzymes involve two zinc ions and their ligands, making it hard to obtain a reliable quantum mechanical description and then to obtain an effective free energy sampling. Furthermore, the options for the reaction path are quite complicated. To progress in this direction we started with DFT calculations of the energetics of different mechanistic options of cluster models and then used the results to calibrate empirical valence bond (EVB) models and to generate properly sampled free energy surfaces for different mechanisms in the enzyme. Interestingly, it is found that the catalytic effect depends on the Zn-Zn distance making the mechanistic analysis somewhat complicated. Comparing the activation barriers of paraoxon and the 2NH ester at the beginning and end of the evolutionary path reproduced the observed evolutionary trend. However, although our findings provide an advance in exploring the nature of promiscuous enzymes, they also indicate that modeling the reaction mechanism in the case of enzymes with a binuclear zinc center is far from trivial and presents a challenge for computer-aided enzyme design.

One of the greatest challenges in biotechnology and in biochemistry is the ability to design efficient enzymes. In fact, such an ability would be one of the most convincing manifestations of a full understanding of the origin of enzyme catalysis. Despite some progress on this front, most of the advances have been made by placing the reacting fragments in the proper places rather than by optimizing the preorganization of the environment, which is the key factor in enzyme catalysis. A rational improvement of the preorganization and a consistent assessment of the effectiveness of different design options require approaches capable of evaluating reliably the actual catalytic effect. In this work we examine the ability of the empirical valence bond (EVB) to reproduce the results of directed evolution improvements of the catalysis of diethyl 7-hydroxycoumarinyl by a designed mononuclear zinc metalloenzyme. Encouragingly, our study reproduced the catalytic effect obtained by directed evolution and offers a good start for further studies of this system.

Understanding the nature of the free-energy surfaces for phosphate hydrolysis is a prerequisite for understanding the corresponding key chemical reactions in biology. Here, the challenge has been to move to careful ab initio QM/MM (QM(ai)/MM) free-energy calculations, where obtaining converging results is very demanding and computationally expensive. This work describes such calculations, focusing on the free-energy surface for the hydrolysis of phosphate monoesters, paying special attention to the comparison between the one water (1W) and two water (2W) paths for the proton-transfer (PT) step. This issue has been explored before by energy minimization with implicit solvent models and by nonsystematic QM/MM energy minimization, as well as by nonsystematic free-energy mapping. However, no study has provided the needed reliable 2D (3D) surfaces that are necessary for reaching concrete conclusions. Here we report a systematic evaluation of the 2D (3D) free-energy maps for several relevant systems, comparing the results of QM(ai)/MM and QM(ai)/implicit solvent surfaces, and provide an advanced description of the relevant energetics. It is found that the 1W path for the hydrolysis of the methyl diphosphate (MDP) trianion is 6-9 kcal/mol higher than that the 2W path. This difference becomes slightly larger in the presence of the Mg(2+) ion because this ion reduces the pKa of the conjugated acid form of the phosphate oxygen that accepts the proton. Interestingly, the BLYP approach (which has been used extensively in some studies) gives a much smaller difference between the 1W and 2W activation barriers. At any rate, it is worth pointing out that the 2W transition state for the PT is not much higher that the common plateau that serves as the starting point of both the 1W and 2W PT paths. Thus, the calculated catalytic effects of proteins based on the 2W PT mechanistic model are not expected to be different from the catalytic effects predicted using the 1W PT mechanistic model, which was calibrated on the observed barrier in solution and in which the TS charge distribution was similar to the that of the plateau (as was done in all of our previous EVB studies).

The nature and mechanism of phosphate hydrolysis reactions are of great interest in view of the crucial role of these reactions in key biological processes. Although it is becoming clearer that the ultimate way of resolving mechanistic controversies must involve reliable theoretical studies, it is not widely realized that such studies cannot be performed at present by using most existing automated ways and that only careful systematic studies can lead to meaningful conclusions. The present work clarifies the above point by considering the hydrolysis of phosphate monoesters. The clarification starts by defining the actual issues that should be addressed in careful studies and by highlighting the problems with studies that ignore the need for unique mechanistic definitions (e.g., works that confuse associative and dissociative pathways). We then focus on the analysis of the proton transfer (PT) pathways in phosphate hydrolysis and on recent suggestions that PT involves more than one water molecule. Here we point out that most of the studies that found a proton transfer through several water molecules have not involved a sufficient systematic search of the relevant reaction coordinates. This includes both energy minimization approaches as well as a recent metadynamics (MTD) simulation study. To illustrate the crucial need of exploring the potential surfaces reliably, rather than relying on automated approaches, we present here a very careful study of the free energy landscape along a 3D reaction coordinate (RC) exploring both the standard 2D RC, comprised of the attacking and leaving group reaction coordinates, as well as of the proton transfer (PT) coordinate. Our study points out that QM/MM minimization or MTD studies that concluded that the hydrolysis of phosphate monoesters involves a PT through several water molecules, have not explored carefully the single water (1W) path (that involves a direct PT form the attacking water molecule to the phosphate oxygen). Furthermore, we identified the most likely reason for the difficulty in finding the 1W path by QM/MM minimization methods, as well as by the current MTD simulations. We also discuss the problems with current studies that challenge the phosphate as a base mechanism and emphasize that all recent studies found associative/concerted paths (although many have not realized the meaning of their results). Finally, although we clearly do not have the last word about the 1W versus 2W paths we believe that we illustrated that the crucial mechanistic problems with alternative pathways should not be resolved by just running black box search approaches.

The development of enzyme mimetic catalysts as well as the analysis of the catalytic effects of such catalysts has been a major challenge for synthetic chemists. One of the impressive examples of artificial catalysts has been the development of a highly charged host compound that provides a significant acceleration to the hydrolysis of orthoformates and other systems. However, the origin of the catalytic effect has not been quantified, and its origin remains somewhat unclear. The understanding of the corresponding supramolecular catalysis has thus become a major challenge, both in terms of computational modeling and in terms of the analysis of the corresponding acid-catalyzed reaction. Here we present a computer simulation study and kinetic analyses that reproduce the experimentally observed catalytic effect, establishing that this effect is due to electrostatic stabilization of the positively charged transition state (relative to the uncharged bound complex). Our study illustrates the crucial need for careful analysis of the complex kinetics of the catalytic effect and the host system, as well as the need for computational modeling in analyzing the catalytic effect and in the potential design of better catalysts. Finally, our finding of the large stabilization of the bound H(3)O(+) points out the very low "local pH" inside the host system even when the solvent is kept at a high pH.

Reliable studies of proton transfer (PT) reactions in solution and in enzymes by combined quantum mechanical/molecular mechanics (QM/MM) approaches with an ab initio description of the quantum region present a major challenge to computational chemists. The main problem is the need for extensive computer time to evaluate the QM energy, which in turn makes it extremely challenging to perform proper configurational sampling. The present work presents a new effective way for performing such calculations by using the frozen density functional (FDFT) approach to generate diabatic surfaces that are used to generate a mapping potential that takes the system from the reactant to the product state. The resulting umbrella sampling/free energy perturbation (US/FEP) mapping is done in full analogy with the approach used in the empirical valence bond (EVB) treatment, moving from the diabatic mapping potential to the adiabatic ground state surface, while an ab initio Hamiltonian is used for the QM part. The present approach provides a particularly effective way for evaluating the free energy associated with both the substrate and the solvent motions. This allows us to obtain a free energy barrier that properly reflects the solute entropy. This advance allows one to obtain ab initio QM/MM (QM(ai)/MM) free energy surfaces for very challenging cases such as the autodissociation of water in water, proton transfer between methanol and water in water, and the effect of Mg2+ ion on such a reaction. We also consider as a benchmark the initial PT reaction in the catalytic cycle of triose phosphate isomerase and obtain excellent results without any adjustable parameters. Our results point out that the present implementation of the FDFT approach provides a very promising approach for evaluating QM(ai)/MM free energy surfaces.