A theoretical study on nitric oxide reductase activity in a ba3-type heme-copper oxidase

The mechanism of nitric oxide reduction in a ba₃-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a₃ and is partly reduced having some nitroxyl anion character (³NO⁻), and it is thus activated toward the attack by the second NO. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to Cu_B is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the NO bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the NO bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the NO bond and form N₂O at a feasible rate. The cleavage of the NO bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.     

Reduction of nitric oxide in bacterial nitric oxide reductase--a theoretical model study

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b(3)-Fe(B) active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe(B) consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu(B) in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b(3) and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe(B) (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe(B) the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b(3) taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe(B) will form a mu-oxo bridge to heme b(3) in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable mu-oxo bridge between heme b(3) and Fe(B) is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe(B) in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.     

Reduction of nitric oxide in bacterial nitric oxide reductase—a theoretical model study

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b₃-Fe_B active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe_B consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu_B in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b₃ and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe_B (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe_B the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b₃ taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe_B will form a μ-oxo bridge to heme b₃ in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable μ-oxo bridge between heme b₃ and Fe_B is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe_B in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.     

Mechanism for N₂O generation in bacterial nitric oxide reductase: a quantum chemical study

The catalytic mechanism of reduction of NO to N(2)O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N(2)O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.     

Insight into the phosphodiesterase mechanism from combined QM/MM free energy simulations

Molecular dynamics simulations employing a combined quantum mechanical and molecular mechanical potential have been carried out to elucidate the reaction mechanism of the hydrolysis of a cyclic nucleotide cAMP substrate by phosphodiesterase 4B (PDE4B). PDE4B is a member of the PDE superfamily of enzymes that play crucial roles in cellular signal transduction. We have determined a two-dimensional potential of mean force (PMF) for the coupled phosphoryl bond cleavage and proton transfer through a general acid catalysis mechanism in PDE4B. The results indicate that the ring-opening process takes place through an S(N)2 reaction mechanism, followed by a proton transfer to stabilize the leaving group. The computed free energy of activation for the PDE4B-catalyzed cAMP hydrolysis is about 13 kcal·mol(-1) and an overall reaction free energy is about -17 kcal·mol(-1), both in accord with experimental results. In comparison with the uncatalyzed reaction in water, the enzyme PDE4B provides a strong stabilization of the transition state, lowering the free energy barrier by 14 kcal·mol(-1). We found that the proton transfer from the general acid residue His234 to the O3' oxyanion of the ribosyl leaving group lags behind the nucleophilic attack, resulting in a shallow minimum on the free energy surface. A key contributing factor to transition state stabilization is the elongation of the distance between the divalent metal ions Zn(2+) and Mg(2+) in the active site as the reaction proceeds from the Michaelis complex to the transition state.     

Probing the general base catalysis in the first step of BamHI action by computer simulations.

BamHI is a type II restriction endonuclease that catalyzes the scission of the phoshodiester bond in the GAGTCC cognate sequence in the presence of two divalent metal ions. The first step of the reaction is the preparation of water for nucleophilic attack by Glu-113, which has been proposed to abstract the proton from the attacking water molecule. Alternatively, the 3'-phosphate group to the susceptible phosphodiester bond has been suggested to play a role as the general base. The two hypotheses have been tested by computer simulations using the semiempirical protein dipoles Langevin dipoles (PDLD/S) method. Deprotonation of water by Glu-113 has been found to be less favorable by 5.7 kcal/mol than metal-catalyzed deprotonation with a concomitant proton transfer to bulk solvent. The preparation of the nucleophile by the 3'-phosphate group is less favorable by 12.3 kcal/mol. These results suggest that both the general base and the substrate-assisted mechanisms in the first step of BamHI action are less likely than the metal-catalyzed reaction. The metal ions in the active site of BamHI make the largest contributions to the reduction of the free energy of hydroxide ion formation. On the basis of these findings we propose that the first step of endonuclease catalysis does not require a general base; rather, the essential attacking nucleophile in BamHI catalytic action is stabilized by the metal ions.     

Theoretical study of the reduction of nitric oxide in an A-type flavoprotein

The mechanism for the reduction of nitric oxide to nitrous oxide and water in an A-type flavoprotein (FprA) in Moorella thermoacetica, which has been proposed to be a scavenging type of nitric oxide reductase, has been investigated using density functional theory (B3LYP). A dinitrosyl complex, [{FeNO}⁷]₂, has previously been proposed to be a key intermediate in the NO reduction catalyzed by FprA. The electrons and protons involved in the reduction were suggested to “super-reduce” the dinitrosyl intermediate to [{FeNO}⁸]₂ or the corresponding diprotonated form, [{FeNO(H)}⁸]₂. In this type of mechanism the electron and/or proton transfers will be a part of the rate-determining step. In the present study, on the other hand, a reaction mechanism is suggested in which N₂O can be formed before the protons and electrons enter the catalytic cycle. One of the irons in the diiron center is used to stabilize the formation of a hyponitrite dianion, instead of binding a second NO. Cleaving the N–O bond in the hyponitrite dianion intermediate is the rate-determining step in the proposed reaction mechanism. The barrier of 16.5 kcal mol⁻¹ is in good agreement with the barrier height of the experimental rate-determining step of 14.8 kcal mol⁻¹. The energetics of some intermediates in the “super-reduction” mechanism and the mechanism proceeding via a hyponitrite dianion are compared, favoring the latter. It is also discussed how to experimentally discriminate between the two mechanisms. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Elucidation of the mechanism of selenoprotein glutathione peroxidase (GPx)-catalyzed hydrogen peroxide reduction by two glutathione molecules: a density functional study

The mechanism of the hydrogen peroxide reduction by two molecules of glutathione catalyzed by the selenoprotein glutatione peroxidase (GPx) has been computationally studied. It has been shown that the first elementary reaction of this process, (E-SeH) + H(2)O(2) --> (E-SeOH) + H(2)O (1), proceeds via a stepwise pathway with the overall barrier of 17.1 kcal/mol, which is in good agreement with the experimental barrier of 14.9 kcal/mol. During reaction 1, the Gln83 residue has been found to play a key role as a proton acceptor, which is consistent with experiments. The second elementary reaction, (E-SeOH) + GSH --> (E-Se-SG) + HOH (2), proceeds with the barrier of 17.9 kcal/mol. The last elementary reaction, (E-Se-SG) + GSH --> (E-SeH) + GS-SG (3), is initiated with the coordination of the second glutathione molecule. The calculations clearly suggest that the amide backbone of the Gly50 residue directly participates in this reaction and the presence of two water molecules is absolutely vital for the reaction to occur. This reaction proceeds with the barrier of 21.5 kcal/mol and is suggested to be a rate-determining step of the entire GPx-catalyzed reaction H(2)O(2) + 2GSH --> GS-SG + 2H(2)O. The results discussed in the present study provide intricate details of every step of the catalytic mechanism of the GPx enzyme and are in good general agreement with experimental findings and suggestions.     

Computational delineation of the catalytic step of a high‐fidelity DNA polymerase

The Bacillus fragment, belonging to a class of high‐fidelity polymerases, demonstrates high processivity (adding ～115 bases per DNA binding event) and exceptional accuracy (1 error in 10⁶ nucleotide incorporations) during DNA replication. We present analysis of structural rearrangements and energetics just before and during the chemical step (phosphodiester bond formation) using a combination of classical molecular dynamics, mixed quantum mechanics molecular mechanics simulations, and free energy computations. We find that the reaction is associative, proceeding via the two‐metal‐ion mechanism, and requiring the proton on the terminal primer O3′ to transfer to the pyrophosphate tail of the incoming nucleotide before the formation of the pentacovalent transition state. Different protonation states for key active site residues direct the system to alternative pathways of catalysis and we estimate a free energy barrier of ～12 kcal/mol for the chemical step. We propose that the protonation of a highly conserved catalytic aspartic acid residue is essential for the high processivity demonstrated by the enzyme and suggest that global motions could be part of the reaction free energy landscape.     

A comparison of the reaction mechanisms of iron- and manganese-containing 2,3-HPCD: an important spin transition for manganese

Homoprotocatechuate (HPCA) dioxygenases are enzymes that take part in the catabolism of aromatic compounds in the environment. They use molecular oxygen to perform the ring cleavage of ortho-dihydroxylated aromatic compounds. A theoretical investigation of the catalytic cycle for HPCA 2,3-dioxygenase isolated from Brevibacterium fuscum (Bf 2,3-HPCD) was performed using hybrid DFT with the B3LYP functional, and a reaction mechanism is suggested. Models of different sizes were built from the crystal structure of the enzyme and were used in the search for intermediates and transition states. It was found that the enzyme follows a reaction pathway similar to that for other non-heme iron dioxygenases, and for the manganese-dependent analog MndD, although with different energetics. The computational results suggest that the rate-limiting step for the whole reaction of Bf 2,3-HPCD is the protonation of the activated oxygen, with an energy barrier of 17.4 kcal/mol, in good agreement with the experimental value of 16 kcal/mol obtained from the overall rate of the reaction. Surprisingly, a very low barrier was found for the O-O bond cleavage step, 11.3 kcal/mol, as compared to 21.8 kcal/mol for MndD (sextet spin state). This result motivated additional studies of the manganese-dependent enzyme. Different spin coupling between the unpaired electrons on the metal and on the evolving substrate radical was observed for the two enzymes, and therefore the quartet spin state potential energy surface of the MndD reaction was studied. The calculations show a crossing between the sextet and the quartet surfaces, and it was concluded that a spin transition occurs and determines a barrier of 14.4 kcal/mol for the O-O bond cleavage, which is found to be the rate-limiting step in MndD. Thus the two 83% identical enzymes, using different metal ions as co-factors, were found to have similar activation energies (in agreement with experiment), but different rate-limiting steps.     

Computational studies on nonenzymatic and enzymatic pyridoxal phosphate catalyzed decarboxylations of 2-aminoisobutyrate

A computational study of nonenzymatic and enzymatic pyridoxal phosphate-catalyzed decarboxylation of 2-aminoisobutyrate (AIB) is presented. Four prototropic isomers of a model aldimine between AIB and 5'-deoxypyridoxal, with acetate interacting with the pyridine nitrogen, were employed in calculations of both gas phase and water model (PM3 and PM3-SM3) decarboxylation reaction paths. Calculations employing the transition state structures obtained for the four isomers allow the demonstration of stereoelectronic effects in transition state stabilization as well as a separation of the contributions of the Schiff base and pyridine ring moieties to this stabilization. The unprotonated Schiff base contribution (approximately 16 kcal/mol) is larger than that of the pyridine ring even when it is protonated (approximately 10 kcal/mol), providing an explanation of the catalytic power of pyruvoyl-dependent amino acid decarboxylases. An active site model of dialkylglycine decarboxylase was constructed and validated, and enzymatic decarboxylation reaction paths were calculated. The reaction coordinate is shown to be complex, with proton transfer from Lys272 to the coenzyme C4' likely simultaneous with C alpha--CO(2)(-) bond cleavage. The proposed concerted decarboxylation/proton-transfer mechanism provides a simple explanation for the observed specificity of this enzyme toward oxidative decarboxylation.     

A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

Oxidation and electronic state dependence of proton transfer in the enzymatic cycle of cytochrome P450eryF

Hydrogen bond networks, consisting of hydrogen bonded waters anchored by polar/acidic amino acid sidechains, are often present in the vicinity of the oxygen binding clefts of P450s. Density functional and quantum dynamics calculations of a O(2) binding cleft network model of cytochrome P450eryF(CYP107A1) indicate that such structural motifs facilitate ultrafast proton transfer from network waters to the dioxygen of the reduced oxyferrous species via a multiple proton translocation mechanism with barriers of 7-10 kcal/mol on its doublet ground state, and that the energies of the proton transfer reactant and constrained proton transfer products have an electronic and oxidation state dependence [J. Am. Chem. Soc. 124 (2002) 1430]. In the present study, the origin of the oxidation state dependence is shown to have its roots in differential proton affinities while the electronic state dependence of the reduced oxyferrous heme has its origins in subtle differences in network topologies near the transition state of the initial proton transfer event. Relaxed potential surface scans and unconstrained proton transfer product optimizations indicate that the proton transfer product in both the singlet oxyferrous heme and the reduced oxyferrous heme species in a quartet state are not viable stable (bound) states relative to the reactant form. While the proton affinity of H(3)O(+) is sufficient for it to protonate both the oxyferrous and the reduced oxyferrous heme species, hydrogen bond network stabilized water is only capable of protonating the reduced oxyferrous form. This interpretation is substantiated by study of the NO bound reduced ferrous heme of P450nor, which is isoelectronic with the oxyferrous heme and has a similar proton affinity. Density functional calculations on a more extensive O(2) binding cleft model support the multiple proton translocation mechanism of transfer but indicates that the significant negative charge density on the bound dioxygen of the reduced oxyferrous heme species, in its doublet ground state, polarizes the associated hydrogen bond network sufficiently so as to result in short, strong, low-barrier hydrogen bonds. The computed O-H-O bond distances are less than 2.55 A and have a near degeneracy of the proton transfer reactant and initial (sudden) proton transfer products. These low-barrier hydrogen bond features, in addition to the finding of a (zero point uncorrected) barrier of 1.3 kcal/mol, indicate that proton transfer from water to the distal oxygen should be rapid, facile and may not require large curvature tunneling as originally suggested by use of a smaller model. An initial assessment of protonation of the reduced oxyferrous heme distal oxygen by a model of 6-deoxyerythronolide B (6-DEB) indicates it to be low barrier (3.8 kcal/mol) and exothermic (-2.9 kcal/mol). The combined results indicate the plausibility of simultaneous diprotonation of the distal oxygen of the reduced oxyferrous heme, leading to O-O bond scission, using the combined water network and 6-DEB substrate protonation agents.     

Molecular dynamics simulations reveal proton transfer pathways in cytochrome C-dependent nitric oxide reductase

Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666-70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb(3) oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.     

Computational evidence for the catalytic mechanism of glutaminyl cyclase. A DFT investigation

The results of a DFT theoretical investigation on the catalytic mechanism of the QC enzyme are presented. A rather large model-system is used. It includes the most important residues that are believed to play a key-role in the catalysis. The computational results show that the rate-determining step of the catalytic process is not the nucleophilic attack leading to the cycle formation (a very easy and fast process with a negligible barrier of 0.8 kcal mol(-1)), but a proton transfer, which is assisted by the Glu201 residue acting as a proton shuttle (general base and general acid). A complex network of hydrogen bonds (involving Asp248 and other residues) contribute to lower the activation barrier for the proton shift which affords the formation of an ammonia molecule bonded to the substrate. The ammonia molecule is a good leaving group which is easily expelled from the substrate in the last step of the catalytic cycle, but remains anchored to the enzyme as a ligand of the zinc cation. The metal plays a key-role in assisting the nucleophilic attack (electrostatic catalysis) since it polarizes the substrate gamma-amide carbonyl group (its electrophilic character increases). Also, the strength of the nucleophilic nitrogen (substrate alpha-amino group) is enhanced by hydrogen bonds involving the Glu201 residue. The computations outline the important role of Trp329 in helping the substrate binding process and stabilizing the cyclization transition state.     

X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases

Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9 kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1 kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants.     

Calorimetric determination of linkage effects involving an acyl-enzyme intermediate

Enthalpy changes of alpha-chymotrypsin acylation by 3-(2-furyl)acryloylimidazole (FAI) were calorimetrically determined as a function of pH. By observing the functional dependence of acylation enthalpies on buffer ionization heats, a complex pH profile was obtained describing proton release accompanying formation of acyl-enzyme. A pKa of 4.0 for FAI ionization and apparent pKa values of 6.8, 7.55 and 8.8 on the enzyme were used to account for the proton release data. A model which accounts for the proton release behavior was used to fit the acylation enthalpy data and values for the apparent dissociation enthalpies of the groups involved were obtained along with a pH-independent intrinsic enthalpy of acylation. This model suggests a group with an apparent pK = 6.8 and delta Hion = 8.7 kcal/mol which is perturbed to a pK of 7.55 and delta Hion = 7.6 kcal/mol on attachment of the acyl moiety to the enzyme. The apparent ionization enthalpy change for the active-inactive transition (pK3 = 8.8; delta H = 3.0 kcal/mol) corresponds with that calculated from the data of Fersht (J. Mol. Biol. 64 (1972) 497). The pH-independent intrinsic enthalpy of acylation (delta H = -7.9 kcal/mol) is corrected for group ionizations linked to the acylation process. Consequently, it more closely reflects molecular processes of interest such as substrate binding, covalent bond rearrangement, and product release.     

Stereoelectronic control in peptide bond formation. Ab initio calculations and speculations on the mechanism of action of serine proteases.

Ab initio molecular orbital calculations have been performed on the reaction profile for the addition/elimination reaction between ammonia and formic acid, proceeding via a tetrahedral intermediate: NH3 + HCO2H----H2NCH(OH)2----NH2CHO + H2O. Calculated transition state energies for the first addition step of the reaction revealed that a lone pair on the oxygen of the OH group, which is antiperiplanar to the attacking nitrogen, stabilized the transition state by 3.9 kcal/mol, thus supporting the hypothesis of stereoelectronic control for this reaction. In addition, a secondary, counterbalancing stereoelectronic effect stabilizes the second step, water elimination, transition state by 3.1 kcal/mol if the lone pair on the leaving water oxygen is not antiperiplanar to the C-N bond. The best conformation for the transition states was thus one with a lone pair antiperiplanar to the adjacent scissile bond and also one without a lone-pair orbital on the scissile bond oxygen or nitrogen antiperiplanar to the adjacent polar bond. The significance of these stereoelectronic effects for the mechanism of action of serine proteases is discussed.     

Water-assisted proton transfer in ferredoxin I

The role of water molecules in assisting proton transfer (PT) is investigated for the proton-pumping protein ferredoxin I (FdI) from Azotobacter vinelandii. It was shown previously that individual water molecules can stabilize between Asp(15) and the buried [3Fe-4S](0) cluster and thus can potentially act as a proton relay in transferring H(+) from the protein to the μ(2) sulfur atom. Here, we generalize molecular mechanics with proton transfer to studying proton transfer reactions in the condensed phase. Both umbrella sampling simulations and electronic structure calculations suggest that the PT Asp(15)-COOH + H(2)O + [3Fe-4S](0) → Asp(15)-COO(-) + H(2)O + [3Fe-4S](0) H(+) is concerted, and no stable intermediate hydronium ion (H(3)O(+)) is expected. The free energy difference of 11.7 kcal/mol for the forward reaction is in good agreement with the experimental value (13.3 kcal/mol). For the reverse reaction (Asp(15)-COO(-) + H(2)O + [3Fe-4S](0)H(+) → Asp(15)-COOH + H(2)O + [3Fe-4S](0)), a larger barrier than for the forward reaction is correctly predicted, but it is quantitatively overestimated (23.1 kcal/mol from simulations versus 14.1 from experiment). Possible reasons for this discrepancy are discussed. Compared with the water-assisted process (ΔE ≈ 10 kcal/mol), water-unassisted proton transfer yields a considerably higher barrier of ΔE ≈ 35 kcal/mol.     

A comparative theoretical study for the methanol dehydrogenation to CO over Pt3 and PtAu2 clusters

The density functional theory (DFT) calculations are carried out to study the mechanism details and the ensemble effect of methanol dehydrogenation over Pt(3) and PtAu(2) clusters, which present the smallest models of pure Pt clusters and bimetallic PtAu clusters. The energy diagrams are drawn out along both the initial O-H and C-H bond scission pathways via the four sequential dehydrogenation processes, respectively, i.e., CH(3)OH → CH(2)OH → CH(2)O → CHO → CO and CH(3)OH → CH(3)O → CH(2)O → CHO → CO, respectively. It is revealed that the reaction kinetics over PtAu(2) is significantly different from that over Pt(3). For the Pt(3)-mediated reaction, the C-H bond scission pathway, where an ensemble composed of two Pt atoms is required to complete methanol dehydrogenation, is energetically more favorable than the O-H bond scission pathway, and the maximum barrier along this pathway is calculated to be 12.99 kcal mol(-1). In contrast, PtAu(2) cluster facilitates the reaction starting from the O-H bond scission, where the Pt atom acts as the active center throughout each elementary step of methanol dehydrogenation, and the initial O-H bond scission with a barrier of 21.42 kcal mol(-1) is the bottom-neck step of methanol decomposition. Importantly, it is shown that the complete dehydrogenation product of methanol, CO, can more easily dissociate from PtAu(2) cluster than from Pt(3) cluster. The calculated results over the model clusters provide assistance to some extent for understanding the improved catalytic activity of bimetal PtAu catalysts toward methanol oxidation in comparison with pure Pt catalysts.