•Calculation of the Markovnikov transition state for H2O and HX (X = F, Cl, Br).•Validation of transition state using intrinsic reaction coordinate and vibrational frequency.•Quantitative agreement of transition state energy based on partition function.•Dipolar nature of transition state validated using Hirshfeld charge and Fukui function.•Explanation of Markovnikov regioselectivity for H2O based on dipolar transition state.The properties of the transition states for the electrophilic addition reactions of four molecules of the type HX (X = Br, Cl, F and OH) to 1-propene form 2-X propane by the Markovnikov mechanism have been calculated using density functional theory (DFT). A comparative study of the transition state along the reaction path for both M and AM addition was studied in order to understand the origins of regioselectivity of H2O. The quadrapolar nature of the transition state is arises because of a sequential mechanism, in which the addition across the π-bond occurs in two steps, first H+ and then X−. The M reaction mechanism is consistent with a H+X− dipole which induces an oppositely polarized C1−C2+ dipole in the transition state, resulting in a quadrupole. The C1−C2+ dipole in the M mechanism is consistently larger than the C1+C2− dipole of the AM transition state for all species studied.

•Use of steered molecular dynamics to determine molecular binding trajectory in a protein•Comparison of umbrella sampling and jarzynski equality methods for validation•Systematic test of effect of restraints on calculated binding free energy•Comparison of calculated binding free energy with experiment in dehaloperoxidase•General method applicable to full range of substrates and inhibitors that bind to dehaloperoxidaseThe binding free energy of 4-bromophenol (4-BP), an inhibitor that binds in the internal binding site in dehaloperoxidase-hemoglobin (DHP) was calculated using Molecular Dynamics (MD) methods combined with pulling or umbrella sampling. The effects of systematic changes in the pulling speed, pulling force constant and restraint force constant on the calculated potential of mean force (PMF) are presented in this study. The PMFs calculated using steered molecular dynamics (SMD) were validated by umbrella sampling (US) in the strongly restrained regime. A series of restraint force constants ranging from 1000 down to 5 kJ/(mol nm2) were used in SMD simulations. This range was validated using US, however noting that weaker restraints give rise to a broader sampling of configurations. This comparison was further tested by a pulling simulation conducted without any restraints, which was observed to have a value closest to the experimentally measured free energy for binding of 4-BP to DHP based on ultraviolet–visible (UV–vis) and resonance Raman spectroscopies. The protein-inhibitor system is well suited for fundamental study of free energy calculations because the DHP protein is relatively small and the inhibitor is quite rigid. Simulation configuration structures are compared to the X-ray crystallography structures of the binding site of 4-BP in the distal pocket above the heme.

The application of fluoride anion as a probe for investigating the internal substrate binding has been developed and applied to dehaloperoxidase–hemoglobin (DHP) from Amphitrite ornata. By applying the fluoride titration strategy using UV–vis spectroscopy, we have studied series of halogenated phenols, other substituted phenols, halogenated indoles, and several natural amino acids that bind internally (and noncovalently) in the distal binding pocket of the heme. This approach has identified 2,4-dibromophenol (2,4-DBP) as the tightest binding substrate discovered thus far, with approximately 20-fold tighter binding affinity than that of 4-bromophenol (4-BP), a known internally binding inhibitor in DHP. Combined with resonance Raman spectroscopy, we have confirmed that competitive binding equilibria exist between fluoride anion and internally bound molecules. We have further investigated the hydrogen bonding network of the active site of DHP that stabilizes the exogenous fluoride ligand. These measurements demonstrate a general method for determination of differences in substrate binding affinity based on detection of a competitive fluoride binding equilibrium. The significance of the binding that 2,4-dibromophenol binds more tightly than any other substrate is evident when the structural and mechanistic data are taken into consideration.

The time-resolved kinetics of substrate oxidation and cosubstrate H2O2 reduction by dehaloperoxidase-hemoglobin (DHP) on a seconds-to-minutes time scale was analyzed for peroxidase substrates 2,4,6-tribromophenol (2,4,6-TBP), 2,4,6-trichlorophenol (2,4,6-TCP), and ABTS. Substrates 2,4,6-TBP and 2,4,6-TCP show substrate inhibition at high concentration due to the internal binding at the distal pocket of DHP, whereas ABTS does not show substrate inhibition at any concentration. The data are consistent with an external binding site for the substrates with an internal substrate inhibitor binding site for 2,4,6-TBP and 2,4,6-TCP. We have also compared the kinetic behavior of horseradish peroxidase (HRP) in terms of kcat, KmAH2 and KmH2O2 using the same kinetic scheme. Unlike DHP, HRP does not exhibit any measurable substrate inhibition, consistent with substrate binding at the edge of heme near the protein surface at all substrate concentrations. The binding of substrates and their interactions with the heme iron were further compared between DHP and HRP using a competitive fluoride binding experiment, which provides a method for quantitative measurement of internal association constants associated with substrate inhibition. These experiments show the regulatory role of an internal substrate binding site in DHP from both a kinetic and competitive ligand binding perspective. The interaction of DHP with substrates as a result of internal binding actually stabilizes that protein and permits DHP to function under conditions that denature HRP. As a consequence, DHP is a tortoise, a slow but steady enzyme that wins the evolutionary race against the HRP-type of peroxidase, which is a hare, initially rapid, but flawed for this application because of the protein denaturation under the conditions of the experiment.

The ground state reaction path for formation of the pyrimidine hydrates was calculated using a nudged elastic band (NEB) approach, combined with a calculation of the transition state, and implemented using a numerical basis set in the density functional theory (DFT) code DMol3. The model systems used for study consist of 1-methyl pyrimidines with a H2O molecule as the reactant, and the corresponding C5-hydro-C6-hydroxypyrimidine as the product. The barrier to addition of water across the C5–C6 π-bond ranges from 43–48 kcal mol−1 in the 1-methylpyrimidines (1-MP) studied. Similar but slightly smaller barriers of 34–45 kcal mol−1 were found for the tautomers of the 1-MPs, i.e. the enols of uridine and thymine and imine of cytosine. Comparison of these calculations with previous computational and experimental work suggests that a hot ground state formed by the rapid internal conversion of pyrimidines has sufficient energy to permit crossover from the common form to the tautomeric form of the pyrimidine at the transition state. The hot ground state mechanism can account for the experimentally observed yield and thermal reversion of pyrimidine photohydrates, while simultaneously explaining the effect of photohydrates on the mutation rate.

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Hydroquinone (H2Q) has been observed to compete with the oxidation of substrates 2,4,6-tribromophenol (2,4,6-TBP) and 2,4,6-trichlorophenol (2,4,6-TCP) catalyzed by the dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata in the presence of H2O2. This competition is observed as a lag phase during which H2Q is preferentially oxidized to 1,4-benzoquinone (1,4-BQ) while totally inhibiting either 2,4,6-TBP or 2,4,6-TCP oxidation. The inhibition by H2Q is distinct from that of the native competitive inhibitor 4-bromophenol (4-BP) since H2Q is itself oxidized and its product 1,4-BQ is not an inhibitor. Thus, once H2Q is completely consumed, the inhibition is removed, and normal substrate turnover is initiated, which explains the lag phase. To probe the mechanism of lag phase, the reactions between H2Q and DHP were both studied both in the presence and in the absence of H2O2. The reversible reactions between ferric/oxyferrous DHP A and H2Q/1,4-BQ are shown to involve a proton-coupled electron transfer (PCET) mechanism, where the distal histidine His55 serves as the proton acceptor. The pKa of the distal histidine His55 has been determined by resonance Raman spectroscopy in order to corroborate its involvement in this mechanism. Consistent with the proposed mechanism, kinetic assays have shown that H2Q serves as a substrate for DHP that follows the Michaelis–Menten kinetics. Unlike H2Q, the product 1,4-BQ has a relatively large Ki value and therefore has negligible inhibition. This study sheds light on understanding the difference between substrate and inhibitor binding sites and regulatory implication for the peroxidase and oxygen-transporter functions in DHP. It also provides information on PCET in DHP, which is important for resolving the switching between the ferric peroxidase catalytic function and the ferrous oxygen transport function.

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The ground state reaction path for formation of the pyrimidine hydrates was calculated using a nudged elastic band (NEB) approach, combined with a calculation of the transition state, and implemented using a numerical basis set in the density functional theory (DFT) code DMol3. The model systems used for study consist of 1-methyl pyrimidines with a H2O molecule as the reactant, and the corresponding C5-hydro-C6-hydroxypyrimidine as the product. The barrier to addition of water across the C5–C6 π-bond ranges from 43–48 kcal mol−1 in the 1-methylpyrimidines (1-MP) studied. Similar but slightly smaller barriers of 34–45 kcal mol−1 were found for the tautomers of the 1-MPs, i.e. the enols of uridine and thymine and imine of cytosine. Comparison of these calculations with previous computational and experimental work suggests that a hot ground state formed by the rapid internal conversion of pyrimidines has sufficient energy to permit crossover from the common form to the tautomeric form of the pyrimidine at the transition state. The hot ground state mechanism can account for the experimentally observed yield and thermal reversion of pyrimidine photohydrates, while simultaneously explaining the effect of photohydrates on the mutation rate.