Common Reactivity and Properties of Heme Peroxidases: A DFT Study of Their Origin.

Electronic structure calculations using the density-functional theory (DFT) have been performed to analyse the effect of water molecules and protonation on the heme group of peroxidases in different redox (ferric, ferrous, compounds I and II) and spin states. Shared geometries, spectroscopic properties at the Soret region, and the thermodynamics of peroxidases are discussed. B3LYP and M06-2X density functionals with different basis sets were employed on a common molecular model of the active site (Fe-centred porphine and proximal imidazole). Computed Gibbs free energies indicate that the corresponding aquo complexes are not thermodynamically stable, supporting the five-coordinate Fe(III) centre in native ferric peroxidases, with a water molecule located at a non-bonding distance. Protonation of the ferryl oxygen of compound II is discussed in terms of thermodynamics, Fe-O bond distances, and redox properties. It is demonstrated that this protonation is necessary to account for the experimental data, and computed Gibbs free energies reveal pKa values of compound II about 8.5-9.0. Computation indicates that the general oxidative properties of peroxidase intermediates, as well as their reactivity towards water and protons and Soret bands, are mainly controlled by the iron porphyrin and its proximal histidine ligand.

Effect of ionizing radiation on human myeloperoxidase: Reaction with hydrated electrons.

Myeloperoxidase (MPO) is a myeloid-lineage restricted enzyme largely expressed in the azurophilic granules of neutrophils. It catalyses the formation of reactive oxygen species, mainly hypochlorous acid, contributing to anti-pathogenic defense. Disorders in the production or regulation of MPO may lead to a variety of health conditions, mainly of inflammatory origin, including autoimmune inflammation. We have studied the effect of ionizing radiation on the activity of MPO, as measured by the capacity retained by the enzyme to produce hypochlorous acid as reactive oxygen species after exposure to successive doses of solvated electrons, the strongest possible one-e- reducing agent in water. Chlorination activity was still present after a very high irradiation dose, indicating that radiation damage does not take place at the active site, hindered in the core of MPO structure. Decay kinetics show a dependence on the wavelength, supporting that the process must occur at peripheral functional groups situated on external and readily accessible locations of the enzyme. These results are relevant to understand the mechanism of resistance of our innate anti-pathogenic defense system and also to get insight into potential strategies to regulate MPO levels as a therapeutic target in autoimmune diseases.

A theoretical study on the mechanism of the base-promoted decomposition of N-chloro,N-methylethanolamine.

The first step of the base-promoted decomposition of N-chloro,N-methylethanolamine in aqueous solution (CH3N(Cl)CH2CH2OH + HO- --> imine + Cl- + H2O (+ CH2O) --> amine + aldehyde) is investigated at the MP2/6-31++G(d,p) computing level. Solvation is included by using both a microsolvated model, in which two explicit water molecules simulate the specific solvent effects, and a hybrid cluster-continuum model, by applying a polarized continuum on the previous results, to account for the bulk effect of the solvent. Four alternative pathways (bimolecular fragmentation, Hofmann, Zaitsev and intramolecular eliminations) are possible for the rate-limiting step of this base-promoted decomposition. These reactive processes are bimolecular asynchronous concerted reactions. The common feature of the four pathways is the proton transfer to HO- being more advanced than all other molecular events, whereas imine formation is delayed. Non-reactive cyclic arrangements involving one of the explicit water molecules are found at transition structures of Hofmann and Zaitsev eliminations, such water molecule acting both as H+ donor and acceptor. Although MP2 calculations misjudge the absolute activation Gibbs free energy values, this computational level adequately predicts the enhancement in the decomposition rate due to the presence of the -OH group.

Myeloperoxidase-catalyzed chlorination: the quest for the active species.

Myeloperoxidase (MPO) is a dominating enzyme of circulating polymorphonuclear neutrophils that catalyzes the two-electron oxidation of chloride, thereby producing the strong halogenating agent hypochlorous acid (ClO(-)/HOCl). In absence of MPO the tripeptide Pro-Gly-Gly reacts with HOCl faster than the amino acid taurine (2-aminoethanesulfonic acid, Tau), while the MPO-mediated chlorination shows reverse order. A comparative study of the enzymatic oxidation of both substrates at pH 4.0-6.0, varying H2O2 concentration is presented. Initial and equilibrium rates studies have been carried on, reaction rates in the latter being slower due to the chemical equilibrium between MPO-I and MPO-II-HO2. A maximum of chlorination rate is observed for Pro-Gly-Gly and Tau when [H2O2] approximately 0.3-0.7 mM and pH approximately 4.5-5.0. Several mechanistic possibilities are considered, the proposed one implies that chlorination takes place via two pathways. One, for bulkier substrates, involves chlorination by free HOCl outside the heme cavity; ClO(-) is released from the active center, diffuses away the heme cavity, and undergoes protonation to HOCl. The other implies the existence of compound I-Cl(-) complex (MPO-I-Cl), capable of chlorinating smaller substrates in the heme pocket. Electronic structure calculations show the size of Pro-Gly-Gly comparable to the available gap in the substrate channel, this tripeptide being unable to reach the active site, and its chlorination is only possible by free HOCl outside the enzyme.

Myeloperoxidase-catalyzed taurine chlorination: initial versus equilibrium rate.

Myeloperoxidase (MPO) catalyzes the two-electron oxidation of chloride, thereby producing hypochlorous acid (HOCl). Taurine (2-aminoethane-sulfonic acid, Tau) is thought to act as a trap of HOCl forming the long-lived oxidant monochlorotaurine [(N-Cl)-Tau], which participates in pathogen defense. Here, we amend and extend previous studies by following initial and equilibrium rate of formation of (N-Cl)-Tau mediated by MPO at pH 4.0-7.0, varying H(2)O(2) concentration. Initial rate studies show no saturation of the active site under assay conditions (i.e. [H(2)O(2)] > or = 2000 [MPO]). Deceleration of Tau chlorination under equilibrium is quantitatively described by the redox equilibrium established by H(2)O(2)-mediated reduction of compound I to compound II. At equilibrium regime the maximum chlorination rate is obtained at [H(2)O(2)] and pH values around 0.4mM and pH 5. The proposed mechanism includes known acid-base and binding equilibria taking place at the working conditions. Kinetic data ruled out the currently accepted mechanism in which a proton participates in the molecular step (MPO-I+Cl(-)) leading to the formation of the chlorinating agent. Results support the formation of a chlorinating compound I-Cl(-) complex (MPO-I-Cl) and/or of ClO(-), through the former or even independently of it. ClO(-) diffuses away and rapidly protonates to HOCl outside the heme pocket. Smaller substrates will be chlorinated inside the enzyme by MPO-I-Cl and outside by HOCl, whereas bulkier ones can only react with the latter.

Understanding the mechanism of base-assisted decomposition of (N-halo),N-alkylalcoholamines.

The base-assisted decomposition of (N-X),N-methylethanolamine (X = Cl, Br) takes place mainly through two concurrent processes: a fragmentation and an intramolecular elimination. The global process follows second order kinetics, first order relative to both (N-X),N-methylethanolamine and base. Interaction of the base with the ionizable hydroxylic hydrogen triggers the reaction. The intramolecular elimination pathway leads to formaldehyde and 2-aminoethanol as reaction products via base-assisted proton transfer from the methyl to the partially unprotonated hydroxylic oxygen, with loss of halide. Meanwhile, the fragmentation pathway leads to methylamine and two equivalents of formaldehyde via bimolecular base-promoted concerted breakage of the molecule into formaldehyde, halide ion and N-methylmethanimine. Kinetic evidences allow a crude estimation of the concertedness and characterization of the transition structure for both processes, which are slightly asynchronous, the proton transfer to the base taking place ahead of the rest of the molecular events. The degree of asynchroneity increases as the bases become weaker. Electronic structure calculations, at the B3LYP/6-31++G** level, on the fragmentation pathway support the proposed mechanism.