Why Is the Oxidation State of Iron Crucial for the Activity of Heme-Dependent Aldoxime Dehydratase? A QM/MM Study

Aldoxime dehydratase is a heme-containing enzyme that utilizes the ferrous rather than the ferric ion to catalyze the synthesis of nitriles by dehydration of the substrate. We report a theoretical study of this enzyme aimed at elucidating its catalytic mechanism and understanding this oxidation state preference (Fe²⁺ versus Fe³⁺). The uncatalyzed dehydration reaction was modeled by including three and four water molecules to assist in the proton transfer, but the computed barriers were very high at both the DFT (B3LYP) and coupled cluster CCSD­(T) levels. The enzymatic dehydration of <i>Z</i>-acetaldoxime was explored through QM/MM calculation using two different QM regions and covering all three possible spin states. The reaction starts by substrate coordination to Fe²⁺ via its nitrogen atom to form a six-coordinated singlet reactant complex. The ferrous heme catalyzes the N–O bond cleavage by transferring one electron to the antibond in the singlet state, while His320 functions as a general acid to deliver a proton to the leaving hydroxide, thus facilitating its departure. The key intermediate is identified as an Fe^III(CH₃CHN^•) species (triplet or open-shell singlet), with the closed-shell singlet Fe^II(CH₃CHN⁺) being about 6 kcal/mol higher. Subsequently, the same His320 residue abstracts the α-proton, coupled with electron transfer back to the iron center. Both steps are calculated to have feasible barriers (14–15 kcal/mol), in agreement with experimental kinetic studies. For the same mode of substrate coordination, the ferric heme does not catalyze the N–O bond cleavage, because the reaction is endothermic by about 40 kcal/mol, mainly due to the energetic penalty for oxidizing the ferric heme. The alternative binding option, in which the anionic aldoxime coordinates to the ferric ion via its oxyanion, also results in a high barrier (around 30 kcal/mol), mainly because of the large endothermicity associated with the generation of a suitable base (neutral His320) for proton abstraction

On the Effect of Varying Constraints in the Quantum Mechanics Only Modeling of Enzymatic Reactions: The Case of Acetylene Hydratase

Quantum mechanics only (QM-only) studies of enzymatic reactions employ a coordinate-locking scheme, in which certain key atoms at the periphery of the chosen cluster model are fixed to their crystal structure positions. We report a case study on acetylene hydratase to assess the uncertainties introduced by this scheme. Random displacements of 0.1, 0.15, and 0.2 Å were applied at the ten terminal atoms fixed in the chosen 116-atom cluster model to generate sets of ten distorted structures for each given displacement. The relevant stationary points were reoptimized under these modified constraints to determine the variations of the computed energies and geometries induced by the displacements of the fixed atoms. Displacements of 0.1 Å cause a relatively minor perturbation that can be accommodated during geometry optimization, resulting in rather small changes in key bond distances and relative energies (typically of the order of 0.01 Å and 1 kcal/mol), whereas displacements of 0.2 Å lead to larger fluctuations (typically twice as high) and may sometimes even cause convergence to different local minima during geometry optimization. A literature survey indicates that protein crystal structures with a resolution higher than 2.0 Å are normally associated with a coordinate error of less than 0.1 Å for the backbone atoms. Judging from the present results for acetylene hydratase, such uncertainties seem tolerable in the design of QM-only models with more than 100 atoms, which are flexible enough to adapt during geometry optimization and thus keep the associate uncertainties in the computed energies and bond distances at tolerable levels (around 1 kcal/mol and 0.01 Å, respectively). On the other hand, crystal structures with significantly lower resolution should be used with great caution when setting up QM-only models because the resulting uncertainties in the computational results may become larger than acceptable. The present conclusions are mostly based on systematic DFT­(B3LYP) calculations with a medium-size basis set. Test calculations on selected structures confirm that similar results are obtained for larger basis sets, different functionals (ωB97X, BMK, M06), and upon including solvation and zero-point corrections, even though the fluctuations in the computed relative energies become somewhat larger in some cases

Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations

FosA is a manganese-dependent enzyme that utilizes a Mn²⁺ ion to catalyze the inactivation of the fosfomycin antibiotic by glutathione (GSH) addition. We report a theoretical study on the catalytic mechanism and the factors governing the regioselectivity and chemoselectivity of FosA. Density functional theory (DFT) calculations on the uncatalyzed reaction give high barriers and almost no regioselectivity even when adding two water molecules to assist the proton transfer. According to quantum mechanics/molecular mechanics (QM/MM) calculations on the full solvated protein, the enzyme-catalyzed glutathione addition reaction involves two major chemical steps that both proceed in the sextet state: proton transfer from the GSH thiol group to the Tyr39 anion and nucleophilic attack by the GSH thiolate leading to epoxide ring-opening. The second step is rate-limiting and is facilitated by the presence of the high-spin Mn²⁺ ion that functions as a Lewis acid and stabilizes the leaving oxyanion through direct coordination. The barrier for C1 attack is computed to be 8.9 kcal/mol lower than that for C2 attack, in agreement with the experimentally observed regioselectivity of the enzyme. Further QM/MM calculations on the alternative water attack predict a concerted mechanism for this reaction, where the deprotonation of water, nucleophilic attack, and epoxide ring-opening take place via the same transition state. The calculated barrier is 8.3 kcal/mol higher than that for GSH attack, in line with the observed chemoselectivity of the enzyme, which manages to catalyze the addition of GSH in the presence of water molecules around its active site. The catalytic efficiency, regioselectivity, and chemoselectivity of FosA are rationalized in terms of the influence of the active-site protein environment and the different stabilization of the distorted substrates in the relevant transition states

Comparison of QM-Only and QM/MM Models for the Mechanism of Tungsten-Dependent Acetylene Hydratase

We report a comparison of QM-only and QM/MM approaches for the modeling of enzymatic reactions. For this purpose, we present a QM/MM case study on the formation of vinyl alcohol in the catalytic cycle of tungsten-dependent acetylene hydratase. Three different QM regions ranging from 32 to 157 atoms are designed for the reinvestigation of the previously suggested one-water attack mechanism. The QM/MM calculations with the minimal QM region <b>M1</b> (32 atoms) yield a two-step reaction profile, with an initial nucleophilic attack followed by the protonation of the formed vinyl anion intermediate, as previously proposed on the basis of QM-only calculations on cluster model <b>M2</b> (116 atoms); however, the overall QM/MM barrier with <b>M1</b> is much too high, mainly due to an overestimate of the QM/MM electrostatic repulsions. QM/MM calculations with QM region <b>M2</b> (116 atoms) fail to reproduce the published QM-only results, giving a one-step profile with a very high barrier. This is traced back to the strong electrostatic influence of the two neighboring diphosphate groups that were neglected in the QM-only work but are present at the QM/MM level. These diphosphate groups and other electrostatically important nearby residues are included in QM region <b>M3</b> (157 atoms). QM/MM calculations with <b>M3</b> recover the two-step mechanism and yield a reasonable overall barrier of 16.7 kcal/mol at the B3LYP/MM level. They thus lead to a similar overall mechanistic scenario as the previous QM-only calculations, but there are also some important variations. Most notably, the initial nucleophilic attack becomes rate limiting at the QM/MM level. A modified two-water attack mechanism is also considered but is found to be less favorable than the previously proposed one-water attack mechanism. Detailed residue interaction analyses and comparisons between QM/MM results with electronic and mechanical embedding and QM-only results without and with continuum solvation show that the protein environment plays a key role in determining the mechanistic preferences in acetylene hydratase. The combined use of QM-only and QM/MM methods provides a powerful approach for the modeling of enzyme catalysis

Phosphate Hydrolysis by the Fe₂–Ca₃‑Dependent Alkaline Phosphatase PhoX: Mechanistic Insights from DFT calculations

PhoX is a pentanuclear metalloenzyme that employs two ferric ions and three calcium ions to catalyze the hydrolysis of phosphomonoesters. On the basis of the X-ray structure of PhoX (Science 2014, 345, 1170−1173), a model of the active site is designed, and quantum chemical calculations are used to investigate the reaction mechanism of this enzyme. The calculations support the experimental suggestion, in which the two high spin ferric ions interact in an antiferromagnetic fashion. The two step mechanism proposed by experimentalists has been investigated. The nucleophilic attack of a trinuclear bridging oxo group on the phosphorus center was calculated to be the first step, which is concomitant with the departure of the phenolate, which is stabilized by a calcium ion. The second step is a reverse attack by a water molecule activated by a calcium-bound hydroxide, leading to the regeneration of the bridging oxo group. The second step was calculated to have a barrier of 27.6 kcal/mol. The high barrier suggests that the alternative mechanism involving phosphate release directly from the active site seems to be more likely. All five metal ions are involved in the catalysis by stabilizing the pentacoordinated trigonal bipyramidal transition states

Which Oxidation State Leads to O–O Bond Formation in Cp*Ir(bpy)Cl-Catalyzed Water Oxidation, Ir(V), Ir(VI), or Ir(VII)?

Density functional calculations are used to revisit the reaction mechanism of water oxidation catalyzed by the Cp*Ir­(bpy)Cl (Cp* = pentamethyl­cyclopentadienyl, bpy = 2,2′-bipyridine) complex. One of the experimentally suggested active species [(bpy)­Ir­(H₂O)₂(HCOO)­Cl]⁺ can undergo very facile intramolecular formate oxidation at higher oxidation state even though it can also promote O–O bond formation. Therefore, [(bpy)­Ir­(H₂O)₂(CH₃COO)­Cl]⁺ is here proposed to be the most likely precatalyst as acetate was also experimentally observed after Cp* oxidation. O–O bond formation takes place at the high formal oxidation states of Ir^VI and Ir^VII, rather than that of Ir^V, as suggested before. Three sequential proton-coupled electron transfer oxidations result in the formation of a highly oxidized intermediate, [(bpy)­Ir^VIO­(OH)­(CH₃COO)­Cl]⁺. From this formal Ir^VI intermediate, O–O bond formation takes place by a water attack on the Ir^VI=O moiety assisted by the acetate ligand, which abstracts a proton during the attack. The barrier was calculated to be very facile, being 14.7 kcal/mol, in good agreement with experimental kinetic results, which gave a barrier of around 18 kcal/mol. The attack leads to the formation of an Ir^IV-peroxide intermediate, which undergoes proton-coupled electron transfer to form an Ir^III–O₂ intermediate. Finally, O₂ can be released, coupled with the binding of another water molecule, to regenerate the catalytic Ir^III species. Water oxidation at Ir^VII has a slightly higher barrier, but it may also contribute to the activity. However, water oxidation at Ir^V has a significantly higher barrier. Acetate oxidation by C–H activation was found to have a much higher barrier, suggesting that [(bpy)­Ir­(H₂O)₂(CH₃COO)­Cl]⁺ is a remarkably stable catalyst. The possible catalytic species [(bpy-dc)­Ir^III(H₂O)₃Cl]²⁺ without acetate coordination has also been considered and also gave a reasonably feasible barrier for the water oxidation. O–O bond formation at Ir^VII is slightly preferred compared with at Ir^VI, which is different from the case with acetate

Theoretical Studies on the Photochemistry of Pentose Aminooxazoline, a Hypothetical Intermediate Product in the Prebiotic Synthetic Scenario of RNA Nucleotides

2-Aminooxazole is generally considered a prebiotic precursor of ribonucleotides on the early earth. Its pentose compound, pentose aminooxazoline, has been suggested to be a key intermediate in the prebiotic synthetic scenario. In this article, detailed mechanism of the photochemistry of pentose aminooxazoline has been studied by performing density functional theory and multireference complete active space self-consistent field calculations. Parallel to the “ring-puckering” process, which leads to ultrafast nonradiative deactivation, several other photodissociation channels are explored in detail. In addition, the influences of the pentose structure and solvation effects with both implicit and explicit water models have been uncovered for both neutral and protonated forms. The current theoretical results provide very important information not only for the photostability of RNA nucleotides but also for an in-depth understanding of the synthesis of other prebiotic nucleotides

Theoretical Study of the Mechanism of the Nonheme Iron Enzyme EgtB

EgtB is a nonheme iron enzyme catalyzing the C–S bond formation between γ-glutamyl cysteine (γGC) and <i>N</i>-α-trimethyl histidine (TMH) in the ergothioneine biosynthesis. Density functional calculations were performed to elucidate and delineate the reaction mechanism of this enzyme. Two different mechanisms were considered, depending on whether the sulfoxidation or the S–C bond formation takes place first. The calculations suggest that the S–O bond formation occurs first between the thiolate and the ferric superoxide, followed by homolytic O–O bond cleavage, very similar to the case of cysteine dioxygenase. Subsequently, proton transfer from a second-shell residue Tyr377 to the newly generated iron–oxo moiety takes place, which is followed by proton transfer from the TMH imidazole to Tyr377, facilitated by two crystallographically observed water molecules. Next, the S–C bond is formed between γGC and TMH, followed by proton transfer from the imidazole CH moiety to Tyr377, which was calculated to be the rate-limiting step for the whole reaction, with a barrier of 17.9 kcal/mol in the quintet state. The calculated barrier for the rate-limiting step agrees quite well with experimental kinetic data. Finally, this proton is transferred back to the imidazole nitrogen to form the product. The alternative thiyl radical attack mechanism has a very high barrier, being 25.8 kcal/mol, ruling out this possibility

Which Oxidation State Initiates Dehalogenation in the B12-Dependent Enzyme NpRdhA: Co^II, Co^I, or Co⁰?

The quantum chemical cluster approach was used to elucidate the reaction mechanism of debromination catalyzed by the B12-dependent reductive dehalogenase NpRdhA. Various pathways, involving different oxidation states of the cobalt ion and different protonation states of the model, have been analyzed in order to find the most favorable one. We find that the reductive C–Br cleavage takes place exclusively at the Co^I state via a heterolytic pathway in the singlet state. Importantly, the C–H bond formation and the C–Br bond cleavage proceeds via a concerted transition state, as opposed to the stepwise pathway suggested before. C–Br cleavage at the Co^II state has a very high barrier, and the reduction of Co^I to Co⁰ is associated with a very negative potential; thus, reductive dehalogenation at Co^II and Co⁰ can be safely ruled out. Examination of substrates with different halogen substitutions (F, Cl, Br, I) shows that the dehalogenation reactivity follows the order C–I > C–Br > C–Cl > C–F, and the barrier for defluorination is so high that NpRdhA cannot catalyze that reaction

Origins of Stereoselectivity in Peptide-Catalyzed Kinetic Resolution of Alcohols

The origin of the stereoselectivity of the tetrapeptide-catalyzed kinetic resolution of <i>trans</i>-2-<i>N</i>-acetamidocyclohexanol is investigated by means of density functional theory calculations. Transition states for the functionalization of both (<i>R</i>,<i>R</i>) and (<i>S</i>,<i>S</i>) substrates were optimized considering all possible conformers. Due to the flexibility of the peptidic catalyst, a large number of transition states had to be located, and analysis of the geometries and energies allowed for the identification of the main factors that control the stereoselectivity