Mimicking Elementary Reactions of Manganese Lipoxygenase Using Mn-hydroxo and Mn-alkylperoxo Complexes

Abstract

Manganese lipoxygenase (MnLOX) is an enzyme that converts polyunsaturated fatty acids to alkyl hydroperoxides. In proposed mechanisms for this enzyme, the transfer of a hydrogen atom from a substrate C-H bond to an active-site Mn^III-hydroxo center initiates substrate oxidation. In some proposed mechanisms, the active-site Mn^III-hydroxo complex is regenerated by the reaction of a Mn^III-alkylperoxo intermediate with water by a ligand substitution reaction. In a recent study, we described a pair of Mn^III-hydroxo and Mn^III-alkylperoxo complexes supported by the same amide-containing pentadentate ligand (^6Medpaq). In this present work, we describe the reaction of the Mn^III-hydroxo unit in C-H and O-H bond oxidation processes, thus mimicking one of the elementary reactions of the MnLOX enzyme. An analysis of kinetic data shows that the Mn^III-hydroxo complex [Mn^III(OH)(^6Medpaq)]⁺ oxidizes TEMPOH (2,2'-6,6'-tetramethylpiperidine-1-ol) faster than the majority of previously reported Mn^III-hydroxo complexes. Using a combination of cyclic voltammetry and electronic structure computations, we demonstrate that the weak Mn^III-N(pyridine) bonds lead to a higher Mn^III/II reduction potential, increasing the driving force for substrate oxidation reactions and accounting for the faster reaction rate. In addition, we demonstrate that the Mn^III-alkylperoxo complex [Mn^III(OO^tBu)(^6Medpaq)]⁺ reacts with water to obtain the corresponding Mn^III-hydroxo species, thus mimicking the ligand substitution step proposed for MnLOX.

Keywords: alkylperoxo; hydrogen-atom transfer; ligand substitution; lipoxygenase; manganese enzymes.

Scheme 1

Proposed mechanisms for MnLOX.

Scheme 2

Reaction of the Mn^III-hydroxo complex [Mn^III(OH)(^6Medpaq)]⁺ with ^tBuOOH to generate the Mn^III-alkylperoxo complex [Mn^III(OO^tBu)(^6Medpaq)]⁺. The structure of the protonated Hdpaq^6Me ligand is shown on the far left.

Figure 1

Electronic absorption spectra showing the reaction of a 2.5 mM solution of [Mn^II(H₂O)(^6Medpaq)](OTf) in CH₃CN (red trace) with dioxygen at 25 °C. The dashed traces show the reaction progress; the blue trace represents the final spectrum. The inset shows the growth in absorbance at 510 nm over time.

Figure 2

The electronic absorption spectrum of 1.0 mM [Mn^III(OH)(^6Medpaq)]⁺ (blue trace) and 1.0 mM [Mn^III(OH)(dpaq)]⁺ in MeCN at 25 °C shown for comparison (red trace).

Figure 3

MO energy level diagram for [Mn^III(OH)(dpaq)]⁺ and [Mn^III(OH)(^6Medpaq)]⁺ based on the Kohn-Sham orbitals from DFT calculations. α and β refer to spin-up and spin-down MOs, respectively.

Figure 4

TD-DFT-computed electronic absorption spectra for [Mn^III(OH)(dpaq)]⁺ (left) and [Mn^III(OH)(^6Medpaq)]⁺ (right). The sticks indicate electronic transitions; EDDMs of selected transitions are included as an inset. Red and blue colors in the EDDMs denote gain and loss of electron density, respectively. The DFT-computed structures of the Mn^III-hydoxo complexes are shown above the absorption spectra.

Figure 5

Reactions of 1.25 mM [Mn^III(OH)(^6Medpaq)](OTf) with 10 equiv. TEMPOH at −35 °C in MeCN (initial and final spectra are the red and blue traces, respectively). Inset: The decay of the 510 nm band over time (black trace) and fit to pseudo-first-order kinetic model (red trace).

Figure 6

Pseudo-first-order rate constants k_obs (s⁻¹) as a function of TEMPOH concentration for a 1.25 mM solution of [Mn^III(OH)(^6Medpaq)](OTf) in MeCN at −35 °C. The second-order rate constant (k₂) was calculated from the slope of the linear fit.

Scheme 3

Thermodynamic Square Scheme for Decomposing the O−H BDFE of a Mn^III-OH/Mn^II-OH₂ Couple.

Figure 7

A plot of ln k₂ vs. Mn^II-aqua O−H BDFE (kcal/mol).

Figure 8

A plot of ln k₂ vs. calculate E_1/2 vs. Fc⁺/Fc (V) and pK_a.

Figure 9

Electronic absorption spectra monitoring the reaction of an anaerobic sample of 2 mM [Mn^III(OO^tBu)(^6Medpaq)]⁺ in MeCN with 100 equiv. of H₂O at 298 K. (Inset) time course for the spectral changes.

Figure 10

Pseudo-first-order rate constants k_obs (s⁻¹) as a function of H₂O concentration for an anaerobic solution of 1 mM [Mn^III(OO^tBu)(^6Medpaq)]⁺ in MeCN at 25 °C. The second-order rate constant (k₂) was calculated from the slope of the linear fit.