Proton-Electron Transfer to the Active Site Is Essential for the Reaction Mechanism of Soluble Δ9-Desaturase

Abstract

A full understanding of the catalytic action of non-heme iron (NHFe) and non-heme diiron (NHFe₂) enzymes is still beyond the grasp of contemporary computational and experimental techniques. Many of these enzymes exhibit fascinating chemo-, regio-, and stereoselectivity, in spite of employing highly reactive intermediates which are necessary for activations of most stable chemical bonds. Herein, we study in detail one intriguing representative of the NHFe₂ family of enzymes: soluble Δ⁹ desaturase (Δ⁹D), which desaturates rather than performing the thermodynamically favorable hydroxylation of substrate. Its catalytic mechanism has been explored in great detail by using QM(DFT)/MM and multireference wave function methods. Starting from the spectroscopically observed 1,2-μ-peroxo diferric P intermediate, the proton-electron uptake by P is the favored mechanism for catalytic activation, since it allows a significant reduction of the barrier of the initial (and rate-determining) H-atom abstraction from the stearoyl substrate as compared to the "proton-only activated" pathway. Also, we ruled out that a Q-like intermediate (high-valent diamond-core bis-μ-oxo-[Fe^IV]₂ unit) is involved in the reaction mechanism. Our mechanistic picture is consistent with the experimental data available for Δ⁹D and satisfies fairly stringent conditions required by Nature: the chemo-, stereo-, and regioselectivity of the desaturation of stearic acid. Finally, the mechanisms evaluated are placed into a broader context of NHFe₂ chemistry, provided by an amino acid sequence analysis through the families of the NHFe₂ enzymes. Our study thus represents an important contribution toward understanding the catalytic action of the NHFe₂ enzymes and may inspire further work in NHFe₍₂₎ biomimetic chemistry.

Figure 1.

Proposed oxygenated intermediates of selected NHFe₂ enzymes. Δ⁹D = Δ⁹ desaturase; sMMO = soluble methane monooxygenase; BoxB = benzoyl coenzyme A epoxidase; AurF = p-aminobenzoate N-oxygenase; T4MO = toluene 4-monooxygenase; Rbr = rubrerythrin; RNR = ribonucleotide reductase; CmlI = arylamine oxygenase.

Figure 2.

Truncated cluster model of the Δ⁹D active site utilized in the WFT calculations.

Figure 3.

The spectroscopically observed 1,2-μ-peroxo-[Fe^III]₂ intermediate P is inactive toward the C–H bond cleavage. Thus, several activation pathways are proposed: (i) the proton-assisted rearrangement producing the μ-η²:η¹-hydroperoxo-[Fe^III]₂ species P’ (1a), (ii) the O–O bond cleavage to yield the bis-μ-oxo-[Fe^IV]₂ complex putatively corresponding to the intermediate Q in sMMO (1b), and (iii) concomitant proton and electron transfers to yield the complex, which would be reminiscent of the intermediate X of the Class Ia RNR enzyme (1c). The energies are calculated at the QM(B3LYP*-D3)/MM level of theory and referenced to the energy of the P intermediate.

Figure 4.

A possible pathway for the protonation of the active site of the P intermediate. The energies are calculated at the QM(B3LYP*-D3)/MM level of theory and referenced to the energy of the P intermediate. Note that 1a₀ structure is already protonated at the His203 residue and, therefore, does not correspond to the referential P intermediate from Figure 3.

Figure 5.

A different character of the TS for C₁₀–H bond cleavage is observed for the DFT calculations (heterolytic cleavage; Left) and multiconfigurational WFT-based methods (homolytic cleavage; Right).

Figure 6.

Activation of the C₁₀–H bond by the Q intermediate. The energies are calculated at the QM(B3LYP*-D3)/MM level of theory and referenced to the energy of the P intermediate.

Figure 7.

Plausible PET-assisted activation (stepwise PT/ET mechanism) of the P intermediate. Energies are calculated at the QM(B3LYP*-D3)/MM level of theory and referenced to the P intermediate. Note that coupled (concerted) proton-electron transfer might be a possible mechanism, bypassing the ‘high-energy’ P’ intermediate and converting P to X directly.

Figure 8.

The energetics associated with all of the studied pathways, calculated at the QM(B3LYP*-D3)/MM level of theory. Raw data are shown in Table S3. Black: A direct C₁₀–H bond activation by P intermediate. First, the O–O bond of the P intermediate is cleaved with a calculated barrier of ~25 kcal mol⁻¹ (the rate-determining step), followed by a C₁₀–H bond activation (~22 kcal mol⁻¹). Red: The proton-assisted activation pathway (1a). The protonation of P generates a P’ intermediate (12.8 kcal mol⁻¹ above P), which may activate C₁₀–H bond of a substrate. The barrier of ~33 kcal mol⁻¹ is, however, too high to be operative. Blue: Cleavage of the peroxo O–O bond (pathway 1b). The ‘Q’ like [Fe^IV]₂O₂ intermediate is suggested to be inactive due to the rate-determining barrier of C₁₀–H bond activation of ~29 kcal mol⁻¹. Green: The activation of P by proton-electron uptake (1c). Here, either concerted or step-wise proton-electron transfer (dashed or solid line) is proposed, leading to a formation of an intermediate X analog (~4 kcal mol⁻¹ above P). The energy of the rate-determining step within the 1c pathway (i.e., the C₁₀–H bond cleavage) is consistent with the barrier derived from the experimental data (calcd. vs. expt.: ~13 kcal mol⁻¹ vs. 14.8 kcal mol⁻¹). The second C₉–H bond is then activated with a small activation energy of ~7 kcal mol⁻¹.