Mechanistic Studies of a Flavin Monooxygenase: Sulfur Oxidation of Dibenzothiophenes by DszC

Abstract

Flavin monoxygenases (FMOs) are enzymes of increasing biotechnological (e.g., crude oil biodesulfurization) and pharmacological (e.g., drug metabolism) interest that perform the oxidation of soft nucleophiles and play key roles in the excretion of xenobiotics or in sulfur amino acid metabolism. DszC is a key FMO involved in sulfur oxidation of dibenzothiophenes (DBTs) through the 4S metabolic pathway of some bacteria. This pathway can be a cheaper and greener alternative for sulfur removal, as DBTs are the major source of crude oil sulfur. Here, we investigate the reaction mechanism of DszC with quantum mechanics/molecular mechanics methods (SCS-MP2/def2-TZVPP:ff10//B3LYP/6-31G(d):ff10). We observe that the reaction mechanism of DBT oxidation occurs in three stages: (1) spin-forbidden formation of a C4a-hydroperoxyflavin (C4aOOH) intermediate; (2) oxidation of DBT to DBTO, upon nucleophilic attack of the DBT-sulfur on the distal oxygen of C4aOOH; and (3) proton transfer from the N5H of the flavin group to the His92-imidazole via Ser163-hydroxyl, releasing a water molecule and oxidized flavin mononucleotide. The overall reaction is computed to be exergonic (−38.7 kcal·mol–1), and the rate-limiting step is the oxidation of DBT to DBTO (ΔG ‡ = 19.7 kcal·mol–1, consistent with the experimental turnover of 1.6 min–1). We observe that oxygen activation is a nearly spontaneous process that occurs through a proton-coupled electron transfer to produce a hydroperoxyl radical, followed by a triplet-singlet spin-forbidden inversion to form the C4aOOH intermediate. In agreement with other studies, His391 is a key acid to activate O2 and form the covalent bond. Further clarifying previous mutagenesis results, we also propose that His92 and Tyr96 are key residues for the mechanism: His92 acts as acid to deprotonate N5H in flavin via Ser163; and Tyr96 enhances the oxidation of DBT-sulfur by anchoring the proximal oxygen of C4aOOH, and acts as acid to form the water byproduct and regenerate the flavin cofactor. These are important results to clarify the chemistry of flavin monoxygenases and to open doors for the rational design of DszC mutants with improved catalytic activity.

Reaction Mechanism and Determinants for Efficient Catalysis by DszB, a Key Enzyme for Crude Oil Bio-desulfurization

“…Here, we aim to understand the intricacies of the electrophilic aromatic substitution mechanism of DszB using molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) methods, within the ONIOM formalism, using a similar methodology applied to other enzymes of this pathway. ,, After constructing a suitable QM/MM model, we followed this mechanism with the enzyme in many different conformations to better understand the relationship between the active site preorganization and the activation barrier. , The results shed light on the mechanism of the whole catalytic cycle and the preorganization determinants that promote the emergence of low activation barriers.…”

Theoretical Insight into a Nonadiabatic Proton-Coupled Electron Transfer Mechanism of Reduced Flavin Oxygenation

“…However, later, a positively charged residue around the flavin was found to be recurrent but crucial for maintaining the reactivity of flavin-dependent proteins . Specifically, the PCET is put forward in some specific flavin-dependent oxidases and mono-oxygenases, ,, which provide a new view for oxygen activation in flavin chemistry. In the PCET mechanism, the electron transfer (step 1 in the SET mechanism) is coupled with the proton transfer (step 3 in the SET mechanism).…”

“…In most investigated flavin-dependent proteins, the oxygenation reaction follows the multiple site-electron proton transfer mechanism, which is a special PCET pathway where the electron–proton acceptor (oxygen for this case) simultaneously accepts the electron and the proton from different donors (reduced flavin and some residue). The positively charged residue is the proton source. ,,, So the mutations of the vital residue would result in a dramatic decrease in reactivity. − As discussed above, for the oxygenation reaction of free reduced flavin, the proton is transferred from the reduced flavin itself to the oxygen because there are no other proton sources. Therefore, the reaction kinetics of oxygen activation in flavin-dependent proteins and in bulk solutions vary considerably. , Additionally, it has been reported that the hydrogen bond connecting to N 5 of the flavin can stabilize the oxygenated intermediate in flavin-dependent proteins. , So it is rational to speculate that the oxygenated product from self-proton transfer of free reduced flavin is less stable than it is in the protein.…”

“…The positively charged residue is the proton source. 16,53,54,57 So the mutations of the vital residue would result in a dramatic decrease in reactivity. 24−27 As discussed above, for the oxygenation reaction of free reduced flavin, the proton is transferred from the reduced flavin itself to the oxygen because there are no other proton sources.…”

Improved Efficiency of the Desulfurization of Oil Sulfur Compounds in Escherichia coli Using a Combination of Desensitization Engineering and DszC Overexpression

“…The K I values of the DszC mutants were slightly higher than that of the WT protein, indicating that their substrate inhibition was alleviated (Table 2). The DszC substrate binding pocket has been previously described in detail, [33][34][35][36]43 and the HBP binding site should not be the same as the substrate binding site because the inhibition of DszC by HBP is noncompetitive. 23 Although the true HBP binding site could not be verified in this study, the acquisition of the AKWC mutant made a significant contribution to the feedback inhibition desensitization of DszC.…”