QM/MM Modeling of the Flavin Functionalization in the RutA Monooxygenase

Abstract

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re-side and si-side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

Keywords: QM/MM; RutA enzyme; absorption spectra; flavin; molecular oxygen; monooxygenases; quantum chemistry.

Scheme 1

Reduced FMN (left), oxidized FMN (right), and atom numbering in the isoalloxazine ring of flavin (center).

Figure 1

A general view of the model system considered in this work. Inset—the isoalloxazine ring of FMN and the nearest molecular groups at the re-side (Asn134 and Thr105) and at the si-side (water molecules). In this and other figures, carbon atoms are shown in green, oxygen in red, nitrogen in blue, and hydrogen in white.

Figure 2

Pathway-1: (a) Triplet state complex Fl_red-O₂ (Complex-1) formed at the si-side. (b) Singlet state Fl_C4aOO⁻ species. In this and other figures, distances are given in Å.

Figure 3

Results of the CASSCF(14/10)/cc-pVDZ calculations for Complex-1 (Figure 2a). The legends on the right side show dominant electronic configurations and their weights in the triplet state and singlet state CASSCF wavefunctions composed of the active orbitals illustrated in ten boxes. The occupancies of the fifth and sixth orbitals, mainly assigned to dioxygen, are distinguished by the red color.

Figure 4

Pathway-2: (a) Triplet state complex Fl_red-O₂ (Complex-2) formed at the si-side. (b) Singlet state system with the hydroperoxide anion. (c) A possible route to the reaction products, Fl_ox…H₂O₂…OH⁻(Wat6). Magenta arrows show a proton transfer path from Wat6.

Figure 5

Minimum energy points on potential energy surfaces along pathway-3. (a) Triplet state complex. (b) Oxidized flavin and hydrogen peroxide with the nearby hydroxyl species. (c) Oxidized flavin, hydrogen peroxide, and the remote hydroxyl species.

Figure 6

Results of the CASSCF(14/10)/cc-pVDZ calculations for Complex-3 (Figure 5a). The legends on the right side show a dominant electronic configuration and its weight in the triplet state and singlet state CASSCF wavefunctions composed of the active orbitals illustrated in ten boxes. The occupancies of the sixth and ninth orbitals in the triplet state or the sixth and seventh orbitals in the singlet state, mainly assigned to dioxygen, are distinguished by the red color.

Figure 7

Pathway-4: (a) The initial triplet state, Complex-4. (b) Singlet state minimum energy point with the hydroperoxyl.

Figure 8

Singlet state minimum energy structures along Pathway-4. (a) The N(5)-oxide flavin-oxygen adduct. (b) The C(8)-hydrated N(5)-oxide flavin-oxygen adduct.

Scheme 2

Left—flavin-N(5)-oxide; right—flavin-C(8)-hydrated N(5)-oxide.

Figure 9

Structures along pathway-5: (a) Triplet state minimum energy point with the dioxygen moiety (Complex-5). (b) Structure with the flavin-C(6)-hydroperoxide adduct. (c) Structure with the C(6)-C(7)-epoxide adduct.

Figure 10

The QM subsystem for QM/MM calculations. Water molecules are shown as sticks. The carbon atoms at the broken covalent bonds (C_β for Thr105, Asn134, Trp139, and C_γ for Glu292) are highlighted in yellow.