Reaction coordinate of isopenicillin N synthase: oxidase versus oxygenase activity

Abstract

Isopenicillin N synthase (IPNS) can have both oxidase and oxygenase activity depending on the substrate. For the native substrate, ACV, oxidase activity exists; however, for the substrate analogue ACOV, which lacks an amide nitrogen, IPNS exhibits oxygenase activity. The potential energy surfaces for the O-O bond elongation and cleavage were calculated for three different reactions: homolytic cleavage via traditional Fenton chemistry, heterolytic cleavage, and nucleophilic attack. These surfaces show that the hydroperoxide-ferrous intermediate, formed by O(2)-activated H atom abstraction from the substrate, can exploit different reaction pathways and that interactions with the substrate govern the pathway. The hydrogen bonds from hydroperoxide to the amide nitrogen of ACV polarize the sigma* orbital of the peroxide toward the proximal oxygen, facilitating heterolytic cleavage. For the substrate analogue ACOV, this hydrogen bond is no longer present, leading to nucleophilic attack on the substrate intermediate C-S bond. After cleavage of the hydroperoxide, the two reaction pathways proceed with minimal barriers, resulting in the closure of the beta-lactam ring for the oxidase activity (ACV) or formation of the thiocarboxylate for oxygenase activity (ACOV).

Figure 1. Models Used to Calculate Three Reaction Pathways of Fe^II-Hydroperoxide

The arrow in Model 3 indicates the extra constraint added to prohibit interaction of the distal oxygen with the carbon of the substrate.

Figure 2. Molecular Orbital Diagram of IPNS-ACV-Fe^II-Hydroperoxide

Spin unrestricted contours given for representative molecular orbitals. Note the 4 unoccupied β d orbitals indicating Fe^II, and the unoccupied α, β pair of the σ* orbital of peroxide with the α, β unoccupied pair of C-S π* orbitals of the substrate π bond. The observed d_xy character (24%) in the C-S π* bond reflects backbonding.

Figure 3. Geometric Structures of IPNS-Fe^II-Hydroperoxide Complexes with O-O Bond Elongation

The three models undergo homolytic cleavage (top, 3), heterolytic O-O bond cleavage (middle, 1) and nucleophilic attack by the peroxide (bottom, 2).

Figure 4. Donor and Acceptor Orbitals for Homolytic Cleavage, Heterolytic Cleavage and Nucleophilic Attack

In the homolytic cleavage reaction, one β electron is transferred from the Fe d to the hydroperoxide σ* orbital. Heterolytic cleavage and nucleophilic attack involve the transfer of an α/β electron pair. For clarity, only the alpha donor and acceptor orbitals of these electron pairs are shown.

Figure 5. Energetics of O-O Bond Cleavage

The relative electronic energies are plotted for the homolytic cleavage (3, red circles), heterolytic cleavage (1, blue triangles) and nucleophilic attack (2, black squares) models as the O-O bond is elongated. Note that the nucleophilic attack model is only valid for O-O bond cleavage in ACOV and that for the ACV substrate, an additional barrier to break the amide-peroxide hydrogen bond is present.

Figure 6. Alpha Hydroperoxide σ* Orbitals

This orbital polarizes toward the proximal oxygen in Model 1, directing the reaction to heterolytic O-O bond cleavage, and toward the distal oxygen in Model 3, directing its reaction to homolytic O-O bond cleavage. By modeling the dipole interaction of the ACV amide N-H, the polarization of the σ* bond in Model 3 is reversed

Figure 7. Donor and Acceptor Orbitals and Product for Beta-Lactam Ring Closure

Deprotonation of the ACV amide leaves a lone pair on the amide N with the correct orientation for SN₂ nucleophilic attack at the C-S double bond.

Scheme 1. The four electron oxidative double ring closure of ACV to form Isopenicillin N

Scheme 2. Three Reaction Pathways of Fe^II-Hydroperoxide

Scheme 3. Rotation of the Peroxide for Nucleophilic Attack

Scheme 4. Nucleophilic Attack by the distal and proximal oxygen of the hydroperoxide