Dioxygen Binding Is Controlled by the Protein Environment in Non-heme FeII and 2-Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene-Forming Enzyme

Abstract

This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme Fe^II /2OG enzymes: a class 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene-forming enzyme (EFE), which catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially bind 2OG by using an off-line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in-line mode, whereas EFE is catalytically productive for ethylene production from 2OG in the off-line mode. We used classical molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics (QM/MM-MetD) simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in-line Fe^III -OO⋅^- intermediate in PHF8 and the off-line Fe^III -OO⋅^- intermediate in EFE.

Keywords: QM/MM metadynamics; dioxygen diffusion; ethylene-forming enzymes; histone demethylation; molecular dynamics.

Figure 1.

Dioxygen (O₂) transport in PHF8. (A) Dioxygen (O₂) transport in PHF8 via tunnel-1 obtained from diffusion dynamics. The tunnel residues are not labeled here for clarity but are shown in detail in Figures S5–S7. (B) The approach of dioxygen to the Fe(II)-center for likely formation of an in-line Fe(III)-OO^−. complex following 2OG rearrangement. (C) The dioxygen approach for likely formation of an off-line Fe(III)-OO^−. complex. The Fe(II)-center, dioxygen, 2OG, and H3K9me2 substrates are shown as balls and sticks.

Figure 2.. Dioxygen (O₂) transport in EFE.

(A) Dioxygen (O₂) transport in EFE via tunnel-1 obtained from diffusion dynamics; the tunnel residues are not labeled here for clarity and are shown in detail in Figures S10–S12. (B) The off-line dioxygen approach through tunnel-1 in EFE for binding to the Fe(II)-center forms an off-line Fe(III)-OO^−. complex. (C) The in-line approach of dioxygen through tunnel-2 for binding to the Fe(II)-center in EFE, leading to formation of an in-line Fe(III)-OO^−. complex with 2OG rearrangement. The Fe(II)-center, dioxygen, 2OG, and H3K9me2 substrate are shown as balls and sticks.

Figure 3.. QM/MM MetD free energy surfaces for dioxygen binding in PHF8.

Free energy versus Fe-O distance plots are shown for generation of (A) the in-line Fe(III)-OO^.− complex and (B) the off-line Fe(III)-OO^.− complex. Representative structures for (C) in-line dioxygen binding RC, (D) in-line dioxygen binding TS, and (E) in-line dioxygen binding PD are obtained from QM/MM MetDs.

Figure 4.. Free energy surfaces for dioxygen binding in EFE.

Free energy versus Fe-O distance plots for generation of (A) an in-line Fe(III)-OO^.− complex and (B) an off-line Fe(III)-OO^.− complex. Representative structures for (C) off-line dioxygen binding RC, (D) off-line dioxygen binding TS, and (E) off-line dioxygen binding PD were obtained from QM/MM MetD.