PHF8 (KDM7B) is a human non-heme 2oxoglutarate (2OG) JmjC domain oxygenase that catalyzes the demethylation of the di/mono-N ε -methylated K9 residue of histone H3. Altered PHF8 activity is linked to genetic diseases and cancer; thus, it is an interesting target for epigenetic modulation. We describe the use of combined quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of PHF8, including dioxygen activation, 2OG binding modes, and substrate demethylation steps. A PHF8 crystal structure manifests the 2OG C-1 carboxylate bound to iron in a nonproductive orientation, i.e., trans to His247. A ferryl−oxo intermediate formed by activating dioxygen bound to the vacant site in this complex would be nonproductive, i.e., "off-line" with respect to reaction with N ε -methylated K9. We show rearrangement of the "off-line" ferryl−oxo intermediate to a productive "in-line" geometry via a solvent exchange reaction (called "ferryl-flip") is energetically unfavorable. The calculations imply that movement of the 2OG C-1 carboxylate prior to dioxygen binding at a five-coordination stage in catalysis proceeds with a low barrier, suggesting that two possible 2OG C-1 carboxylate geometries can coexist at room temperature. We explored alternative mechanisms for hydrogen atom transfer and show that second sphere interactions orient the N ε -methylated lysine in a conformation where hydrogen abstraction from a methyl C−H bond is energetically more favorable than hydrogen abstraction from the N−H bond of the protonated N ε -methyl group. Using multiple HAT reaction path calculations, we demonstrate the crucial role of conformational flexibility in effective hydrogen transfer. Subsequent hydroxylation occurs through a rebound mechanism, which is energetically preferred compared to desaturation, due to second sphere interactions. The overall mechanistic insights reveal the crucial role of iron-center rearrangement, second sphere interactions, and conformational flexibility in PHF8 catalysis and provide knowledge useful for the design of mechanism-based PHF8 inhibitors.

The ethylene-forming enzyme (EFE) is a non-heme Fe(II), 2-oxoglutarate (2OG), and L-arginine (L-Arg)-dependent oxygenase that catalyzes dual reactions: the generation of ethylene from 2OG and the C5 hydroxylation of L-Arg. Using an integrated molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) approach that references previous experimental studies, we tested the hypothesis that synergy between the conformation of L-Arg and the coordination mode of 2OG directs the reaction toward ethylene formation or L-Arg hydroxylation. The dynamics of EFE•Fe(III)•OO •− •2OG•L-Arg show that L-Arg can exist in conformation A (productive for hydroxylation) and conformation B (unproductive for hydroxylation). QM/MM calculations show that when 2OG is bound in an off-line mode and L-Arg is present in conformation A, the Fe(III)-OO •− intermediate undergoes the standard O 2 activation mechanism involving ferryldependent hydroxylation. With the same off-line 2OG coordination, but with conformation B of L-Arg, a unique pathway produces a half-bond ferric-bicarbonate intermediate that decomposes to ethylene, two CO 2 , and a ferrous-bicarbonate species. The results demonstrate that when 2OG is coordinated in off-line mode to the Fe center, the L-Arg conformation acts as a switch that directs the reaction toward ethylene formation or hydroxylation. Analysis of the electronic structure shows that the L-Arg conformation defines the precise location of an unpaired β electron in the Fe(III)-OO − complex, either in a π* ∥ orbital that triggers ethylene formation or a π* ⊥ orbital that cascades to L-Arg hydroxylation. A change in 2OG coordination from off-line to in-line reduces stabilization of the 2OG C1 carboxylate such that neither conformation of L-Arg produces the ethylene-forming half-bond ferric-bicarbonate intermediate. Thus, L-Arg conformation-dependent changes in the electronic structure of the Fe(III)-OO •− orbitals, together with the 2OG binding mode-associated stabilization of the C1-carboxylate, distinguish whether the EFE reaction proceeds via the ethylene-forming pathway or catalyzes a hydroxylation mechanism.

The Nξ-methyl lysine status of histones is important in the regulation of eukaryotic transcription. The Fe(II) and 2-oxoglutarate (2OG) - dependent JmjC domain enzymes are the largest family of histone...