Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity

Can an external electric field switch between ethylene formation and l-arginine hydroxylation in the ethylene forming enzyme?

The non-heme Fe(ii) and 2-oxoglutarate (2OG) dependent ethylene-forming enzyme (EFE) catalyzes both ethylene generation and l-Arg hydroxylation. Despite experimental and computational progress in understanding the mechanism of EFE, no EFE variant has been optimized for ethylene production while simultaneously reducing the l-Arg hydroxylation activity. In this study, we show that the two l-Arg binding conformations, associated with different reactivity preferences in EFE, lead to differences in the intrinsic electric field (IntEF) of EFE. Importantly, we suggest that applying an external electric field (ExtEF) along the Fe-O bond in the EFE·Fe(iii)·OO−˙·2OG·l-Arg complex can switch the EFE reactivity between l-Arg hydroxylation and ethylene generation. Furthermore, we explored how applying an ExtEF alters the geometry, electronic structure of the key reaction intermediates, and the individual energy contributions of second coordination sphere (SCS) residues through combined quantum mechanics/molecular mechanics (QM/MM) calculations. Experimentally generated variant forms of EFE with alanine substituted for SCS residues responsible for stabilizing the key intermediates in the two reactions of EFE led to changes in enzyme activity, thus demonstrating the key role of these residues. Overall, the results of applying an ExtEF indicate that making the IntEF of EFE less negative and stabilizing the off-line binding of 2OG is predicted to increase ethylene generation while reducing l-Arg hydroxylation

Can Second Coordination Sphere and Long-Range Interactions Modulate Hydrogen Atom Transfer in a Non-Heme Fe(II)-Dependent Histone Demethylase?

Fe(II)-dependent oxygenases employ hydrogen atom transfer (HAT) to produce a myriad of products. Understanding how such enzymes use dynamic processes beyond the immediate vicinity of the active site to control the selectivity and efficiency of HAT is important for metalloenzyme engineering; however, obtaining such knowledge by experiments is challenging. This study develops a computational framework for identifying second coordination sphere (SCS) and especially long-range (LR) residues relevant for catalysis through dynamic cross-correlation analysis (DCCA) using the human histone demethylase PHF8 (KDM7B) as a model oxygenase. Furthermore, the study explores the mechanistic pathways of influence of the SCS and LR residues on the HAT reaction. To demonstrate the plausibility of the approach, we investigated the effect of a PHF8 F279S clinical mutation associated with X-linked mental retardation, which has been experimentally shown to ablate PHF8-catalyzed demethylation. In agreement, the molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies showed a change in the H31-14K9me2 substrate orientation and an increased HAT barrier. We systematically analyzed the pathways by which the identified SCS and LR residues may influence HAT by exploring changes in H3K9me2 substrate orientation, interdomain correlated motions, HAT transition state stabilization, reaction energetics, electron transfer mechanism, and alterations in the intrinsic electric field of PHF8. Importantly, SCS and LR variations decrease key motions of α9-α12 of the JmjC domain toward the Fe(IV)-center that are associated with tighter binding of the H31-14K9me2 substrate. SCS and LR residues alter the intrinsic electric field of the enzyme along the reaction coordinate and change the individual energetic contributions of residues toward TS stabilization. The overall results suggest that DCCA can indeed identify non-active-site residues relevant for catalysis. The substitutions of such dynamically correlated residues might be used as a tool to tune HAT in non-heme Fe(II)- and 2OG-dependent enzymes

Catalysis by KDM6 Histone Demethylases - A Synergy between the Non-Heme Iron(II) Center, Second Coordination Sphere, and Long-Range Interactions

KDM6A (UTX) and KDM6B (JMJD3) are human non-heme Fe(II) and 2-oxoglutarate (2OG) dependent JmjC oxygenases that catalyze the demethylation of trimethylated lysine 27 in the N-terminal tail of histone H3, a post-translational modification that regulates transcription. We performed Combined Quantum Mechanics/ Molecular Mechanics and Molecular Dynamics study on the catalytic mechanism of KDM6A/B. The results reveal that the transition state for the rate-limiting hydrogen atom transfer (HAT) reaction in KDM6A/B catalysis is stabilized by polar (Asn217) and aromatic (Trp369)/non-polar (Pro274) residues in contrast to KDM4 and KDM7 demethylases where charged residues (Glu, Arg, Asp) are involved. KDM6A employs both σ- and π-electron transfer pathways for HAT, whereas KDM6B employs the σ-electron pathway. Differences in hydrogen bonding of the Fe-chelating Glu252(KDM6B) contribute to the lower energy barriers in KDM6B vs. KDM6A. The study reveals a dependence of the activation barrier of the rebound hydroxylation on the Fe-O-C angle in the transition state of KDM6A. Anti-correlation of the Zn-binding domain with the active site residues is a key factor distinguishing KDM6A/B from KDM7/4s. The results reveal the importance of communication between the Fe center, second coordination sphere, and long-range interactions in catalysis by KDMs and, by implication, other 2OG oxygenases

Dioxygen Binding Is Controlled by the Protein Environment in Non-heme Fe\u3csup\u3eII\u3c/sup\u3e and 2-Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene-Forming Enzyme

This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme FeII/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 FeIII-OO⋅− intermediate in PHF8 and the off-line FeIII-OO⋅− intermediate in EFE