How Do Variants of Residues in the First Coordination Sphere, Second Coordination Sphere, and Remote Areas Influence the Catalytic Mechanism of Non-Heme Fe(II)/2-Oxoglutarate Dependent Ethylene-Forming Enzyme?

Abstract

The ethylene-forming enzyme (EFE) is a Fe(II)/2-oxoglutarate (2OG) and l-arginine (l-Arg)-dependent oxygenase that primarily decomposes 2OG into ethylene while also catalyzing l-Arg hydroxylation. While the hydroxylation mechanism in EFE is similar to other Fe(II)/2OG-dependent oxygenases, the formation of ethylene is unique. Various redesign strategies have aimed to increase ethylene production in EFE, but success has been limited, highlighting the need for alternate approaches. It is crucial to incorporate an accurate and comprehensive description of the integrative and multidimensional effects of the protein environment to enhance the redesign strategy in metalloenzymes, particularly in EFE. This involves understanding the role of the second coordination sphere (SCS) and long-range (LR) interacting residues, correlated motions, electronic structure, intrinsic electric field (IntEF), as well as the stabilization of transition states and reaction intermediates. In this study, we employ a molecular dynamics-based quantum mechanics/molecular mechanics approach to examine the integrative effects of the protein environment on reactions catalyzed by EFE variants from the first coordination sphere (FCS, D191E), SCS (A198V and R171A) and LR (E215A). The study uncovers how substitutions at different positions in EFE similarly impact the ethylene-forming reaction while posing distinct effects on the hydroxylation reaction. Results predict the effect of the variants in controlling the 2OG coordination mode in the Fe(II) center. Specifically, the study suggests that D191E uniquely prefers transitioning from an off-line to an in-line 2OG coordination mode before dioxygen binding. However, studies on the 2OG flip in the presence of off-line approaching dioxygen and dioxygen binding in the D191E variant indicate that the 2OG flip might not be feasible in the 5C Fe(II) state. Calculations show the possibility of a hydrogen atom transfer (HAT)-assisted oxygen flip in EFE and its variants (other than D191E). MD simulations elucidate the characteristic dynamic change in the α7 region in the D191E variant that might contribute to its increased hydroxylation reaction. Results indicate the possibility of forming an in-line ferryl from the IM2 (Fe(III)-partial bond intermediate) in the D191E variant. This alternative pathway from IM2 may also exist in WT EFE and other variants, which are yet to be explored. The study also delineates the impact of substitutions on the electronic structure and IntEF. Overall, the calculations support the idea that understanding the integrative and multidimensional effects of the protein environment on the reactions catalyzed by EFE variants provides the basics for improved enzyme redesign protocols of EFE to increase ethylene production. The results of this study will also contribute to the development of alternate redesign strategies for other metalloenzymes.

Figure 1

(A) Schematic representation of the various reactions catalyzed by EFE. (B) The structure of the wild-type (WT) EFE shows the position of the chosen EFE variants for the study. The variants are shown in green (D191E), red (A198V), magenta (E215A), and blue (R171A). The active site is zoomed in on the right. The position of variants and the active site of EFE are shown in the modeled 5V2Y PDB file, representing the Fe(III)·OO^•– complex.

Figure 2

Proposed reaction pathway for the divergent reactions catalyzed by WT EFE. The hydroxylation pathway is shown in blue arrows, and the ethylene and 3-HP formation is in magenta. Here, R represents the substrate l-Arg.

Figure 3

Orientation of the l-Arg substrate in WT (yellow), A198V (red), D191E (green), E215A (magenta), and R171A (blue) versions of EFE.

Figure 4

(A) Dynamic cross-correlation analysis (DCCA) of WT EFE. Differential DCCA (d-DCCA) comparison of the WT enzyme versus (B) A198V, (C) D191E, (D) E215A, and (E) R171A variants of EFE. The positive values shown in cyan represent the positively correlated motions. The negative values in pink represent anticorrelated motions. The identified flexible regions in the WT EFE are shown in circles.

Figure 5

QM/MM potential energy profile for the dioxygen activation reaction in WT EFE and its variants. The energies are calculated using UB3LYP/def2-TZVP with ZPE. The relative energies are shown in kcal/mol. The ChemDraw images for the corresponding reactants and intermediates are provided below the potential energy surface.

Figure 6

Orientation of the TS1 complex during dioxygen activation in WT (yellow), A198V (red), D191E (green), E215A (magenta), and R171A (blue) versions of EFE.

Figure 7

Natural orbitals showing the Fe–O bond character in EFE variants.

Figure 8

Energetically stabilizing (orange) and destabilizing (violet) residues during the dioxygen activation reaction in the (A) A198V, (B) D191E, (C) E215A, and (D) R171A EFE variants are shown in stick mode.

Figure 9

QM/MM potential energy surface for the (A) HAT-assisted oxygen flip in the 5C Fe(IV) center and (B) 2OG rotation in the 5C Fe(II) center. The energies are calculated using UB3LYP/def2-TZVP with ZPE. The relative energies are shown in kcal/mol. The ChemDraw images for the corresponding reactants and intermediates are provided below the potential energy surface.

Figure 10

QM/MM potential energy profile for the off-line dioxygen binding in the Fe(II)-center of the D191E EFE in the septet (red) and quintet (black) surfaces. The calculated energies are at the B1 level, and the relative energies are shown in kcal/mol.

Figure 11

QM/MM potential energy surface for the HAT and rebound hydroxylation reactions in WT EFE and its variants. The energies are calculated using UB3LYP/def2-TZVP with ZPE. The relative energies are shown in kcal/mol. The ChemDraw images for the corresponding reactants and intermediates are provided below the potential energy surface.

Figure 12

Spin natural orbitals (SNOs) for the HAT transition states in variant EFE proteins.

Figure 13

Energetically stabilizing (orange) and destabilizing (violet) residues during the HAT reaction in the EFE variants (A) A198V, (B) D191E, (C) E215A, and (D) R171A are shown in stick mode.