Atomic and Electronic Structure Determinants Distinguish between Ethylene Formation and l-Arginine Hydroxylation Reaction Mechanisms in the Ethylene-Forming Enzyme

Abstract

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.

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1

“…In an alternative pathway, the Fe III −OH species may undergo the OH‐rebound to the C21 radical site via a slightly larger barrier of 5.2 kcal mol −1 , affording to the hydroxylated substrate which is 27.6 kcal mol −1 more stable than the intermediate IC1 (Figure S13). The calculated rebound barrier is in line with previous studies [60–73] . The hydroxylated species would lead to the side product 8 through elimination (refer to Scheme 3), as observed in experiments.…”

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1

“…Thec alculated rebound barrier is in line with previous studies. [60][61][62][63][64][65][66][67][68][69][70][71][72][73] The hydroxylated species would lead to the side product 8 through elimination (refer to Scheme 3), as observed in experiments. Clearly,t he dioxygen attack is kinetically favorable over the rebound pathway,w hile the rebound pathway is more thermodynamically favorable.Inthe situation of low concen- S3 and Figure S15).…”

Hybrid radical-polar pathway for excision of ethylene from 2-oxoglutarate by an iron oxygenase

“…5 is supported by both the random ethylene stereochemistry and the two O atom transfers-from O 2 to the C1-derived CO 2 [via (bi)carbonate] and from the C1-carboxylate to a hydroxylated product-that have not previously been seen in the reaction of an Fe/2OG oxygenase. These outcomes appear to flow from the recently proposed C1-C2 O 2 -insertion step, which provides an entirely new way to envisage access to activated iron and fragmented 2OG intermediates and/or products in this class of enzymes (16,17). The mechanism of the net insertion [the two computational studies advanced different hypotheses (16,17)], precisely how this step is promoted by the EFE active site, and whether it is specific to EFE, occurs in other Fe/2OG systems, or may be elicited by enzyme engineering all remain to be established.…”

“…These outcomes appear to flow from the recently proposed C1-C2 O 2 -insertion step, which provides an entirely new way to envisage access to activated iron and fragmented 2OG intermediates and/or products in this class of enzymes (16,17). The mechanism of the net insertion [the two computational studies advanced different hypotheses (16,17)], precisely how this step is promoted by the EFE active site, and whether it is specific to EFE, occurs in other Fe/2OG systems, or may be elicited by enzyme engineering all remain to be established. In addition, although couplings of substrate carbon radicals to Fe(III)coordinated alkylthiolate, halide, nitrite, and azide have been implicated in studies of related enzymes (25-27), we are not aware of any precedent for the proposed radical coupling to carbonate.…”