Structures and Mechanisms of the Non-Heme Fe(II)- and 2‑Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist

The ethylene-forming enzyme (EFE)
from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear
nonheme Fe­(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily.
EFE converts 2OG into ethylene plus three CO2 molecules
while also catalyzing the C5 hydroxylation of l-arginine
(l-Arg) driven by the oxidative decarboxylation of 2OG to
form succinate and CO2. Here we report 11 X-ray crystal
structures of EFE that provide insight into the mechanisms of these
two reactions. Binding of 2OG in the absence of l-Arg resulted
in predominantly monodentate metal coordination, distinct from the
typical bidentate metal-binding species observed in other family members.
Subsequent addition of l-Arg resulted in compression of the
active site, a conformational change of the carboxylate side chain
metal ligand to allow for hydrogen bonding with the substrate, and
creation of a twisted peptide bond involving this carboxylate and
the following tyrosine residue. A reconfiguration of 2OG achieves
bidentate metal coordination. The dioxygen binding site is located
on the metal face opposite to that facing l-Arg, thus requiring
reorientation of the generated ferryl species to catalyze l-Arg hydroxylation. Notably, a phenylalanyl side chain pointing toward
the metal may hinder such a ferryl flip and promote ethylene formation.
Extensive site-directed mutagenesis studies supported the importance
of this phenylalanine and confirmed the essential residues used for
substrate binding and catalysis. The structural and functional characterization
described here suggests that conversion of 2OG to ethylene, atypical
among Fe­(II)/2OG oxygenases, is facilitated by the binding of l-Arg which leads to an altered positioning of the carboxylate
metal ligand, a resulting twisted peptide bond, and the off-line geometry
for dioxygen coordination.