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Thermodynamics of Iron(II) and Substrate Binding to the Ethylene-Forming Enzyme
journal contribution
posted on 2018-09-05, 00:00 authored by Mingjie Li, Salette Martinez, Robert P. Hausinger, Joseph P. EmersonThe
ethylene-forming enzyme (EFE), like many other 2-oxoglutarate
(2OG)-dependent nonheme iron(II) oxygenases, catalyzes the oxidative
decarboxylation of 2OG to succinate and CO2 to generate
a highly reactive iron species that hydroxylates a specific alkane
C–H bond, in this case targeting l-arginine (Arg)
for hydroxylation. However, the prominently observed reactivity of
EFE is the transformation of 2OG into ethylene and three molecules
of CO2. Crystallographic and biochemical studies have led
to several proposed mechanisms for this 2-fold reactivity, but the
detailed reaction steps are still obscure. Here, the thermodynamics
associated with iron(II), 2OG, and Arg binding to EFE are studied
using calorimetry (isothermal titration calorimetry and differential
scanning calorimetry) to gain insight into how these binding equilibria
organize the active site of EFE, which may have an impact on the O2 activation pathways observed in this system. Calorimetric
data show that the addition of iron(II), Arg, and 2OG increases the
stability over that of the apoenzyme, and there is distinctive cooperativity
between substrate and cofactor binding. The energetics of binding
of 2OG to Fe·EFE are consistent with a unique monodentate binding
mode, which is different than the prototypical 2OG coordination mode
in other 2OG-dependent oxygenases. This difference in the pre-O2 activation equilibria may be important for supporting the
alternative ethylene-forming chemistry of EFE.