Thermodynamics of Iron(II) and Substrate Binding to the Ethylene-Forming Enzyme

Abstract

The 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.

Figure 1.

Key features of the EFE monomer structure. (A) The protein binds iron(II) [substituted with manganese(II) in this structure, Protein Data Bank entry 5V2Y] in the center of the characteristic cupin barrel. (B) Active site cavity with bound substrates. The H189, H268, and D191 metal ligands are colored green. The carbon atoms of 2OG are colored yellow, and the carbon atoms of l-arginine (Arg) are colored magenta. The residues stabilizing 2OG are indicated with orange carbon atoms, whereas the residues stabilizing Arg are shown with their purple carbon atoms. Throughout this study, the protein-derived amino acids will be abbreviated with single-letter amino acid codes, whereas the substrate Arg will be designated by its three-letter code for the sake of clarity.

Figure 2.

CD spectra of 5 μM EFE complexes: EFE apoprotein (black), Fe·EFE (purple), EFE·Arg (orange), Fe·EFE·Arg (red), Fe· EFE·2OG (green), and the quaternary complex (blue) consisting of EFE with bound iron(II), Arg, and 2OG.

Figure 3.

Representative raw heat and integrated isotherm for iron(II) binding to EFE in 25 mM TES buffer (pH 7.4) at 25 °C.

Figure 4.

Heat capacity curves for the thermal denaturation of EFE species: EFE apoprotein (black dashes), Fe·EFE (green dots), EFE·Arg (solid orange line), Fe·EFE·Arg (red dashes and dots), Fe·EFE 2OG (blue dashes), and the quaternary complex (solid purple line) consisting of EFE with bound iron(II), Arg, and 2OG.

Figure 5.

Summary of enthalpy changes measured by DSC. δΔH terms associated with each equilibrium are shown in parentheses and have units of kilocalories per mole. Exothermic processes are shown with blue equilibrium arrows, whereas the two endothermic processes are shown with red arrows.

Figure 6.

Comparison of the energetic reaction coordinates from enzyme apoprotein to the quaternary complex (4°) of EFE (blue) and the related enzyme TauD (red). Iron(II)·protein refers to the iron(II)-bound forms of EFE and TauD, and “3°” refers to either of two types of tertiary complexes associated with these enzymes, where dashed lines show the profile for the substrate.

Scheme 1.

Reactions Catalyzed by the Ethylene-Forming Enzyme (EFE)

Scheme 2.

Proposed Equilibria in EFE Prior to the Steps of O₂ Activation