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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 CO(2) 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 CO(2). 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 O(2) 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-O(2) activation equilibria may be important for supporting the alternative ethylene-forming chemistry of EFE.

Stopped-flow mixing of the Desulfovibrio vulgaris two-iron superoxide reductase (2Fe-SOR) containing the ferrous active site with superoxide generates a dead time intermediate whose absorption spectrum is identical to that of a putative ferric-hydroperoxo intermediate previously observed by pulse radiolysis. The dead time intermediate is shown to be a product of reaction with superoxide and to be generated at a much higher proportion of active sites than by pulse radiolysis. This intermediate decays smoothly to the resting ferric active site ( approximately 30 s-1 at 2 degrees C and pH 7) with no other detectable intermediates. Deuterium isotope effects demonstrate that solvent proton donation occurs in the rate-determining step of dead time intermediate decay and that neither of the conserved pocket residues, Glu47 or Lys48, functions as a rate-determining proton donor between pH 6 and pH 8. Fluoride, formate, azide, and phosphate accelerate decay of the dead time intermediate and for azide or fluoride lead directly to ferric-azido or -fluoro complexes of the active site, which inhibit Glu47 ligation. A solvent deuterium isotope effect is observed for the azide-accelerated decay, and the decay rate constants are proportional to the concentrations and pKa values of HX (X- = F-, HCO2-, N3-). These data indicate that the protonated forms of the anions function analogously to solvent as general acids in the rate-determining step. The results support the notion that the ferrous SOR site reacts with superoxide by an inner sphere process, leading directly to the ferric-hydroperoxo intermediate, and demonstrate that the decay of this intermediate is subject to both specific- and general-acid catalysis.

Superoxide reductases (SORs) contain a novel square pyramidal ferrous [Fe(NHis)(4)(SCys)] site that rapidly reduces superoxide to hydrogen peroxide. Here we report extensive pulse radiolysis studies on recombinant two-iron SOR (2Fe-SOR) from Desulfovibrio vulgaris. The results support and elaborate on our originally proposed scheme for reaction of the [Fe(NHis)(4)(SCys)] site with superoxide [Coulter, E. D., Emerson, J. E., Kurtz, D. M., Jr., and Cabelli, D. E. (2000) J. Am. Chem. Soc. 122, 11555-11556]. This scheme consists of second-order diffusion-controlled formation of an intermediate absorbing at approximately 600 nm, formulated as a ferric-(hydro)peroxo species, and its decay to the carboxylate-ligated ferric [Fe(NHis)(4)(SCys)] site with loss of hydrogen peroxide. The second-order rate constant for formation of the 600-nm intermediate is essentially pH-independent (pH 5-9.5), shows no D(2)O solvent isotope effect at pH 7.7, and decreases with increasing ionic strength. These data indicate that formation of the intermediate does not involve a rate-determining protonation, and are consistent with interaction of the incoming superoxide anion with a positive charge at or near the ferrous [Fe(NHis)(4)(SCys)] site. The rate constant for decay of the 600-nm intermediate follows the pH-dependent rate law: k(2)(obs) = k(2)'[H(+)] + k(2)' ' and shows a significant D(2)O solvent isotope effect at pH 7.7. The values of k(2)' and k(2)' ' indicate that the 600-nm intermediate decays via diffusion-controlled protonation at acidic pHs and a first-order process involving either water or a water-exchangeable proton on the protein at basic pHs. The formation and decay rate constants for an E47A variant of 2Fe-SOR are not significantly perturbed from their wild-type values, indicating that the conserved glutamate carboxylate does not directly displace the (hydro)peroxo ligand of the intermediate at basic pHs. The kinetics of a K48A variant are consistent with participation of the lysyl side chain in directing the superoxide toward the active site and in directing the protonation pathway of the ferric-(hydro)peroxo intermediate toward release of hydrogen peroxide.