18O kinetic isotope effects in non-heme iron enzymes: probing the nature of Fe/O2 intermediates

Abstract

Contrasted here are the competitive 18O/16O kinetic isotope effects (18O KIEs) on kcat/Km(O2) for three non-heme iron enzymes that activate O2 at an iron center coordinated by a 2-His-1-carboxylate facial triad: taurine dioxygenase (TauD), (S)-(2)-hydroxypropylphosphonic acid epoxidase (HppE), and 1-aminocyclopropyl-1-carboxylic acid oxidase (ACCO). Measured 18O KIEs of 1.0102 +/- 0.0002 (TauD), 1.0120 +/- 0.0002 (HppE), and 1.0215 +/- 0.0005 (ACCO) suggest the formation in the rate-limiting step of O2 activation of an FeIII-peroxohemiketal, FeIII-OOH, and FeIV O species, respectively. The comparison of the measured 18O KIEs with calculated or experimental 18O equilibrium isotope effects (18O EIEs) provides new insights into the O2 activation through an inner-sphere mechanism at a non-heme iron center.

Figure 1

Isotope fractionation plots for TauD (●), HppE (■), and ACCO (◆). The fits for obtaining ¹⁸O KIEs are shown in solid, dashed, and dotted lines, respectively. Conditions: TauD: 0.2 μM TauD, 0.4–0.6 mM O₂, 2 mM taurine, 2 mM αKG, 0.2 mM ascorbate, 50 mM BisTris pH 6.2, 30 °C; HppE: 10μM HppE, 0.4–0.6 mM O₂, 1 mM S-HPP, 11 μM FMN, 1.5 mM NADH, 20 mM Tris-HCl pH 7.5, 25 °C; ACCO: 5 μM ACCO, 0.3–0.5 mM O₂, 3 mM ACC, 20 mM ascorbate, 20 mM NaHCO₃, 100 mM NaCl, 100 mM MOPS pH 7.2, 25 °C.

Scheme 1

Proposed mechanisms of O₂ activation for TauD, HppE, and ACCO (RDS = proposed rate determining step of k_cat/K_m(O₂), R-H = taurine, Asc = ascorbate).