Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase

Abstract

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI complex reacts rapidly and reversibly with O2 to form an intermediate, G, with a g = (2.05, 1.98, 1.90) EPR signal. The rhombic g-tensor and observed hyperfine coupling to 57Fe are rationalized in terms of a (superoxo)diiron(III/III) structure with coordination of the superoxide to a single iron. G decays to H, the intermediate previously detected in the reaction with unlabeled substrate. This step is associated with a kinetic isotope effect of > or =5, showing that the superoxide-level complex does indeed cleave a C-H(D) bond of MI.

Scheme 1.

Reaction catalyzed by MIOX and the mechanism used to simulate kinetics of MIOX^II/III·MI, G, and H in the FQ EPR experiment depicted in Figs. 2 and 3. The rate constants for MI binding and dissociation, decay of H, and release of DG are taken from a previous study (4). For rate constants lacking error limits, more experiments are required to estimate such limits.

Fig. 1.

Stopped-flow absorption evidence for a substrate ²H-KIE in the MIOX reaction. Concentrations after mixing were ≈0.3 mM MIOX^II/III·MI (0.5 mM total MIOX), 0.10 mM O₂, and 25 mM D₆-MI (solid traces) or H₆-MI (dashed traces). (A) The change in absorbance at 390 nm with time for the two reactions. (B) Kinetic-difference absorption spectra (changes with respect to the first reliable spectrum at 1.9 ms).

Fig. 2.

X-band EPR spectra at 10 K of FQ samples from the reaction at 5°C of MIOX^II/III·D₆-MI with excess O₂. Concentrations after mixing were 0.22 mM MIOX^II/III·MI, 0.95 mM O₂, and 33 mM D₆-MI. Reaction times were 0 ms (A), 24 ms (B), 60 ms (C), and 260 ms (D). Note the different scaling of A.

Fig. 3.

Kinetics of MIOX^II/III·D₆-MI (black), G (red), and H (blue) determined (as described in Supporting Text) from the FQ EPR experiment of Fig. 2. Concentrations are expressed in terms of the fraction of the initial [MIOX^II/III·D₆-MI]. The solid lines are simulations according to Scheme 1.

Fig. 4.

Photolytic decay of G at 77 K and its use to resolve the EPR spectrum and ⁵⁷Fe hyperfine coupling for G. (A) The spectrum is of the 0.043-s sample from the experiment of Fig. 2 and was obtained after the sample had been stored in the dark in liquid nitrogen for 1 week. (B) The spectrum was acquired after subsequent exposure of the same sample to laboratory fluorescent light for 40 min in liquid nitrogen. (C) The solid line is the change caused by the exposure (A − B). The dotted trace is a simulation of this difference spectrum. (D) The difference spectrum from equivalent analysis of a G-containing sample prepared with ⁵⁷Fe (solid line) and simulations generated by applying to the theoretical spectrum for ⁵⁶Fe-G two isotropic ⁵⁷Fe hyperfine-coupling tensors of either 7 G (corresponding to the superoxide-bridged formulation for G; dashed trace) or 13 G and 25 G [corresponding to Fe_A and Fe_B, respectively, of the favored terminal (superoxo)diiron(III/III) formulation for G; dotted trace]. The experimental spectra were acquired at 10 K.

Fig. 5.

Accumulation of G early in the H₆-MI reaction (red spectra) and photolysis to quantify it relative to [G] in an identical sample from the D₆-MI reaction (blue spectra). The reaction was carried out at 0.5°C. Concentrations were 0.20 mM MIOX^II/III·MI, 1.0 mM O₂, and 33 mM MI. The reaction time was 0.020 s. (A) The samples before light exposure. (B) The same samples after light exposure as in Fig. 4. (C) The difference spectra (A − B). Note the different scaling in C. The spectra were acquired at 12.5 K. Ten scans were accumulated for each.

Scheme 2.

Possible mechanisms for conversion of MI to DG initiated by the formally (superoxo)diiron(III/III) intermediate G. Both pathways would be initiated by abstraction of hydrogen, most likely as a hydrogen atom, from C1 (A). (B Upper) The first class would involve formation of the 1-hydroperoxy-MI intermediate. (B Lower) The second class would involve formation of a (possibly) coordinated gem-diol(ate) intermediate by formal hydroxylation.