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

doi:10.1073/pnas.0508473103

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

*Departments of Biochemistry and Molecular Biology,
^†Veterinary and Biomedical Sciences, and
^‡Chemistry, Pennsylvania State University, University Park, PA 16802

Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved March 2, 2006 (received for review September 28, 2005)

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 ≥5, showing that the superoxide-level complex does indeed cleave a C–H(D) bond of MI.

Footnotes

^§To whom correspondence may be addressed. E-mail: jmb21{at}psu.eduor ckrebs{at}psu.edu
Author contributions: G.X., C.K., and J.M.B. designed research; G.X., Y.D., L.M.H., E.W.B., C.K., and J.M.B. performed research; K.S.P., R.J.A., and C.C.R. contributed new reagents/analytic tools; G.X., Y.D., L.M.H., E.W.B., C.K., and J.M.B. analyzed data; and C.K. and J.M.B. wrote the paper.
↵ ¶ When J _Fe-Fe is in the range from 0.25 × J _{Fe-superoxide} to 1 × J _{Fe-superoxide}, c _A = c _B = +2/3 is observed. Values of J _Fe-Fe outside of this range are incompatible with the EPR data: for J _Fe-Fe < 0.25 × J _{Fe-superoxide}, the ground state is S = 3/2; for J _Fe-Fe > 1 × J _{Fe-superoxide}, the S = 1/2 ground-state has spin projection factors of 0, which would result in no ⁵⁷Fe-hyperfine coupling in the EPR spectrum.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

MI,

myo-inositol;

MIOX,

MI oxygenase;

DG,

d-glucuronate;

D6-MI,

1,2,3,4,5,6-[2H]6-MI;

FQ,

freeze-quench;

2H-KIE,

deuterium kinetic isotope effect;

H6-MI,

1,2,3,4,5,6-[1H]6-MI.