myo -Inositol oxygenase (MIOX) activates O ₂ 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 C ₁ by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[ ² H] ₆ -MI (D ₆ -MI), has now permitted initial characterization of the C–H-cleaving intermediate. The MIOX·1,2,3,4,5,6-[ ² H] ₆ -MI complex reacts rapidly and reversibly with O ₂ 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 ⁵⁷ Fe 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.