myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to D-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by Mössbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O2 has been identified. L-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O2, the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O 2, Both observations implicate the mixed-valent, diiron-(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III)-MI] with limiting O2 in the presence of excess, saturating MI reveal the following cycle: (1) reacts rapidly with O2 to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III)-MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O2 stoichiometry in the reaction, 0.8 ± 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O2. The DG/O2 yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III)·MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O2 from the II/II manifold.
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