Circular dichroism, magnetic circular dichroism, and variable temperature variable field magnetic circular dichroism studies of biferrous and mixed-valent myo-inositol oxygenase: insights into substrate activation of O2 reactivity.

myo-Inositol oxygenase (MIOX) catalyzes the 4e(-) oxidation of myo-inositol (MI) to D-glucuronate using a substrate activated Fe(II)Fe(III) site. The biferrous and Fe(II)Fe(III) forms of MIOX were studied with circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The MCD spectrum of biferrous MIOX shows two ligand field (LF) transitions near 10000 cm(-1), split by ~2000 cm(-1), characteristic of six coordinate (6C) Fe(II) sites, indicating that the modest reactivity of the biferrous form toward O2 can be attributed to the saturated coordination of both irons. Upon oxidation to the Fe(II)Fe(III) state, MIOX shows two LF transitions in the ~10000 cm(-1) region, again implying a coordinatively saturated Fe(II) site. Upon MI binding, these split in energy to 5200 and 11200 cm(-1), showing that MI binding causes the Fe(II) to become coordinatively unsaturated. VTVH MCD magnetization curves of unbound and MI-bound Fe(II)Fe(III) forms show that upon substrate binding, the isotherms become more nested, requiring that the exchange coupling and ferrous zero-field splitting (ZFS) both decrease in magnitude. These results imply that MI binds to the ferric site, weakening the Fe(III)-µ-OH bond and strengthening the Fe(II)-µ-OH bond. This perturbation results in the release of a coordinated water from the Fe(II) that enables its O2 activation.

myo-Inositol oxygenase: a radical new pathway for O(2) and C-H activation at a nonheme diiron cluster.

The enzyme myo-inositol oxygenase (MIOX) catalyzes conversion of myo-inositol (cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to d-glucuronate (DG), initiating the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides. Recent kinetic, spectroscopic and crystallographic studies have shown that the enzyme activates its substrates, MI and O(2), at a carboxylate-bridged nonheme diiron(ii/iii) cluster, making it the first of many known nonheme diiron oxygenases to employ the mixed-valent form of its cofactor. Evidence suggests that: (1) the Fe(iii) site coordinates MI via its C1 and C6 hydroxyl groups; (2) the Fe(ii) site reversibly coordinates O(2) to produce a superoxo-diiron(iii/iii) intermediate; and (3) the pendant oxygen atom of the superoxide ligand abstracts hydrogen from C1 to initiate the unique C-C-bond-cleaving, four-electron oxidation reaction. This review recounts the studies leading to the recognition of the novel cofactor requirement and catalytic mechanism of MIOX and forecasts how remaining gaps in our understanding might be filled by additional experiments.

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

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.

A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

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 O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), 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 O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) 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/O(2) 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 O(2). The DG/O(2) 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 O(2) from the II/II manifold.