. Recent studies have suggested that the enzyme, which was shown nearly 50 years ago to require iron (1, 2), contains a coupled dinuclear nonheme iron cluster (5), making MIOX the most recent addition to the nonheme diiron oxygenase͞oxidase family that also includes bacterial hydrocarbon hydroxylases (e.g., soluble methane monooxygenase), plant fatty acyl desaturases (e.g., stearoyl acyl carrier protein ⌬ 9 desaturase), and protein R2 of class I ribonucleotide reductase (R2) (6-10). Mössbauer and EPR spectra showed that treatment of recombinant Mus musculus MIOX isolated in its iron-free form from Escherichia coli with Fe(II) and O 2 leads to formation of an antiferromagnetically coupled diiron cluster in either the II͞III or III͞III oxidation state, depending on the O 2 ͞MIOX ratio and the presence or absence of a reductant (ascorbate or cysteine). Binding of MI was shown to perturb the spectra of both oxidation states in a manner consistent with direct coordination of the substrate to the cluster (5).All nonheme diiron oxygenases and oxidases characterized before MIOX activate O 2 with the II͞II oxidation state of the cofactor (11,12). For several of the reactions, (peroxo)diiron(III͞III) intermediates have been demonstrated. These complexes are generally proposed to undergo O-O-bond cleavage to generate high-valent iron complexes that cleave strong C-H or O-H bonds of their oxidation targets (8,(11)(12)(13)(14). Indeed, the diiron(III͞IV) cluster, X (15, 16), and the diiron(IV͞IV) cluster, Q (8, 13, 17), have been directly characterized in the R2 and soluble methane monooxygenase reactions, respectively. In each of the previously characterized diiron-oxygenase͞oxidase reactions, a diiron(III͞III) ''product'' state of the cluster is generated at the end of the oxidation sequence. Subsequent events require reduction of the cluster back to the diiron(II͞II) state by additional proteins, with electrons provided ultimately by NAD(P)H. This redox cycling of the cofactor and provision of two electrons by the nicotinamide ''cosubstrate'' ensure that at most two electrons can be extracted from the substrate. The MIOX reaction, a four-electron oxidation, would seem to require a different mechanism.Indeed, a recent study concluded that the mixed-valent, II͞III state of the cofactor, rather than the conventional II͞II state, activates O 2 for DG production in the MIOX reaction (4). Single-turnover experiments showed that the fully reduced enzyme (MIOX II/II ) reacts with limiting O 2 in the presence of saturating MI to generate the mixed-valent enzyme as a stable product with unit stoichiometry and with only a low yield of DG. By contrast, the

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.

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.Bacterial multi-component monooxygenases [BMMs; e.g., soluble methane monooxygenase (sMMO), toluene/o-xylene monooxygenase (ToMO), and phenol hydroxylase], plant fatty acyl desaturases (e.g., stearoyl acyl carrier protein Δ 9 -desaturase) and the R2 subunits of conventional class I ribonucleotide reductases (RNR-R2s) all use carboxylate-bridged dinuclear iron clusters to activate O 2 for cleavage of strong C-H or O-H bonds. 1-5 Each of these reaction begins with the reduction of O 2 to the peroxide oxidation state by the diiron(II/ II) form of the cofactor. In several cases, peroxide-bridged diiron(III/III) intermediates have been directly characterized. 6-11 Several of the peroxide complexes are known or believed to undergo O-O-bond cleavage to generate high-valent iron complexes that cleave the target C/ O-H bonds. 1-5 For example, the diiron(III/IV) complex, X, in the RNR-R2 reaction oxidizes a tyrosine residue by one electron, cleaving the phenolic O-H bond and activating the protein for participation in nucleotide reduction with its partner subunit, RNR-R1. [12][13][14][15][16][17][18][19][20][21] Similarly, the diiron(IV/IV) complex, Q, in the sMMO reaction cleaves a C-H bond of methane to initiate its hydroxylation. 1,3,7,[22][23][24] In each of these reactions, a diiron(III/III) form of the cluster is generated at the end of the oxidation sequence. For the reactions that are catalytic, a complete "turnover" therefore requires reduction of the cluster back to the O 2 -reactive diiron(II/II) state by additional proteins, with electrons provided ultimately by NAD(P)H. 1,3,4 Although this Please send correspondence to: J.

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 O2. 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) MIOX(II/III)·MI 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.

It was possible to immobilize the laccase on carboxylated polyvinyl alcohol by activation with N-hydroxysuccinimide in HAc-NaAc buffer. The immobilized laccase is both stable and reusable. SIGNIFICANCE IMPACT OF THE STUDY: The results indicate that this immobilized laccase can be used in the removal of poisonous effluent from pulp bleaching mills.