. 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

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-B-dependent proinflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-B cascade, IB-kinase ␤ (IKK␤) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKK␤ in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of seleniumsupplemented macrophages resulted in a time-dependent increase in 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) formation, an endogenous inhibitor of IKK␤ activity. Further analysis revealed that inhibition of IKK␤ activity in seleniumsupplemented cells correlated with the Michael addition product of 15d-PGJ 2 with Cys-179 of IKK␤, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ 2 -dependent activation of the peroxisome proliferator-activated nuclear receptor-␥ in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ 2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ 2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ 2 represents a previously undescribed code for redox regulation of gene expression by selenium.

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.

Selenium (Se) is an important element required for the optimal functioning of the immune system. Particularly in macrophages, which play a pivotal role in immune regulation, Se acts as a major antioxidant in the form of selenoproteins to mitigate the cytotoxic effects of reactive oxygen species. Here we describe the role of Se as an anti-inflammatory agent and its effect on the macrophage signal transduction pathways elicited by bacterial endotoxin, LPS. Our studies demonstrate that supplementation of Se to macrophages (Se-deficient) leads to a significant decrease in the LPS-induced expression of two important pro-inflammatory genes, cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-alpha) via the inhibition of MAP kinase pathways. Furthermore, Se-deficiency in mice exacerbated the LPS-mediated infiltration of macrophages into the lungs suggesting that Se status is a crucial host factor that regulates inflammation. In summary, our results indicate that Se plays an important role as an anti-inflammatory agent by tightly regulating the expression of pro-inflammatory genes in immune cells.

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.