Browsing by Subject "Dioxygenase"

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    Kinetic and spectroscopic studies of cobalt- and manganese-substituted extradiol-cleaving homoprotocatechuate 2,3-dioxygenases
    (2013-02) Fielding, Andrew Jay
    Homoprotocatechuate (HPCA) 2,3-dioxygenase (HPCD) is an Fe(II)-dependent extradiol-cleaving dioxygenase, which oxidatively cleaves the aromatic C(2)-C(3) bond of its catecholic substrate. Here we compare the reactivity of Fe-HPCD with its Mn(II)- and Co(II)-substituted analogues. While Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD exhibits significantly higher KMO2 and kcat values. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. These kinetic differences and the spectroscopic properties of Co-HPCD have proven to be useful in further exploring the unique O2 activation mechanism associated with the extradiol dioxygenase family. Employing the electron-poor substrate analogue 4-nitrocatechol (4NC), which is expected to slow down the rate of catechol oxidation, we were able to trap and characterize the initial O2-adduct in the single-turnover reaction of 4-nitrocatechol by Co-HPCD. This intermediate exhibits an S = 1/2 EPR signal typical of low-spin Co(III)−superoxide complexes. Both the formation and decay of the low-spin Co(III)−superoxide intermediate are slow compared to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O2 binding. Possible effects of the observed spin-state transition upon the rate of O2 binding and catechol oxidation are discussed. Two transient intermediates were detected in the reaction of the [M-HPCD(4XC)] enzyme-substrate complexes (M = Mn or Co, and 4XC = 4-halocatechols, where X = F, Cl, and Br) with O2. The first intermediate (Co4XlCInt1) exhibited an S = 1/2 EPR signal associated with an organic radical species. Based on the UV-Vis and EPR data, Co4XCInt1 was assigned to a unique low-spin [Co(III)(4XSQ*)(hydro)peroxo] species where the semiquinone radical is localized onto C4 of the ring. M4XCInt2 was observed to have a high-spin metal(II) center by EPR and exhibit intense chromophores similar to the independently synthesized halogenated quinones (4XQ). Based on the UV-Vis and EPR data, M4XCInt2 is assigned to a [M(II)(4XQ)(hydro)peroxo] species. The M4XCInt2 species were further characterized by resonance Raman spectroscopy. Resonance enhanced vibrations between 1350-1450 cm-1 suggest that M4XCInt2 is a metal-semiquinone species, conflicting with the initial assignment of these intermediates as a quinone species. Based on the EPR and resonance Raman data, M4XCInt2 might be assigned to a [M(II)(SQ*)O2*-] diradical species.