Laccase is a multicopper oxidase which contains four coppers, one type 1, one type 2, and a coupled binuclear type 3 pair, the type 2 and type 3 copper centers together forming a trinuclear copper cluster. The type 1 mercury derivative of laccase (T1Hg Lc) has the type 1 center substituted with a redox inactive Hg 2+ ion and an intact trinuclear copper cluster. Reaction of reduced T1Hg Lc with dioxygen produces an oxygen intermediate which has now been studied in detail. Isotope ratio mass spectrometry (IRMS) has shown that both oxygen atoms of O 2 are bound in the intermediate. EPR and SQUID magnetic susceptibility studies have shown that the intermediate is diamagnetic. The results combined with X-ray absorption edge data indicate that the intermediate contains a bound peroxide and that the two electrons have derived from the type 3 center which is antiferromagnetically coupled. EXAFS data show that there is no short Cu-oxo bond in the intermediate and that there is a new bridging interaction in the intermediate, with two coppers being separated by 3.4 Å, that is not present in the resting enzyme. Circular dichroism (CD) and magnetic circular dichroism (MCD) studies in the ligand field region confirm that the two type 3 coppers are oxidized and antiferromagnetically coupled and that the type 2 copper is reduced. In addition, the charge transfer (CT) absorption spectrum of the intermediate supports a µ-1, 1 hydroperoxide description based on a comparison to Cu(II)-peroxo model spectra. The decay of the T1Hg Lc oxygen intermediate is pH dependent, slow, and proceeds through an additional intermediate with an MCD spectrum in the CT region analogous to that of the oxygen intermediate in the native enzyme which is at least one electron further reduced. These studies lead to a spectroscopically effective model for peroxide bound to the trinuclear copper cluster site in the intermediate, and provide significant insight into the molecular mechanism of the catalytic reduction of dioxygen to water by the multicopper oxidases.
The first enzyme-based catalyst that is superior to platinum in the four-electron electroreduction of oxygen to water is reported. The smooth Pt cathode reached half and 90% of the mass transport-limited current density at respective overpotentials of -0.4 and -0.58 V in 0.5 M sulfuric acid, and only at even higher overpotentials in pH 7.2 phosphate buffer. In contrast, the smooth "wired" bilirubin oxidase cathode reached half and 90% of the mass transport-limited current density at respective overpotentials as low as -0.2 and -0.25 V. The mass transport-limited current density for the smooth "wired" enzyme cathode in PBS was twice that with smooth Pt in 0.5 M sulfuric acid. Under 1 atm O2 pressure, O2 was electroreduced to water on a polished carbon cathode, coated with the "wired" BOD film, in pH 7.2 saline buffer (PBS) at an overpotential of -0.31 V at a current density of 9.5 mA cm-2. At the same overpotential, the current density of the polished platinum cathode in 0.5 M H2SO4 was 16-fold lower, only 0.6 mA cm-2.
Iron(III)-superoxo intermediates are believed to play key roles in oxygenation reactions by non-heme iron enzymes. We now report that a non-heme iron(II) complex activates O(2) and generates its corresponding iron(IV)-oxo complex in the presence of substrates with weak C-H bonds (e.g., olefins and alkylaromatic compounds). We propose that a putative iron(III)-superoxo intermediate initiates the O(2)-activation chemistry by abstracting a H atom from the substrate, with subsequent generation of a high-valent iron(IV)-oxo intermediate from the resulting iron(III)-hydroperoxo species.
The mechanisms of heterolytic versus homolytic O−O bond cleavage of H2O2, tert-butyl
hydroperoxide (t-BuOOH), 2-methyl-1-phenyl-2-propyl hydroperoxide (MPPH), and m-chloroperoxybenzoic
acid (m-CPBA) by iron(III) porphyrin complexes have been studied by carrying out catalytic epoxidations of
cyclohexene in protic solvent. In these reactions, various iron(III) porphyrin complexes containing electron-withdrawing and -donating substituents on phenyl groups at the meso position of the porphyrin ring were
employed to study the electronic effect of porphyrin ligands on the heterolytic versus homolytic O−O bond
cleavage of the hydroperoxides. In addition, various imidazoles were introduced as axial ligands to investigate
the electronic effect of axial ligands on the pathways of hydroperoxide O−O bond cleavage. Unlike the previous
suggestions by Traylor, Bruice, and co-workers, the hydroperoxide O−O bonds were found to be cleaved
both heterolytically and homolytically and partitioning between heterolysis and homolysis was significantly
affected by the electronic nature of the iron porphyrin complexes (i.e., electronic properties of porphyrin and
axial ligands). Electron-deficient iron porphyrin complexes show a tendency to cleave the hydroperoxide O−O
bonds heterolytically, whereas electron-rich iron porphyrin complexes cleave the hydroperoxide O−O bonds
homolytically. The heterolytic versus homolytic O−O bond cleavage of the hydroperoxides was also found to
be significantly affected by the substituent of the hydroperoxides, ROOH (R = C(O)R‘, H, C(CH3)3, and
C(CH3)2CH2Ph for m-CPBA, H2O2, t-BuOOH, and MPPH, respectively), in which the tendency of O−O bond
heterolysis was in the order of m-CPBA > H2O2 > t-BuOOH > MPPH. This result indicates that the O−O
bond of hydroperoxides containing electron-donating tert-alkyl groups such as t-BuOOH and MPPH tends to
be cleaved homolytically, whereas electron-withdrawing substituents such as an acyl group in m-CPBA facilitates
O−O bond heterolysis. Since we have observed that the homolytic O−O bond cleavage of hydroperoxides
prevails in the reactions performed with electron-rich iron porphyrin complexes and with hydroperoxides
containing electron-donating substituents such as the tert-alkyl group, we suggest that the homolytic O−O
bond cleavage is facilitated when more electron density resides on the O−O bond of (Porp)Fe(III)-OOR
intermediates. We also present convincing evidence that the previous assertion that the reactions of iron(III)
porphyrin complexes with hydrogen peroxide and tert-alkyl hydroperoxides invariably proceed by heterolytic
O−O bond cleavage in protic solvent and that the failure to obtain high epoxide yields in iron porphyrin
complex-catalyzed epoxidation of olefins by hydroperoxides is due to the mechanism of heterolytic O−O
bond cleavage followed by a fast hydroperoxide oxidation is highly unlike.
One primary goal in biomimetic research is to understand mechanisms of dioxygen activation, structures of reactive intermediates, and reactivities of the intermediates involved in catalytic oxidation reactions by metalloenzymes, such as heme and nonheme iron oxygenases. In this communication, we have reported the first example of generating nonheme iron(III)-hydroperoxo and iron(IV)-oxo complexes by activating O(2) with a biologically important electron donor, an NADH analogue, and an acid. The formation of iron(III)-hydroperoxo and iron(IV)-oxo complexes was found to depend on the supporting ligands. We have also demonstrated that high-spin nonheme iron(II) complexes with a low oxidation potential are able to bind and activate O(2) to generate the iron-oxygen intermediates.
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