Tyrosinase is a copper monooxygenase that catalyzes oxygenation of phenols to catechols (phenolase activity) and the subsequent two-electron oxidation of catechols to the corresponding o-quinones (catecholase activity). 1 Chemical and spectroscopic studies have indicated that the enzyme has a dinuclear copper active site nearly identical to that found in hemocyanin, 1,2 where a side-on type (µ-η 2 :η 2 ) peroxo species 3 is generated by the reaction of the reduced dicopper(I) form and O 2 . 1 As a pioneering work by Karlin and co-workers in Cu/O 2 chemistry, aromatic ligand hydroxylation in a dinuclear Cu(I) complex by O 2 was first reported in early 1980s. 4 The mechanistic studies have indicated that the aromatic ligand hydroxylation reaction involves an electrophilic attack on the arene ring by a (µ-η 2 :η 2 -peroxo)-dicopper(II) intermediate. 5 After their finding, several examples of the aromatic ligand hydroxylation have been reported using similar type of m-xylyl dinucleating ligands. 6 With respect to the intermolecular reactions between phenols and the peroxo intermediate, however, most of the reactions so far reported afford a C-C coupling dimer as a major product. 7 Casella and co-workers have recently reported the first synthetic (µ-η 2 :η 2 -peroxo)dicopper-(II) complex which can react with an exogenous phenolate to yield the corresponding catechol. 8,9 Unfortunately, the low yield of the product (20% based on the dicopper complex) has precluded the kinetic and mechanistic investigation on the reaction between the peroxo intermediate and the phenolate. 8 As such, the mechanism for the catechol formation via intermolecular reactions between the peroxo intermediate and phenol derivatives has yet to be clarified. 10 We report herein that efficient conversion of phenol derivatives to the corresponding catechols is achieved for the first time by intermolecular reactions of a (µ-η 2 :η 2 -peroxo)dicopper(II) complex, supported by tridentate ligand L Py2Bz (N,N-bis[2-(2-pyridyl)-ethyl]-R,R-dideuteriobenzylamine), 11 with lithium salts of phenols. The mechanistic studies on the catechol formation have been performed to provide valuable mechanistic insight into the phenolase activity of the enzyme.Treatment of the copper(I) complex, [Cu I (L Py2Bz )](PF 6 ), with dioxygen in anhydrous acetone at -94°C afforded a brown color solution which exhibited a strong absorption band at 364 nm ( ) 26400 M -1 cm -1 ) together with a small one at 530 nm (1500 M -1 cm -1 ) and a resonance Raman band at 737 cm -1 that shifted to 697 cm -1 upon 18 O-substitution. 12,13 The frozen acetone solution of the intermediate was ESR silent at 77 K, and a Cu:O 2 ) 2:1 stoichiometry was obtained for formation of the intermediate by manometry. These results unambiguously indicate that the oxygenated intermediate is a (µ-η 2 :η 2 -peroxo)dicopper(II) complex as suggested previously by Karlin et al. 11 This compound is quite stable (no self-decomposition) at the low-temperature enabling us to examine the reaction with external subst...

Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF(3)) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O(2) dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O(2) binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O(2). These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure-function relationship of the protein.

Oxygenation of copper(I) with tetradentate tripodal ligands (L) comprised of a tris(aminoethyl)amine (tren) skeleton having sterically bulky substituent(s) on the terminal nitrogens has been investigated, whereAll the copper(I) complexes reacted with dioxygen at low temperatures to produce superoxocopper(II) and/ or trans-("-1,2-peroxo)-dicopper(II) complexes depending on the steric bulkiness of the terminal nitrogens and the reaction conditions. The reaction of a copper( þ ($0:24 mM) and higher dioxygen concentration (P(O 2 ) = $1 atm), the superoxo species is predominantly formed, whereas at a higher concentration of [Cu(L Me,Bn )] þ ($1 mM) and lower dioxygen concentration (P(O 2 ) = $0:02 atm) the formation of the peroxo species is observed.

Copper(I)-dioxygen reactivity has been examined using a series of 2-(2-pyridyl)ethylamine bidentate ligands (R1)Py1(R2,R3). The bidentate ligand with the methyl substituent on the pyridine nucleus (Me)Py1(Et,Bz) (N-benzyl-N-ethyl-2-(6-methylpyridin-2-yl)ethylamine) predominantly provided a (mu-eta(2):eta(2)-peroxo)dicopper(II) complex, while the bidentate ligand without the 6-methyl group (H)Py1(Et,Bz) (N-benzyl-N-ethyl-2-(2-pyridyl)ethylamine) afforded a bis(mu-oxo)dicopper(III) complex under the same experimental conditions. Both Cu(2)O(2) complexes gradually decompose, leading to oxidative N-dealkylation reaction of the benzyl group. Detailed kinetic analysis has revealed that the bis(mu-oxo)dicopper(III) complex is the common reactive intermediate in both cases and that O[bond]O bond homolysis of the peroxo complex is the rate-determining step in the former case with (Me)Py1(Et,Bz). On the other hand, the copper(I) complex supported by the bidentate ligand with the smallest N-alkyl group ((H)Py1(Me,Me), N,N-dimethyl-2-(2-pyridyl)ethylamine) reacts with molecular oxygen in a 3:1 ratio in acetone at a low temperature to give a mixed-valence trinuclear copper(II, II, III) complex with two mu(3)-oxo bridges, the UV-vis spectrum of which is very close to that of an active oxygen intermediate of lacase. Detailed spectroscopic analysis on the oxygenation reaction at different concentrations has indicated that a bis(mu-oxo)dicopper(III) complex is the precursor for the formation of trinuclear copper complex. In the reaction with 2,4-di-tert-butylphenol (DBP), the trinuclear copper(II, II, III) complex acts as a two-electron oxidant to produce an equimolar amount of the C[bond]C coupling dimer of DBP (3,5,3',5'-tetra-tert-butyl-biphenyl-2,2'-diol) and a bis(mu-hydroxo)dicopper(II) complex. Kinetic analysis has shown that the reaction consists of two distinct steps, where the first step involves a binding of DBP to the trinuclear complex to give a certain intermediate that further reacts with the second molecule of DBP to give another intermediate, from which the final products are released. Steric and/or electronic effects of the 6-methyl group and the N-alkyl substituents of the bidentate ligands on the copper(I)-dioxygen reactivity have been discussed.

(mu-Hydroxo or oxo)(mu-1,2-peroxo)diiron(III) complexes having a tetradentate tripodal ligand (L) containing a carboxylate sidearm [Fe2(mu-OH or mu-O)(mu-O2)(L)2]n+ were synthesized as models for peroxo-intermediates of non-heme diiron proteins and characterized by various physicochemical measurements including X-ray analysis, which provide fundamental structural and spectroscopic insights into the peroxodiiron(III) complexes.

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 Å resolution) and reduced form (at 1.8 Å resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His 87 (equivalent to His 90 in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals -stacking between Phe 12 and His 90 , suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His 90 (the "adjacent" site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.

A novel hydroperoxo-copper(II) complex with a square-planar geometry has been prepared, which has exhibited a higher selectivity and catalytic reactivity for dimethyl sulfide, in contrast to that with a trigonal-bipyramidal one.

Bis(mu-oxo)dinickel(III) complexes supported by a series of bis[2-(2-pyridyl)ethyl]amine ligands have been successfully generated by treating the corresponding bis(mu-hydroxo)dinickel(II) complexes or bis(mu-methoxo)dinickel(II) complex with an equimolar amount of H(2)O(2) in acetone at low temperature. The bis(mu-oxo)dinickel(III) complexes exhibit a characteristic UV-vis absorption band at approximately 410 nm and a resonance Raman band at 600-610 cm(-1) that shifted to 570-580 cm(-1) upon (18)O-substitution. Kinetic studies and isotope labeling experiments using (18)O(2) imply the existence of intermediate(s) such as peroxo dinickel(II) in the course of formation of the bis(mu-oxo)dinickel(III) complex. The bis(mu-oxo)dinickel(III) complexes supported by the mononucleating ligands (L1(X) = para-substituted N,N-bis[2-(2-pyridyl)ethyl]-2-phenylethylamine; X = OMe, Me, H, Cl) gradually decompose, leading to benzylic hydroxylation of the ligand side arm (phenethyl group). The kinetics of the ligand hydroxylation process including kinetic deuterium isotope effects (KIE), p-substituent effects (Hammett plot), and activation parameters (Delta H(H)(*) and Delta S(H)(*)) indicate that the bis(muxo)dinickel(III) complex exhibits an ability of hydrogen atom abstraction from the substrate moiety as in the case of the bis(mu-oxo)dicopper(III) complex. Such a reactivity of bis(mu-oxo)dinickel(III) complexes has also been suggested by the observed reactivity toward external substrates such as phenol derivatives and 1,4-cyclohexadiene. The thermal stability of the bis(mu-oxo)dinickel(III) complex is significantly enhanced when the dinucleating ligand with a longer alkyl strap is adopted instead of the mononucleating ligand. In the m-xylyl ligand system, no aromatic ligand hydroxylation occurred, showing a sharp contrast with the reactivity of the (mu-eta(2):eta(2)-peroxo)dicopper(II) complex with the same ligand which induces aromatic ligand hydroxylation via an electrophilic aromatic substitution mechanism. Differences in the structure and reactivity of the active oxygen complexes between the nickel and the copper systems are discussed on the basis of the detailed comparison of these two systems with the same ligand.