Dinuclear copper(II) complexes with the new ligand 1,6-bis[[bis(1-methyl-2-benzimidazolyl)methyl]amino]-n-hexane (EBA) have been synthesized, and their reactivity as models for tyrosinase has been investigated in comparison with that of previously reported dinuclear complexes containing similar aminobis(benzimidazole) donor groups. The complex [Cu2(EBA)(H2O)4]4+, five-coordinated SPY, with three nitrogen donors from the ligand and two water molecules per copper, can be reversibly converted into the bis(hydroxo) complex [Cu2(EBA)(OH)2]2+ by addition of base (pK a1 = 7.77, pK a2 = 9.01). The latter complex can also be obtained by air oxidation of [Cu2(EBA)]2+ in methanol. The X-ray structural characterization of [Cu2(EBA)(OH)2]2+ shows that a double μ-hydroxo bridge is established between the two Cu(II) centers in this complex. The coordination geometry of the coppers is distorted square planar, with two benzimidazole donors and two hydroxo groups in the equatorial plane, and an additional, lengthened and severely distorted axial interaction (∼2.5 Å) with the tertiary amine donor. The small size and the quality of the single crystal as well as the fair loss of crystallinity during data collection required the use of synchrotron radiation at 100 K. [Cu2(EBA)(OH)2][PF6]2:  orthorhombic Pca21 space group, a = 22.458(2) Å, b = 10.728(1) Å, c = 19.843(2) Å, R = 0.089. Besides OH-, the [Cu2(EBA)(H2O)4]4+ complex binds azide as a bridging ligand, with the μ-1,3 mode. Azide can also displace μ-OH in [Cu2(EBA)(OH)2]2+ as a bridging ligand. In general, the binding constants indicate that the long alkyl chain of EBA is less easily folded in the structures containing bridging ligands than the m-xylyl residue present in the previously reported dicopper(II) complexes. Electrochemical experiments show that [Cu2(EBA)(H2O)4]4+ undergoes a single, partially chemically reversible, two-electron reduction to the corresponding dicopper(I) congener at positive potential values (E 0‘ = 0.22 V, vs SCE). Interestingly, however, coordination to azide ion makes the reduction process proceed through two separated one-electron steps. The catalytic activity of [Cu2(EBA)(H2O)4]4+ in the oxidation of 3,5-di-tert-butylcatechol has been examined in methanol/aqueous buffer, pH 5.1. The mechanism of the catalytic cycle parallels that of tyrosinase, where no hydrogen peroxide is released and dioxygen is reduced to water. Low-temperature (−80 °C) spectroscopic experiments show that oxygenation of the reduced complex [Cu2(EBA)]2+ does not produce a stable dioxygen adduct and leads to a μ-oxodicopper(II) species in a fast reaction.

The inhibition of the catechol oxidase activity exhibited by three dinuclear copper(II) complexes, derived from different diaminotetrabenzimidazole ligands, by kojic acid [5-hydroxy-2-(hydroxymethyl)-gamma-pyrone] has been studied. The catalytic mechanism of the catecholase reaction proceeds in two steps and for both of these inhibition by kojic acid is of competitive type. The inhibitor binds strongly to the dicopper(II) complex in the first step and to the dicopper-dioxygen adduct in the second step, preventing in both cases the binding of the catechol substrate. Binding studies of kojic acid to the dinuclear copper(II) complexes and a series of mononuclear analogs, carried out spectrophotometrically and by NMR, enable us to propose that the inhibitor acts as a bridging ligand between the metal centers in the dicopper(II) catalysts.

The activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and 1 H and 19 F NMR spectroscopy. Whereas 3-and 4-fluorophenol react with tyrosinase to give products that undergo a rapid polymerization process, 2-fluorophenol is not reactive and actually acts as a competitive inhibitor in the enzymatic oxidation of 3,4-dihydroxyphenylalanine (L-dopa). The tyrosinase-mediated polymerization of 3-and 4-fluorophenols has been studied in detail. It proceeds through a phenolic coupling pathway in which the common reactive fluoroquinone, produced stereospecifically by tyrosinase, eliminates an inorganic fluorine ion. The enzymatic reaction studied as a function of substrate concentration shows a prominent lag that is completely depleted in the presence of L-dopa. The kinetic parameters of the reactions can be correlated to the electronic and steric effects of the fluorine substituent position. Whereas the fluorine electron withdrawing effect appears to control the binding of the substrates (K m for 3-and 4-fluorophenols and K I for 2-fluorophenol), the k cat parameters do not follow the expected trend, indicating that in the transition state some additional steric effect rules the reactivity. Tyrosinases (Tys)1 are monooxygenating enzymes that catalyze the ortho-hydroxylation of monophenols and the subsequent oxidation of diphenols to quinones (1). The reaction is widespread in nature, from bacteria to fungi, plants, and mammals. In mammals, when L-tyrosine acts as the substrate, the formed quinones are reactive precursors in the synthesis of melanin pigments. In fruits, vegetables, and mushrooms, Ty is a fundamental enzyme in the browning process that occurs during product storage and upon bruising. Ty contains a dinuclear type 3 copper center, in which two copper ions are closely spaced and coordinated each by three histidines through the N-⑀ nitrogen atoms (1). This type of site has been found and structurally characterized also in hemocyanins, which act as oxygen carriers in arthropods and mollusks (2-4), and in catechol oxidases, which perform the oxidation of o-diphenols to quinones (5). The reasons why these proteins perform different functions, although their catalytic sites appear to be similar both in structure and oxygen binding ability, remains to be clarified (6). During activity, the type 3 site of Ty can exist in three main redox forms: ions are normally bridged by a small ligand (7,8). The cloning technique, developed recently, and more detailed kinetic studies (6 -12) have increased the current understanding of Ty mechanism and structure. Additional studies (8, 13-16) on inhibitors also provided structural information. The importance of the latter studies is related to the considerable interest of Tys from the medical, agricultural, and industrial point of view. For instance, Ty inhibitors are of great concern in the cosmetic industry (17) and in food technology as anti-browning agents (18,19). Moreover, the tyrosinases present in soil have been fo...

Deoxymyoglobin has been investigated by NMR spectroscopy to determine the magnetic anisotropy through pseudocontact shifts and the total magnetic susceptibility through Evans measurements. The magnetic anisotropy values were found to be Deltachi(ax)=-2.03+/-0.08 x 10(-32) m(3) and Deltachi(rh)=-1.02+/-0.09 x 10(-32) m(3). The negative value of the axial susceptibility anisotropy originates from the z tensor axis lying in the heme plane, unlike all other heme systems investigated so far. This magnetic axis is almost exactly orthogonal to the axial histidine plane. The other two axes lie essentially in the histidine plane, the closest to the heme normal being tilted by about 36 degrees from it, towards pyrrole A on the side of the proximal histidine. From the comparison with cytochrome c' it clearly appears that the position of the one axis lying in the heme plane is related to the axial histidine orientation. Irrespective of the directions, the magnetic anisotropy is smaller than that of the analogous reduced cytochrome c' and of the order of that of low-spin iron(III). The magnetic anisotropy of the system permits the measurement of residual dipolar couplings, which, together with pseudocontact shifts, prove that the solution structure is very similar to that in the crystalline state. Magnetic measurements, at variance with previous data, demonstrate that there is an orbital contribution to the magnetic moment, micro(eff)=5.5 micro(B). Finally, from the magnetic anisotropy data, the hyperfine shifts of iron ligands could be separated in pseudocontact and contact components, and hints are provided to understand the spin-delocalisation mechanism in S=2 systems by keeping in mind the delocalisation patterns in low-spin S=1/2 and high-spin S= 5/2 iron(III) systems.

The dicopper(II) complex [Cu(2)(L)](4+) (L = alpha,alpha'-bis[bis[2-(1'-methyl-2'-benzimidazolyl)ethyl]amino]-m-xylene) reacts with hydrogen peroxide to give the dicopper(II)-hydroquinone complex in which the xylyl ring of the ligand has undergone a double hydroxylation reaction at ring positions 2 and 5. The dihydroxylated ligand 2,6-bis([bis[2-(3-methyl-1H-benzimidazol-2-yl)ethyl]amino]methyl)benzene-1,4-diol was isolated by decomposition of the product complex. The incorporation of two oxygen atoms from H(2)O(2) into the ligand was confirmed by isotope labeling studies using H(2)(18)O(2). The pathway of the unusual double hydroxylation was investigated by preparing the two isomeric phenolic derivatives of L, namely 3,5-bis([bis[2-(1-methyl-1H-benzimidazol-2-yl)ethyl]amino]methyl)phenol (6) and 2,6-bis([bis[2-(1-methyl-1H-benzimidazol-2-yl)ethyl]amino]methyl)phenol (7), carrying the hydroxyl group in one of the two positions where L is hydroxylated. The dicopper(II) complexes prepared with the new ligands 6 and 7 and containing bridging micro-phenoxo moieties are inactive in the hydroxylation. Though, the dicopper(II) complex 3 derived from 6 and containing a protonated phenol is rapidly hydroxylated by H(2)O(2) and represents the first product formed in the hydroxylation of [Cu(2)(L)](4+). Kinetic studies performed on the reactions of [Cu(2)(L)](4+) and 3 with H(2)O(2) show that the second hydroxylation is faster than the first one at room temperature (0.13 +/- 0.05 s(-1) vs 5.0(+/-0.1) x 10(-3) s(-1)) and both are intramolecular processes. However, the two reactions exhibit different activation parameters (Delta H++ = 39.1 +/- 0.9 kJ mol(-1) and Delta S++ = -115.7 +/- 2.4 J K(-1) mol(-1) for the first hydroxylation; Delta H++ = 77.8 +/- 1.6 kJ mol(-1) and Delta S++ = -14.0 +/- 0.4 J K(-1) mol(-1) for the second hydroxylation). By studying the reaction between [Cu(2)(L)](4+) and H(2)O(2) at low temperature, we were able to characterize the intermediate eta(1):eta(1)-hydroperoxodicopper(II) adduct active in the first hydroxylation step, [Cu(2)(L)(OOH)](3+) [lambda(max) = 342 (epsilon 12,000), 444 (epsilon 1200), and 610 nm (epsilon 800 M(-1)cm(-1)); broad EPR signal in frozen solution indicative of magnetically coupled Cu(II) centers].