Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyze the hydrolysis of a variety of phosphoester substrates within the pH range of 4-7. 1 They are the only binuclear metallohydrolases where the necessity for a heterovalent active site (Fe III -M II , where M ) Fe, Zn, or Mn) for catalysis has been clearly established. To date, the crystal structures of PAPs from red kidney bean (rkbPAP), 2a rat, 2b,c pig, 2d human, 2e and sweet potato 2f have been reported. In the structure of rkbPAP, 2a the Fe III ion is coordinated by a tyrosine, a histidine, and an aspartate, and a Zn II ion is coordinated by two histidines and an asparagine. The Fe III Zn II ions are bridged by two oxygen atoms, one from the carboxylate group of an aspartate and the other from a modeled µ-(hydr)oxo group. Two oxygen atoms from a µ-1,3 phosphate group complete the coordination spheres of the Zn II and Fe III ions.Despite the availability of detailed structural data, the catalytic mechanism of PAPs remains a matter of controversy. For rkbPAP, a mechanism in which, in the first step of the catalytic cycle, the substrate binds in a monodentate fashion to the Zn II ion has been proposed. 2a The enzyme-substrate complex is oriented in such a way that a terminal Fe III -bound hydroxide can efficiently attack the phosphorus atom of the substrate, leading to the release of the alcohol product. 2a The monodentate binding of the substrate to Zn II is corroborated by the fact that the addition of phosphate to the Fe III Zn II derivative of bovine spleen PAP does not affect the spectroscopic properties of the Fe III ion at the pH of optimal activity (pH 6.5). 3 However, for pig 4a and sweet potato PAP, 2f,4b an alternative mechanism in which the substrate forms a µ-1,3 phosphate complex, thus placing the µ-(hydr)oxo bridge in an ideal position to act as the reaction-initiating nucleophile, has also been proposed.Homo-and heterodinuclear Fe III M II complexes which are capable of reproducing the structural, spectroscopic, and functional properties of PAPs can be very informative to evaluate the mechanism(s) of these metalloenzymes. Recently, we reported on the syntheses, characterization, and phosphatase-like activity of the heterodinuclear [LFe III (µ-OAc) 2 Zn II ] + complex (H 2 L ) 2-bis[{(2-pyridylmethyl)-aminomethyl}-6-{(2-hydroxybenzyl)-(2-pyridylmethyl)}aminomethyl]-4-methylphenol), and we have proposed that upon dissolving the complex in an aqueous solution the dissociation of the carboxylate groups leads to the formation of the catalytically active [(OH)Fe III -(µ-OH)Zn II (OH 2 )] species, 5 similar to that proposed to be present in the active site of rkbPAP. 2a Herein we report the X-ray structure, solution studies, and phosphatase activity of the first mixed-valence complex containing the Fe III (µ-OH)Zn II motif (1). The molecular structure of 1 (Figure 1) shows that in the dinuclear [L(OH 2 )Fe-(µ-OH)Zn] 2+ unit the Fe III ion is facially coordinated by the hard tridentate pendant arm of L 2-...

Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the diverse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and divalent metal ions using (71)Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the mu-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.

The design and development of suitable biomimetic catalytic systems capable of mimicking the functional properties of enzymes continues to be a challenge for bioinorganic chemists. In this study, we report on the synthesis, X-ray structures, and physicochemical characterization of the novel isostructural [Fe III Co ] complex as the catalytically active species in diester hydrolysis reactions. Kinetic studies on the hydrolysis of the model substrate bis(2,4-dinitrophenyl)phosphate by 1 and 2 show Michaelis-Menten behavior, with 2 being 35% more active than 1. In combination with k H /k D isotope effects, the kinetic studies suggest a mechanism in which a terminal M III -bound hydroxide is the hydrolysis-initiating nucleophilic catalyst. In addition, the complexes show maximum catalytic activity in DNA hydrolysis near physiological pH. The modest reactivity difference between 1 and 2 is consistent with the slightly increased nucleophilic character of the Ga

Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe III M II center (M II = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric acid esters. The dinuclearhas recently been prepared and is found to closely mimic the coordination environment of the Fe III Zn II active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the Fe III center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted Fe III N 2 O 4 octahedron with a dominant tetragonal compression aligned along the μ-OH-Fe-O phenolate direction. To probe the role of the Fe-O phenolate bond, the phenolate moiety was modified to contain electron-donating or -withdrawing groups (-CH 3 , -H, -Br, -NO 2 ) in the 5-position. The effects of the substituents on the electronic properties of the biomimetic complexes were studied with a range of experimental and computational techniques. This study establishes benchmarks against accurate crystallographic structural information using spectroscopic techniques that are not restricted to single crystals. Kinetic studies on the hydrolysis reaction revealed that the phosphodiesterase activity increases in the order -NO 2 rBr rH rCH 3 when 2,4-bis(dinitrophenyl)phosphate (2,4-bdnpp) was used as substrate, and a linear free energy relationship is found when log(k cat /k 0 ) is plotted against the Hammett parameter σ. However, nuclease activity measurements in the cleavage of double stranded DNA showed that the complexes containing the electron-withdrawing -NO 2 and electron-donating -CH 3 groups are the most active while the cytotoxic activity of the biomimetics on leukemia and lung tumoral cells is highest for complexes with electron-donating groups.

As metal ions are present in the catalytic sites of several enzymes, attention has been focused on the synthesis and characterization of metal complexes able to act as biomimetic functional and structural models for these systems. In this study, a novel dinuclear NiII complex was synthesized, [Ni2(L2)(OAc)2(CH3CN)]BPh4 (2) (HL2=2-[N-(2-(pyridyl-2-yl)ethyl)(1-methylimidazol-2-yl)amin omethyl]-4-methyl-6-[N-(2-(imidazol-4-yl)ethyl)amino methyl]phenol), employing a new unsymmetrical dinucleating ligand containing N,O-donor groups as a model for hydrolases. Complex 2 was characterized by a variety of techniques including: elemental analysis, infrared and UV-vis spectroscopies, molar conductivity, electrochemistry, potentiometric titration, magnetochemistry, and single-crystal X-ray diffractometry. The structural and magnetochemical data of 2 allow us to consider this complex as a structural model for the active site of the ureases, as previously reported for [Ni2(L1)(OAc)2(H2O)]ClO4.H2O (1) (HL1=2-[N-bis-(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-(2-pyridylmethyl)aminomethyl] phenol). The characterization of complexes 1 and 2 (mainly by X-ray diffraction and potentiometric titration) led us to study their reactivities toward the hydrolysis of the substrate bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP). These studies revealed that complexes 1 and 2 show the best catalytic activity reported so far, with acceleration rates 8.8x10(4) and 9.95x10(5) times faster, respectively, than the uncatalyzed hydrolysis of 2,4-BDNPP. Catalytic activity of 2 on 2,4-DNPP showed that the monoester is hydrolyzed 27 times slower than the 2,4-BDNPP diester under identical experimental conditions. Therefore, 1 and 2 can undoubtedly be considered highly efficient functional models of the phosphohydrolases.

The structure and physicochemistry of the [Ni(II)(AAZ)(2)](ClO(4))(2) (1) complex (AAZ = 6-amino-6-methylperhydro-1,4-diazepine), as a system that is able to mimic some important chelate properties of 1,4,7-triazacyclononane, are reported. The syntheses of a new unsymmetric AAZ-functionalized ligand and the structure of its first heterodinuclear Fe(III)Zn(II) complex are also presented.

Herein, we report reactivity studies of the mononuclear water-soluble complex [Mn(II)(HPClNOL)(η 1 -NO 3 )(η 2 -NO 3 )] 1, where HPClNOL ) 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol, toward peroxides (H 2 O 2 and tertbutylhydroperoxide). Both the catalase (in aqueous solution) and peroxidase (in CH 3 CN) activities of 1 were evaluated using a range of techniques including electronic absorption spectroscopy, volumetry (kinetic studies), pH monitoring during H 2 O 2 disproportionation, electron paramagnetic resonance (EPR), electrospray ionization mass spectrometry in the positive ion mode [ESI(+)-MS], and gas chromatography (GC). Electrochemical studies showed that 1 can be oxidized to Mn(III) and Mn(IV). The catalase-like activity of 1 was evaluated with and without pH control. The results show that the pH decreases when the reaction is performed in unbuffered media. Furthermore, the activity of 1 is greater in buffered than in unbuffered media, demonstrating that pH influences the activity of 1 toward H 2 O 2 . . The peroxidase activity of 1 was also evaluated by monitoring cyclohexane oxidation, using H 2 O 2 or tert-butylhydroperoxide as the terminal oxidants. Low yields (<7%) were obtained for H 2 O 2 , probably because it competes with 1 for the catalase-like activity. In contrast, using tert-butylhydroperoxide, up to 29% of cyclohexane conversion was obtained. A mechanistic model for the catalase activity of 1 that incorporates the observed lag phase in O 2 production, the pH variation, and the formation of a Mn(III)-(µ-O) 2 -Mn(IV) intermediate is proposed.

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