Herein, we report the synthesis and characterization of two dinuclear (FeZnII)-Zn-III complexes [Fe(III)Zn(II)LP1] (1) and [Fe(III)Zn(II)LP2] (2), in which LP1 and LP2 are conjugated systems containing one and two pyrene groups, respectively, connected via the diamine -HN(CH2)(4)NH- spacer to the well-known N5O2-donor H2L ligand (H2L = 2-bis{[(2-pyridylmethyl)aminomethyl]-6- [(2-hydroxybenzyl) (2-pyridylmethyl)]aminomethyl}-4-methylphenol). The complex [Fe(III)Zn(II)L1] (3), in which H2L was modified to H(2)L1, with a carbonyl group attached to the terminal phenol group, was included in this study for comparison purposes.(1) Both complexes 1 and 2 were satisfactorily characterized in the solid state and in solution. Extended X-ray absorption fine structure data for 1 and 3 in an acetonitrile solution show that the multiply bridged structure seen in the solid state of 3 is retained in solution. Potentiometric and UV-vis titration of 1 and 2 show that electrostatic interaction between the protonated amino groups and coordinated water molecules significantly decreases the pK(a) of the iron(III)-bound water compared to those of 3. On the other hand, catalytic activity studies using 1 and 2 in the hydrolysis of the activated substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) resulted in a significant increase in the association of the substrate (K-ass congruent to 1/K-M) compared to that of 3 because of electrostatic and hydrophobic interactions between BDNPP and the side-chain diaminopyrene of the ligands H(2)LP1 and H(2)LP2. In addition, the introduction of the pyrene motifs in 1 and 2 enhanced their activity toward DNA and as effective antitumor drugs, although the biochemical mechanism of the latter effect is currently under investigation. These complexes represent interesting examples of how to promote an increase in the activity of traditional artificial metal nucleases by introducing second-coordination-sphere effects.