The bimetallic system is an important strategy for the catalytic hydrolysis of phosphodiester. The purple acid phosphatase (PAPs) enzyme is a typical bimetallic catalyst in this field. Mechanistic details for the hydrolysis cleavage of the DNA dinucleotide analogue BNPP- (BNPP- = bis(p-nitrophenyl) phosphate) by hetero-binuclear [Fe-III(mu-OH)(ZnL)-L-II](2+) complexes (L = 2-[N-bis(2-pyridylmethyl)-aminomethyl]-4-methyl-6-[N'-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl] phenol) were investigated using density functional theory calculations. The catalysts with single-bridged hydroxyl and double-bridged hydroxyl groups were compared. The calculation results show that the doubly hydroxide-bridged complex could better bind to substrates. For the BNPP- hydrolysis, the doubly hydroxide-bridged reactant isomerizes into a single hydroxide-bridged complex, and then the attack is initiated by the hydroxyl group on the iron center. In addition, the catalyst with the electron-donating group (Me) was determined to take precedence over electron-withdrawing groups (Br and NO2 groups) in the hydrolysis reaction. This is because the substituents affect the high-lying occupied molecular orbitals, tuning the Lewis acidity of iron and pK(a) values of the metal-bonded water. These factors influence the hydroxyl nucleophilicity, leading to changes in catalytic activity. To further examine substituent effects, the occupied orbital energies were calculated with several different substituent groups (-CF3, -OMe, -OH, -NH2, and -N(Me)(2)). It was found that the HOMO or HOMO-1 energy decreases with the increase of the sigma(p) value. Further, the catalyst activity of the [Fe-III(mu-OH)(ZnL)-L-II](2+) complexes was found to be mainly affected by the phenolate ligand (B) coordinated to the iron and zinc centers. These fundamental aspects of the hydrolysis reactions of BNPP- catalyzed by [Fe-III(mu-OH)(ZnL)-L-II](2+) complexes should contribute to improved understanding of the mechanism and to catalyst design involving hetero-binuclear metals complexes.