The binuclear structure of Fe-2(DTPB)(mu-O)(mu-Ac)Cl(BF4)2 (DTPB = 1,1,4,7,7-penta (2'-benzimidazol-2-ylmethyl) triazaheptane, Ac = acetate) was characterized by UV-visible absorption and infrared spectra and NMR and ESR. The binding interaction of DNA with the diiron complex was examined spectroscopically. Supercoiled and linear DNA hydrolytic cleavage by the diiron complex is supported by the evidence from anaerobic reactions, free radical quenching, high performance liquid chromatography experiments, and enzymatic manipulation such as T4 ligase ligation, 5'-P-32 end-labeling, and footprinting analysis. The estimation of rate for the supercooiled DNA double strand cleavage shows one of the largest known rate enhancement factors, similar to10(10) against DNA. Moreover, the DNA hydrolysis chemistry needs no coreactant such as hydrogen peroxide, The poor sequence-specific DNA cleavage indicated by the restriction analysis of the pBR322 DNA linearized by the diiron complex might be due to the diiron complex bound to DNA by a coordination of its two ferric ions to the DNA phosphate oxygens, as suggested by spectral characterizations. The hydrolysis chemistry for a variety of binuclear metal complexes including Fe-2(DTPB)(mu-O) (mu-Ac)Cl(BF4)(2) is compared. It is established that the dominant factors for the DNA hydrolysis activities of the binuclear metal complexes are the mu-oxo bridge, labile and anionic ligands, and open coordination site(s). Concerning the hydrolytic mechanisms, the diiron complex Fe-2(DTPB)(mu-O)(mu-Ac)Cl(BF4)(2) might share many points in common with the native purple acid phosphatases.