Mechanistic Studies of Dinuclear Metalloenzymes - A Model Approach

With the goal to study the mechanisms of dinuclear metalloenzymes, new synthetic and computational models for such proteins were prepared and studied. Multidentate ligands based on a central phenolate and with imidazoleand carboxylate-containing donor moieties were synthesized to simulate carboxylate-rich ligand environments. The two ligands that were found to be the most useful, were the symmetric 2,6-Bis[N-(N-(carboxylmethyl)-N-((1-methylimidazol)methyl)amine)methyl]-4-methylphenolate (BCIMP) and the closely related asymmetric 2-(N-isopropyl-N-((1-methylimidazol)methyl)aminomethyl)-6-(N-(carboxyl methyl)-N-((1-methylimidazol)methyl)aminomethyl)-4-methylphenol (ICIMP). To study the mechanism of the dinickel enzyme urease, complexes containing two nickel ions were prepared from the two ligands. Analysis of the crystal structures of the synthesized model complexes indicated that urea prefers a coordinatively unsaturated metal ion for its initial coordination. Reactivity studies of the complexes showed that an open coordination site is important in facilitating hydrolytic catalytic activity. Computational models, based on the native crystal structure of the enzyme, showed that the first coordination of urea most likely occurs on the coordinatively more unsaturated nickel ion, trans to the carbamylated lysine ligand of the urease active site and that a proposal based on the attack of the bridging hydroxide in the enzyme is unlikely due to the high energy barrier for the formation of the tetrahedral intermediate. When ICIMP is reacted with zinc, a tetranuclear zinc complex is formed. Evidence from mass spectrometry indicates that it dissociates to dinuclear complexes in solution. The structure of the tetranuclear zinc complex is similar to the corresponding nickel complex. In addition, the structure of each dizinc unit in tetranuclear complex is structurally similar to the environment around the active site in the enzyme zinc phosphotriesterase. The (dissociated) zinc complex has also been found to be a potent functional model for hydrolytic enzymes. Homoand heteronuclear iron complexes were also synthesized. As a useful precursor, a mononuclear iron complex of ICIMP could be isolated and crystallographically characterized. Based on the mononuclear system, four new heterodinuclear iron complexes were isolated. Of these, the FeZn complex is a potential model complex for the FeZn site of plant purple acid phosphatase.