Understanding the electronic structure of metal sites in metalloproteins is crucial to understanding the contributions of the individual components to the protein's mechanism of action. We wish to examine the effects that alterations in metal types, ligands, and remote residues have on metal - ligand bond strengths, geometries, and reactivities with substrates in the active sites of metalloproteins. Toward this end, we will focus on three major areas: 1) the three superoxide dismutases (SODs) - iron, manganese, and copper/zinc, 2) genetically engineered metalloantibodies, and 3) the carboxylate-histidine-metal motif found in numerous metalloprotein families. In SOD, there are effects on the reactivity of the enzyme that depend on the initial electron affinity, bond cleavage, protonation/deprotonation, as well as other electrostatic forces. We will compare bonding and reaction pathways in the different SODs and the effect of replacing the metal sites with other transition elements, and the effects of alterations of remote residues on the active sites. The metalloantibody inquiries will concentrate on the specificity of binding of various transition metals to genetically engineered metal- binding sites in antibodies. Initially we will look at the active site mimic of carbonic anhydrase engineered into the antifluorescein antibody and compare the binding and reactivity of the zinc and copper derivatives. This work will then be expanded to look at other potential metalloenzyme active sites that can be introduced to antibodies, studying the effects of metal substitution with an eye toward improving the catalytic function of the metalloantibodies. The carboxylate-histidine- metal motif is one that is found in several metalloproteins, including the superoxide dismutases, serine proteases, and cytochrome c peroxidase. We will investigate the significance of the carboxylate group initially by modeling the active sites of SOD, analyzing the electronic structure of the carboxylate and its relevance to the mechanistic behavior of the enzyme in question. We may then extend the work to incorporate other classes of enzymes and compare the results. The techniques used in this work will be a combination of density functional calculations and electrostatic models. The energetics of the metal interacting with ligands in the near-coordination environment will be calculated with quantum mechanical density functional methods, and the result will be incorporated with an electrostatic description of the surrounding protein and the solvent field. The advantage of this approach is that the direct effects of metal-substrate and metal-amino acid residue binding can be derived in detail from the electronic structure allowed by quantum mechanical analysis, while the indirect effect of alteration of remote residues, as in site-directed mutagenesis or protonation/deprotonation due to pH change, can be included efficiently.
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