9418181 Miller The research will employ Fe and Mn-containing superoxide dismutase (SOD) as a model system and exploit the unique ability of nuclear magnetic resonance spectroscopy (NMR) to directly observe protein groups and monitor structure in solution. The objectives are to elucidate the conformational interplay between the metal ion site and the protein of SOD upon substrate analog binding to the metal ion, and to determine why FeSOD protein is inactive with Mn bound instead of Fe, and MnSOD protein is inactive with Fe bound instead of Mn. Isotope edited NMR will be used in conjunction with amino acid-specific isotopic labeling to produce drastically simplified spectra showing signals of labeled amino acids only. Comparisons of spectra of SOD with and without the substrate analog azide bound will identify amino acids affected by azide binding and indicate by what mechanisms the protein is conformationally responsive to events at the metal ion. By monitoring amino acids at the interfaces between domains or subunits, it will be possible to determine whether the conformation change involves potentially general mechanisms for conformational change including (1) relative motions of domains bridged by the metal ion, (2) relative motions of subunits and (3) propagation of conformational effects via hydrogen bonding networks. These studies build on the known structure for SOD and exploit the unique ability of NMR to directly observe protons, provide information on their participation in hydrogen bonds and provide qualitative and quantitative information on both structure and dynamics of proteins in solution unconstrained by crystal packing. The work will also determine why SODs containing the wrong metal ion are inactive, by evaluating their competence for individual elements of catalysis including each of the half reactions, the reduction midpoint potential, proton donation, substrate analog binding and active site structure, thus specifying the chemical reaso n for catalytic incompetence. Site-directed and random mutagenesis will be used to convert MnSOD protein into a FeSOD. Characterization of these SODs will reveal what amino acid changes are important, the extent to which these amino acid changes support activity with the wrong metal, and at what cost to activity with the correct metal. %%% Metalloenzymes combine the reactivity of metal centers with the exquisite specificity and control of enzymes. The Fe-containing superoxide dismutases (FeSODs) and the homologous Mn-containing superoxide dismutases (MnSODs) are metalloenzymes that catalyze the conversion of superoxide to dioxygen and hydrogen peroxide. SOD is relatively small, very stable and soluble but also embodies two central features of metalloenzyme catalysis: the metal site and the protein are believed to affect each other's structure, and each type of SOD, while able to bind either Fe or Mn, is only active with one. Thus, the structure and activity of the enzyme reflect interactions between the protein and the metal ion, and these interactions are metal ion specific. The goal of the research is (1) to determine how much of the protein is affected by binding of small molecules to the metal ion, and (2) to describe the protein structural change in terms of relative movement of the modules that make up the protein structure. The work will also determine why MnSOD protein is not active with Fe bound and FeSOD protein is not active with Mn bound. This new understanding of the protein-metal cooperation that is necessary for metalloenzyme catalytic activity will advance ongoing efforts to design or modify metalloenzymes to catalyze useful reactions in drug synthesis, waste detoxification and chemical sensors. ***
