Title: EPR and Mssbauer Characterization of Mn and Fe Enzymes, Biomimetic Models, and Intermediates. Project Abstract Life for many organisms, including humans, depends on the activation of small stable molecules by metalloproteins to provide selective and rapid chemical transformations. Our goal is to give insight into how specific enzymes function through studies of the atomic level changes that occur at the metal active site as the enzymes turn over their substrate. These studies are augmented with investigations of relevant biomimetic complexes that allow specific states of activated complexes to be studied that cannot be easily trapped during chemical reactions. The results of rapid freeze quench techniques, spectroscopy, and density functional theory calculations will be combined to identify new catalytic intermediates. It is anticipated that our studies will provide a better understanding of the factors that determine the specificity and efficiency of enzymatic reactions.
Three specific aims will be addressed by the proposed studies: ? Characterization of the catalytic mechanism of nitric oxide reductases. Humans possess defense systems that produce NO to combat the invasion of pathogenic bacteria. In response, bacteria can express scavenging NORs to protect the organisms against our defense systems. Knowledge of the NOR mechanism may provide targets for suppressing these defensive responses. ? Characterization of biomimetic complexes for dioxygen and water activation. Enzymes that break O-O bonds are critical for cleaving C-H bonds while those that catalyze dioxygen bond formation are key to photosynthesis. We will investigate biomimetic complexes of iron and manganese with the aim of pursuing reactive molecular states that have been postulated but whose existence has as yet eluded detection. ? Characterization of the catalytic mechanism of thiol dioxygenases. Enzymes involved in sulfur- oxidation and transfer are increasingly being recognized as potential drug targets for development of antimicrobials, therapies for cancer, and inflammatory disease. We will investigate the elemental steps in the reaction mechanisms of enzymes that provide the first step in the biological production of inorganic sulfate, hypotaurine, and taurine.
Life for many organisms, including humans, depends on the activation of small stable molecules by metals to provide selective and rapid transformations. Our goal is to provide insight into how specific enzymes function through studies of the atomic level changes that occur at the metal active site as the enzymes turn over their substrate. These studies are augmented with relevant biomimetic complexes that allow specific states of activated complexes to be studied that cannot be easily trapped during chemical reactions. Biomimetic complexes provide important insight into biochemical function and spectroscopic interpretation. Rapid freeze quench techniques, spectroscopy, and density functional calculations will be combined to characterize new catalytic intermediates. It is anticipated that such studies will provide a better understanding of how nature constructs enzymatic active sites to perform selective and efficient oxidation or reduction of substrates. Three specific aims will be address by the proposed studies. (1) Characterization of the catalytic mechanism of nitric oxide reductases. NO reductases are key enzymes involved in the scavenging of NO. Humans possess defense systems involving the production of NO that can prevent the invasion of pathogenic bacteria. Bacteria can express scavenging NORs that protect the organisms from our defense systems. (2) Characterization of biomimetic complexes for dioxygen and water activation. The enzymes which break and form an O- O bond are critical to cleavage of C-H bonds and in photosynthesis. We will investigate biomimetic complexes that allow a better understanding of the reactive species. (3) Characterization of the catalytic mechanism of thiol dioxygenases. Enzymes involved in sulfur-oxidation and transfer are increasingly being recognized as potential drug targets for development of antimicrobials, therapies for cancer, and inflammatory disease. We will investigate the elemental steps in the reaction mechanisms of enzymes that provide the first step in biological production of inorganic sulfate, hypotaurine, and taurine.
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