Martínez-Rivera, Melissa C

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  • Publication
    Using Model Proteins to Study Tyrosine Oxidation-Reduction: Reversible Voltammograms, Long-Lived Radicals and Detailed Design of the Radical Site
    (2013-01-01) Martínez-Rivera, Melissa C
    Amino acid radicals have been found as key components in a number of biological redox processes. In specific, redox−active tyrosine residues play an essential role in DNA biosynthesis and photosynthesis, among other processes. The thermodynamic and kinetic properties of one−electron redox reactions involving tyrosine have long been obscured by the highly unstable nature of the products of tyrosine oxidation. Remarkable control of these species is achieved within natural proteins. Proteins must, therefore, provide interactions to the amino acid cofactor to generate, control and direct the redox chemistry within the protein milieu. Electrochemical characterization of redox−active tyrosine residues inside natural radical proteins is highly challenging due to potential oxidation of other cofactors and residues. Numerous small−molecule models have been generated in which factors that affect tyrosine/phenol redox chemistry have been elucidated, such as hydrogen−bonding. Although these models have contributed to our knowledge about these systems, these lack the protein environment that certainly renders a stabilizing environment for these species. We have developed a de novo−designed model protein family in which to study these reactions. The family of α3X proteins consists of a well−structured, pH−stable, three−helix bundle with a single redox−active residue within the core of the protein. Construction and characterization of tyrosine−containing (α 3Y, α 3Y−K29H, and α 3Y−K36H) and tyrosine−analogue−containing (2MP− α 3C, 3MP− α 3C, and 4MP− α 3C) proteins are described in this dissertation work. Electrochemical characterization of α 3Y and 2MP− α 3C by Square Wave Voltammetry has allowed us to obtain fully reversible voltammograms and formal reduction potentials for the long−lived neutral radicals formed within these proteins. In the case of α 3Y, we have also found that the protein scaffold is intimately involved in the electron transfer process. We have also achieved the introduction of a tyrosine−histidine interacting pair in two α 3Y variants. This interacting complex is of great interest and will provide insights into how interaction with these basic residues might impact tyrosine redox chemistry within a protein scaffold. In addition, the solution NMR structure of 4MP− α 3C is reported and compared to the previously determined 2MP− α 3C structure. Key structural features are described that are likely to have a major impact on the protein redox properties.