Cys-Tyr Cofactor in Iron and Copper Proteins

University Of Texas At San Antonio, San Antonio TX

Investigators

Abstract

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Aimin Liu from the University of Texas at San Antonio is investigating protein-derived cofactors that contain a cysteine-tyrosine crosslink (Cys-Tyr). These cofactors are generated in a unidirectional protein post-translational modification. They have biological significance because they instill catalytic function in some enzymes, such as galactose oxidase, and enhance the catalytic efficiency of other enzymes, such as thiol dioxygenases. However, the chemistry of the cofactor synthesis and how the Cys-Tyr cofactor contributes to catalysis are not well understood. The proposed research will focus on the elucidation of the mechanism by which the Cys-Tyr crosslink of the cofactor is formed in enzymes. The graduate and undergraduate students who are engaged in the research plan will learn how to determine protein structure through crystallography, advanced spectroscopies, and the rapidly developing technology of substitution of an amino acid in a protein with unnatural amino acids. They will also gain expertise in the study of catalytic function in proteins. This research project seeks to understand the extraordinary stability of the one-electron oxidized free radical form of the Cys-Tyr cofactor in copper-dependent galactose oxidase. The proposed studies will seek to determine the radical spin density distributions of the copper center and aromatic residues in the active site of the enzyme and how these distributions affect proton/electron transfer during catalysis. The research is expected to generate insight into how the Cys-Tyr cofactor in human thiol dioxygenases facilitates catalysis by characterization of substrate-bound complexes and cofactor-bearing structures. These studies will leverage the demonstrated ability of the research team to genetically substitute targeted amino acids with noncanonical amino acids (ncAAs). Although the strategic substitution minimally alters the target amino acid residue, it alters the electronic and chemical structure of the functional groups. The ncAA-containing enzyme variants and their reactions with different substrates will be studied using a combination of chemical, spectroscopic, and structural biology methods. These studies aim to provide insight into the irreversible, self-processing, C-S bond formation and the processes by which the radical is stabilized and the redox activity of the copper active site is tuned. Ultimately, the project sets out to contribute to addressing the fundamental challenge of predicting protein structure and function ways that are distinct from and synergistic with current, best-in-class, prediction methods. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.