Probing Cofactor Biogenesis and Catalytic Mechanism in Iron and Copper Proteins Using Genetically-Substituted Noncanonical Amino Acids

The protein-derived cysteine-tyrosine (Cys-Tyr) redox cofactor is present in a growing number of iron- and copper-containing proteins. Its presence enables or significantly enhances the capacity of the host metalloprotein to mediate a specific redox process. However. the mechanism of Cys-Tyr biosynthesis remains poorly understood due to several intrinsic challenges. particularly its formation from self-processing due to uncoupled oxidation reactions in non-heme iron-dependent enzymes. This proposal is concerned with the mechanism of Cys-Tyr biogenesis in two iron-dependent human thiol dioxygenases. A copper-dependent oxidase from a bacterial source is included in the study for comparison. The human thiol dioxygenases. cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO). play a central role in the biological portion of the sulfur cycle and thiol metabolism. The catalytic activity of these enzymes is important in controlling cysteine and taurine levels. CDO contains a self-processed Cys-Tyr cofactor. It has been a long-standing question whether or not ADO also contains a Cys-Tyr cofactor. One common feature known so far in Cys-Tyr biogenesis in these proteins is that the formation of the thioether (C-S) bond is a metal-dependent process. The work proposed here will utilize site-specific genetic incorporation of unnatural amino acids. protein X-ray crystallography. analytic and spectroscopic studies to (1) determine the molecular basis of how the iron-activated oxidant is directed for cofactor biogenesis in CDO and the role of the Cys-Tyr cofactor in thiol dioxygenation. (2) determine the structure and enzymatic mechanism of ADO. and (3) investigate how the Cys-Tyr radical cofactor is formed in copper-dependent galactose oxidases (GAO) using genetic incorporation of unnatural amino acids to the tyrosine. These studies will elucidate the unknown mechanism of metallocenter-mediated sulfur oxidation and cofactor biogenesis. A better understanding of the irreversible self-processing C-S bond protein modification will also help to meet the fundamental challenge of predicting protein structures and functions.