Protein oxidation involved in Cys-Tyr post-translational modification

Area of Science:

• Biochemistry
• Protein Chemistry
• Redox Biology

Background:

• Post-translational modifications of tyrosine residues can create unique redox-active cofactors.
• The intramolecular thioether-crosslinked 3'-(S-cysteinyl)-tyrosine (Cys-Tyr) is a well-studied modification found in enzymes like galactose oxidase.
• Cys-Tyr linkages are also present in mammalian cysteine dioxygenases and have been observed in orphan proteins like BF4112.

Purpose of the Study:

• To investigate the formation of Cys-Tyr in the orphan protein BF4112.
• To characterize the role of metal ions and specific residues in Cys-Tyr formation.
• To explore potential side reactions and their impact on cofactor synthesis.

Main Methods:

• UV-Vis spectroscopy for characterization of protein modifications.
• Fluorescence-based assays for quantifying Cys-Tyr yield, with guanidinium chloride for specificity.
• Site-directed mutagenesis to replace key tyrosine and cysteine residues.
• Denaturing SDS-PAGE to analyze protein products.

Main Results:

• Cys-Tyr formation in BF4112 was dependent on the presence of Tyr 52, Cys 98, and a Cu²⁺ ion.
• Mutations to Tyr 52 (to Phe) or Cu²⁺ (to Zn²⁺) abolished Cys-Tyr formation.
• Mutations to surface tyrosines and Cys 98 reduced Cys-Tyr yield but revealed a red-shifted emission, indicating surface tyrosyl radical (dityrosine) formation when Cys 98 was replaced by Ser.

Conclusions:

• Protein oxidation, specifically surface tyrosine oxidation, is a significant side reaction during Cys-Tyr cofactor formation in BF4112.
• The study highlights the complexity of post-translational cofactor synthesis and potential competing reaction pathways.
• Understanding these side reactions is crucial for accurately characterizing protein-derived cofactors and their biological roles.