Protein oxidation involved in Cys-Tyr post-translational modification

• Published in: Journal of inorganic biochemistry Vol. 176; pp. 168 - 174
• Main Authors: Hromada, Susan E., Hilbrands, Adam M., Wolf, Elysa M., Ross, Jackson L., Hegg, Taylor R., Roth, Andrew G., Hollowell, Matthew T., Anderson, Carolyn E., Benson, David E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2017
• Subjects: Copper-dioxygen activation; Cys-Tyr; Cysteine - chemistry; Native fluorescence; Outer sphere electron transfer; Oxidation-Reduction; Oxidoreductases - chemistry; Protein Processing, Post-Translational; Protein-derived cofactors; Surface tyrosine oxidation; Tyrosine - chemistry; Protein-derived cofactors; Cys-Tyr; Surface tyrosine oxidation; Native fluorescence; Outer sphere electron transfer; Copper-dioxygen activation
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2017.08.028

Some post-translationally modified tyrosines can perform reversible redox chemistry similar to metal cofactors. The most studied of these tyrosine modifications is the intramolecular thioether-crosslinked 3′-(S-cysteinyl)-tyrosine (Cys-Tyr) in galactose oxidase. This Cu-mediated tyrosine modification in galactose oxidase involves direct electron transfer (inner-sphere) to the coordinated tyrosine. Mammalian cysteine dioxygenase enzymes also contain a Cys-Tyr that is formed, presumably, through outer-sphere electron transfer from a non-heme iron center ~6Å away from the parent residues. An orphan protein (BF4112), amenable to UV spectroscopic characterization, has also been shown to form Cys-Tyr between Tyr 52 and Cys 98 by an adjacent Cu2+ ion-loaded, mononuclear metal ion binding site. Native Cys-Tyr fluorescence under denaturing conditions provides a more robust methodology for Cys-Tyr yield determination. Cys-Tyr specificity, relative to 3,3′-dityrosine, was provided in this fluorescence assay by guanidinium chloride. Replacing Tyr 52 with Phe or the Cu2+ ion with a Zn2+ ion abolished Cys-Tyr formation. The Cys-Tyr fluorescence-based yields were decreased but not completely removed by surface Tyr mutations to Phe (Y4F/Y109F, 50%) and Cys 98 to Ser (25%). The small absorbance and fluorescence emission intensities for C98S BF4112 were surprising until a significantly red-shifted emission was observed. The red-shifted emission spectrum and monomer to dimer shift seen by reducing, denaturing SDS-PAGE demonstrate a surface tyrosyl radical product (dityrosine) when Cys 98 is replaced with Ser. These results demonstrate surface tyrosine oxidation in BF4112 during Cys-Tyr formation and that protein oxidation can be a significant side reaction in forming protein derived cofactors. Native fluorescence allows quantitative determination of cysteine-tyrosine crosslink (Cys-Tyr) formation and with high selectivity in the presence of dityrosine. Mutagenic analysis of Cys-Tyr formation in an orphan protein (BF4112) with a mononuclear copper center showed protein oxidation to dityrosine is a significant side reaction opposed to galactose oxidase. [Display omitted] •Label-free fluorescence assay for cysteine-tyrosine sidechain crosslinks•Dityrosine fluorescence is differentiated from emission from cysteine-tyrosine crosslink•A robust alternative to intramolecular crosslink detection by gel-shift provided•Dityrosine side reaction is significant in the absence of Cys of crosslink•Surface tyrosines oxidized during cysteine-tyrosine crosslink formation