ABSTRACT
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.
