Determination of Thiol Protonation States by Sulfur X-ray Spectroscopy in Biological Systems.

ABSTRACT: Cysteine is one of the most functionally diverse of the proteinogenic amino acids, owing to its reactive thiol side chain that can undergo deprotonation to form a strongly nucleophilic thiolate. However, few techniques can directly interrogate sulfur charge and covalency in cysteine, particularly in proteins. X-ray spectroscopies provide an element specific probe of sulfur. We demonstrate the sensitivity of S Kβ and Kα X-ray emission spectroscopy (XES) to cysteine ionization and compare it to S K-edge X-ray absorption spectroscopy (XAS) in the physiologically relevant biomolecules l-cysteine and N-acetyl-l-cysteine at room temperature in solution phase. Kβ XES and K-edge XAS are most sensitive to chemical changes at the cysteine thiol and can be used to evaluate the composition of thiol/thiolate mixtures. These results provide a foundation for assessing the pK_a of functionally significant cysteine residues in proteins and open the door to time-resolved studies of cysteine-dependent enzymes.

SUBMITTER: Ribson RD 

PROVIDER: S-EPMC11892467 | biostudies-literature | 2025 Mar

REPOSITORIES: biostudies-literature