Probing histidine tautomers by theoretical X-ray absorption spectroscopy for biological and pathological studies

Differentiating histidine tautomeric states is critical for understanding their role in neurodegenerative disease pathogenesis. Shifts in tautomeric equilibrium may contribute to driving protein misfolding and aggregation, suggesting potential therapeutic interventions to mitigate disease progression. Using the second-order restricted active space perturbation theory (RASPT2) method, this study proposes X-ray absorption spectroscopy (XAS) as a viable approach for distinguishing histidine tautomeric states. Pristine histidine exhibits distinct N K-edge spectral features depending on its tautomeric form. To improve tautomer discrimination, functionalization of histidine with various electron-donating or withdrawing groups was explored to modify the electronic environment of the imidazole ring. Substituent effects induce shifts of major peaks, enhancing spectral separability. This strategy highlights XAS labeling as a promising tool for differentiating tautomeric structures in complex biomolecules and will provide novel insights into histidine's role in both biological and pathological processes, ultimately informing therapeutic discovery.