UV‐Vis Spectroscopy Reveals a Correlation Between Y263 and BV Protonation States in Bacteriophytochromes

• Published in: Photochemistry and photobiology Vol. 95; no. 4; pp. 969 - 979
• Main Authors: Rumfeldt, Jessica A., Takala, Heikki, Liukkonen, Alli, Ihalainen, Janne A.
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.07.2019
• Subjects: Amino acids; Biliverdin; Chemical properties; Chromophores; Organic chemistry; Phenols; Phytochromes; Proteins; Protonation; Spectroscopy; Spectrum analysis
• DOI: 10.1111/php.13095
• ISSN: 0031-8655

Red‐light photosensory proteins, phytochromes, link light activation to biological functions by interconverting between two conformational states. For this, they undergo large‐scale secondary and tertiary changes which follow small‐scale Z to E bond photoisomerization of the covalently bound bilin chromophore. The complex network of amino acid interactions in the chromophore‐binding pocket plays a central role in this process. Highly conserved Y263 and H290 have been found to be important for the photoconversion yield, while H260 has been identified as important for bilin protonation and proton transfer steps. Here, we focus on the roles these amino acids are playing in preserving the chemical properties of bilin in the resting Pr state of the photosensory unit of a bacteriophytochrome from Deinococcus radiodurans. By using pH‐dependent UV‐Vis spectroscopy and spectral decomposition modeling, we confirm the importance of H260 for biliverdin protonation. Further, we demonstrate that in the canonical bacteriophytochromes, the pKa value of the phenol group of the Y263 is uncommonly low. This directly influences the protonation of the bilin molecule and likely the functional properties of the protein. Our study expands the understanding of the tight interplay between the nearby amino acids and bilin in the phytochrome family. Biliverdin (grey) and Y263 (teal) undergo parallel deprotonation events, requiring a four‐pKa model to describe the pH‐induced transition. Biliverdin and tyrosine each have two pKas due to reciprocal stabilization of the charges (positive for biliverdin and negative for Y263) that occurs within a selective pH range.