Specific zinc binding to heliorhodopsin.

Heliorhodopsins (HeRs), a recently discovered family of rhodopsins, have an inverted membrane topology compared to animal and microbial rhodopsins. The slow photocycle of HeRs suggests a light-sensor function, although the actual function remains unknown. Although HeRs exhibit no specific binding of monovalent cations or anions, recent ATR-FTIR spectroscopy studies have demonstrated the binding of Zn to HeR from archaeon (TaHeR) and 48C12. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn-binding, suggesting a possible modification of the HeR function by Zn. The present study shows that Zn-binding lowers the thermal stability of TaHeR, and slows back proton transfer to the retinal Schiff base (M decay) during its photocycle. Zn-binding was similarly observed for a TaHeR opsin that lacks the retinal chromophore. We then studied the Zn-binding site by means of the ATR-FTIR spectroscopy of site-directed mutants. Among five and four mutants of His and Asp/Glu, respectively, only E150Q exhibited a completely different spectral feature of the α-helix (amide-I) in ATR-FTIR spectroscopy, suggesting that E150 is responsible for Zn-binding. Molecular dynamics (MD) simulations built a coordination structure of Zn-bound TaHeR, where E150 and protein bound water molecules participate in direct coordination. It was concluded that the specific binding site of Zn is located at the cytoplasmic side of TaHeR, and that Zn-binding affects the structure and structural dynamics, possibly modifying the unknown function of TaHeR.