Detection of a protein-bound water vibration of halorhodopsin in aqueous solution

Protein-bound water molecules play crucial roles in their structure and function, but their detection is an experimental challenge, particularly in aqueous solution at room temperature. By applying attenuated total reflection (ATR) Fourier-transform infrared (FTIR) spectroscopy to a light-driven Cl(−) pump pharaonis halorhodopsin (pHR), here we detected an O-H stretching vibration of protein-bound water molecules in the active center. The pHR(Cl(−)) minus pHR(Br(−)) ATR-FTIR spectra show random fluctuation at 3600–3000 cm(−1), frequency window of water vibration, which can be interpreted in terms of dynamical fluctuation of aqueous water at room temperature. On the other hand, we observed a reproducible spectral feature at 3617 (+)/3630 (−) cm(−1) in the pHR(Cl(−)) minus pHR(Br(−)) spectrum, which is absent in the pHR(Cl(−)) minus pHR(Cl(−)) and in the pHR(Br(−)) minus pHR(Br(−)) spectra. The water O-H stretching vibrations of pHR(Cl(−)) and pHR(Br(−)) at 3617 and 3630 cm(−1), respectively, are confirmed by light-induced difference FTIR spectra in isotope water (H(2)(18)O) at 77 K. The observed water molecule presumably binds to the active center of pHR, and alter its hydrogen bond during the Cl(−) pumping photocycle.