Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR. Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.

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 (H218O) 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.

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In the photocycle of bacteriorhodopsin at neutral pH, the proton transfer fi'om the Schiff base to Asp85 in the L-to-M trarisition causes dcprotonation oF the proton release group (PRG) which is ]ocatcd cTose to the extraceT]ular surface. This leads to unidiTectiona! proton transfer by preventing the reverse proton flows. This prevention is lifted at pH 4-5 below the pKa =-5.7 efthe PRG in M where proton release occurs later after proton uptakc. High proton aMinity to