Proton polarizability of hydrogen-bonded network and its role in proton transfer in bacteriorhodopsin

Room-temperature time-resolved step-scan Fourier Transform Infrared (FTIR) spectroscopy has been used to study the photocycle of native bacteriorhodop sin (bR) suspension in both H2O and D2O. The kinetics of the retinal isomer ization, and that of the protonation/deprotonation of the proton acceptor, Asp85, are compared in the mu s to Ins time domain. It is found that hydrog en/deuterium (H/D) isotope exchange does not significantly affect the kinet ics of the retinal isomerization and relaxation processes. However, the pro tonation/deprotonation processes of Asp85 COO- become slower in D2O. We als o studied the kinetics of the continuum absorbance change in the 1850-1800 cm(-1) frequency region, which has previously been proposed to correspond t o the absorption of the delocalized proton that is involved in the proton t ransport to the surface during the photocycle. An H/D isotope shift of the frequency range of this continuum absorbance has been confirmed by the obse rvation that the band in the 1850-1800 cm(-1) disappears in the photocycle of bR in D2O. These results could support the previous proposal that the in tramolecular proton release pathway consists of an H-bonded network. Our re sults also suggest that the two independent processes, the transfer of a pr oton from the Schiff base to Asp85 and the release of a different proton to the extracellular surface, are closely coupled events.