Proton Polarizability of Hydrogen-Bonded Network and its Role in Proton Transfer in Bacteriorhodopsin

Abstract

Room-temperature time-resolved step-scan Fourier Transform Infrared (FTIR) spectroscopy has been used to study the photocycle of native bacteriorhodopsin (bR) suspension in both H2O and D2O. The kinetics of the retinal isomerization, and that of the protonation/deprotonation of the proton acceptor, Asp85, are compared in the μs to ms time domain. It is found that hydrogen/deuterium (H/D) isotope exchange does not significantly affect the kinetics of the retinal isomerization and relaxation processes. However, the protonation/deprotonation processes of Asp85 COO- become slower in D2O. We also studied the kinetics of the continuum absorbance change in the 1850−1800 cm-1 frequency region, which has previously been proposed to correspond to the absorption of the delocalized proton that is involved in the proton transport to the surface during the photocycle. An H/D isotope shift of the frequency range of this continuum absorbance has been confirmed by the observation 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 intramolecular proton release pathway consists of an H-bonded network. Our results also suggest that the two independent processes, the transfer of a proton from the Schiff base to Asp85 and the release of a different proton to the extracellular surface, are closely coupled events.