Coordinating the Structural Rearrangements Associated with Unidirectional Proton Transfer in the Bacteriorhodopsin Photocycle Induced by Deprotonation of the Proton-Release Group: A Time-Resolved Difference FTIR Spectroscopic Study

摘要

In the photocycle of bacteriorhodopsin at pH7, proton release fromthe proton releasing group (PRG)nto the extracellular medium occurs during formation of the M intermediate. This proton release is inhibited atnacidic pH, below the pKa of the PRG, ∼6 inM, and instead occurs later in the cycle as the initial state is restorednfrom the O intermediate. Here, structural changes related to deprotonation of the PRG have been investigated byntime-resolved FTIR spectroscopy at 25 u0002C. The vibrational features at 2100-1790, 1730-1685, 1661, andn1130-1045 cm-1nhave greater negative intensity in the pureM-minus-BR spectrum and even in theM-minus-BRnspectrum, that is present earlier together with the L-minus-BR spectrum, at pH 7, than in the corresponding M-nminus-BR spectra at pH5 or 4. TheD212Nmutation abolishes the decreases in the intensities of the broad featurenbetween 1730 and 1685 cm-1nand the band at 1661 cm-1n. The 1730-1685 cm-1nfeature may arise from transitionndipole coupling of the backbone carbonyl groups of Glu204, Phe208, Asp212, and Lys216 interacting with Tyr57nand C15-H of the chromophore. The 1661 cm-1nband, which is insensitive to D2O substitution, may arise byninteraction of the backbone carbonyl of Asp212 with C15-H. The 2100-1790 cm-1nfeature with a trough at 1885ncm-1ncould be due to a water cluster. Depletion of these bands upon deprotonation of the PRG is attributable tondisruption of a coordinated structure, held in place by interactions of Asp212. Deprotonation of the PRG isnalso accompanied by disruption of the interaction of the water molecule near Arg82. The liberated Asp212 maynstabilize the protonated state of Asp85 and thus confer unidirectionality to the transport.