Characterization of the primary photochemistry of proteorhodopsin with femtosecond spectroscopy.

Proteorhodopsin is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization, leading to transmembrane translocation of a proton toward the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of proteorhodopsin, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the midinfrared spectral regions. Experiments were performed both at pH 9.5 (a physiologically relevant pH value in which the primary proton acceptor of the protonated Schiff base, Asp(97), is deprotonated) and at pH 6.5 (with Asp(97) protonated). Simultaneous analysis of the data sets recorded in the two spectral regions and at both pH values reveals a multiexponential excited state decay, with time constants of approximately 0.2 ps, approximately 2 ps, and approximately 20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerization pathways that lead to the K-state: one with an effective rate of approximately (2 ps)(-1) and the other with a rate of approximately (20 ps)(-1). At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of approximately 0.7. At pH 6.5, the slower pathway is less productive, which results in an isomerization quantum yield of 0.5. We further observe an ultrafast response of residue Asp(227), which forms part of the counterion complex, corresponding to a strengthening of its hydrogen bond with the Schiff base on K-state formation; and a feature that develops on the 0.2 ps and 2 ps timescale and probably reflects a response of an amide II band in reaction to the isomerization process.