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Characterization of the Primary Photochemistry of Proteorhodopsin with Femtosecond Spectroscopy

Alisa Rupenyan ^*, Ivo H. M. van Stokkum ^*, Jos C. Arents , Rienk van Grondelle ^*, Klaas Hellingwerf ^*  and Marie Louise Groot ^*

^* Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands; and Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1010 WV Amsterdam, The Netherlands

Correspondence: Address reprint requests to Rienk van Grondelle, Dept. of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. E-mail: rienk{at}nat.vu.nl.

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, Asp⁹⁷, is deprotonated) and at pH 6.5 (with Asp⁹⁷ 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 0.2 ps, 2 ps, and 20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerizaton pathways that lead to the K-state: one with an effective rate of (2 ps)^–1 and the other with a rate of (20 ps)^–1. At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of 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 Asp²²⁷, 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.