Characterization of the primary photochemistry of Proteorhodopsin with femtosecond spectroscopy
Alisa Rupenyan 1, Ivo H.M. van Stokkum 1, Joc C. Arents 2, Rienk van Grondelle 1*, Klaas Hellingwerf 2 and Marie Louise Groot 1
1 Vrije Universiteit
2 University of Amsterdam
* To whom correspondence should be addressed. E-mail: rienk{at}nat.vu.nl.
Submitted on September 5, 2007
Revised on October 8, 2007
Accepted on 9 January 2008
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Abstract |
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Proteorhodopsin (pR) is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cisisomerization, leading to trans-membrane translocation of a proton, towards the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of pR, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the mid-infrared 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 multi-exponential 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 upon K-state formation; and a feature that develops on the 0.2 ps and 2 ps time scale and probably reflects a response of an amide II band in reaction the isomerization process.
Key Words:
chromophore, conformational change, photoisomerization, retinal, vibrational spectroscopy
