Biophys J, June 2000, p. 3150-3159, Vol. 78, No. 6

The M Intermediate of Pharaonis Phoborhodopsin Is Photoactive

Sergei P. Balashov,^* Masato Sumi, and Naoki Kamo

 ^*Center for Biophysics and Computational Biology, Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 USA, and  Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan

The retinal protein phoborhodopsin (pR) (also called sensory rhodopsin II) is a specialized photoreceptor pigment used for negative phototaxis in halobacteria. Upon absorption of light, the pigment is transformed into a short-wavelength intermediate, M, that most likely is the signaling state (or its precursor) that triggers the motility response of the cell. The M intermediate thermally decays into the initial pigment, completing the cycle of transformations. In this study we attempted to determine whether M can be converted into the initial state by light. The M intermediate was trapped by the illumination of a water glycerol suspension of phoborhodopsin from Natronobacterium pharaonis called pharaonis phoborhodopsin (ppR) with yellow light (>450 nm) at 50°C. The M intermediate absorbing at 390 nm is stable in the dark at this temperature. We found, however, that M is converted into the initial (or spectrally similar) state with an absorption maximum at 501 nm upon illumination with 380-nm light at 60°C. The reversible transformations ppR  M are accompanied by the perturbation of tryptophan(s) and probably tyrosine(s) residues, as reflected by changes in the UV absorption band. Illumination at lower temperature (160°C) reveals two intermediates in the photoconversion of M, which we termed M' (or M'₄₀₄) and ppR' (or ppR'₄₉₆). A third photoproduct, ppR'₅₀₄, is formed at 110°C during thermal transformations of M'₄₀₄ and ppR'₄₉₆. The absorption spectrum of M'₄₀₄ (maximum at 404 nm) consists of distinct vibronic bands at 362, 382, 404, and 420 nm that are different from the vibronic bands of M at 348, 368, 390, and 415 nm. ppR'₄₉₆ has an absorption band that is shifted to shorter wavelengths by 5 nm compared to the initial ppR, whereas ppR'₅₀₄ is redshifted by at least 3 nm. As in bacteriorhodopsin, photoexcitation of the M intermediate of ppR and, presumably, photoisomerization of the chromophore during the M  M' transition result in a dramatic increase in the proton affinity of the Schiff base, followed by its reprotonation during the M'  ppR' transition. Because the latter reaction occurs at very low temperature, the proton is most likely taken from the counterion (Asp⁷⁵) rather than from the bulk. The phototransformation of M reveals a certain heterogeneity of the pigment, which probably reflects different populations of M or its photoproduct M'. Photoconversion of the M intermediate provides a possible pathway for photoreception in halobacteria and a useful tool for studying the mechanisms of signal transduction by phoborhodopsin (sensory rhodopsin II).

Biophys J, June 2000, p. 3150-3159, Vol. 78, No. 6
© 2000 by the Biophysical Society   0006-3495/00/06/3150/10  $2.00

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