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Correlation of the O-Intermediate Rate with the pK_a of Asp-75 in the Dark, the Counterion of the Schiff Base of Pharaonis Phoborhodopsin (Sensory Rhodopsin II)

Masayuki Iwamoto ^* , Yuki Sudo , Kazumi Shimono , Tsunehisa Araiso ^* and Naoki Kamo ^*

^* Laboratory of Biomolecular Systems, Center for Advanced Science and Technology; and Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Correspondence: Address reprint requests to Masayuki Iwamoto, Division of Molecular Physiology and Biophysics, Dept. of Morphological and Functional Biomedical Science, Faculty of Medical Science, University of Fukui, Matsuoka 910-1193, Japan. Tel.: 81-776-61-8306; Fax: 81-776-61-8101; E-mail: zz04004{at}fmsrsa.fukui-med.ac.jp.

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis. The photocycle rate of ppR is slow compared to that of bacteriorhodopsin, despite the similarity in their x-ray structures. The decreased rate of the photocycle of ppR is a result of the longer lifetime of later photo-intermediates such as M- (ppR_M) and O-intermediates (ppR_O). In this study, mutants were prepared in which mutated residues were located on the extracellular surface (P182, P183, and V194) and near the Schiff base (T204) including single, triple (P182S/P183E/V194T), and quadruple mutants. The decay of ppR_O of the triple mutant was accelerated 20-times from 690 ms for the wild-type to 36 ms. Additional mutation resulting in a triple mutant at the 204th position such as T204C or T204S further decreased the decay half-time to 6.6 or 8 ms, almost equal to that of bacteriorhodopsin. The decay half-times of the ppR_O of mutants (11 species) and those of the wild-type were well-correlated with the pK_a value of Asp-75 in the dark for the respective mutants as spectroscopically estimated, although there are some exceptions. The implications of these observations are discussed in detail.

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