pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. The X-ray crystallographic structure of ppR is very similar to those of the ion-pumping rhodopsins, bacteriorhodopsin (BR) and halorhodopsin (hR). However, the decay processes of the photocycle intermediates such as M and O are much slower than those of BR and hR, which is advantageous for the sensor function of ppR. Iwamoto et al. previously found that, in a quadruple mutant (P182S/P183E/V194T/T204C; denoted as SETC) of ppR, the decay of the O intermediate was accelerated by ×100 times (t1/2 ∼6.6 ms vs 690 ms for the wild type of ppR), being almost equal to that of BR (Iwamoto, M., et al. (2005) Biophys. J. 88, 1215-1223). The mutated residues are located on the extracellular surface (Pro182, Pro183, and Val194) and near the Schiff base (Thr204). The present Fourier-transform infrared (FTIR) spectroscopy of SETC revealed that protein structural changes in the K and M states were similar to those of the wild type. In contrast, the ppRo minus ppR infrared difference spectra of SETC are clearly different from those of the wild type in amide-I (1680-1640 cm-1) and S-H stretching (2580-2520 cm -1) vibrations. The 1673 (+) and 1656 (-) cm-1 bands newly appear for SETC in the frequency region typical for the amide-I vibration of the αII- and αI-helices, respectively. The intensities of the 1673 (+) cm-1 band of various mutants were well correlated with their O-decay half-times. Since the αII-helix possesses a considerably distorted structure, the result implies that distortion of the helix is required for fast O-decay. In addition, the characteristic changes in the S-H stretching vibration of Cys204 were different between SETC and T204C, suggesting that structural change near the Schiff base was induced by mutations of the extracellular surface. We conclude that the lifetime of the O intermediate in ppR is regulated by the distorted α-helix and strengthened hydrogen bond of Cys204.
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