Iwasa Tatsuo, Abe Eisuke, Yakura Yuki, Yoshida Hideaki, Kamo Naoki
Department of Materials Science and Engineering, Muroran Institute of Technology, Muroran, Japan.
Photochem Photobiol. 2007 Mar-Apr;83(2):328-35. doi: 10.1562/2006-06-15-RA-928.
Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor for the photophobic response of Natronomonas pharaonis. Tryptophan 182 (W182) of bacteriorhodopsin (bR) is near the chromophore retinal and has been suggested to interact with retinal during the photoreaction and also to be involved in the hydrogen-bonding network around the retinal. W182 of bR is conserved in ppR as tryptophan 171 (W171). To elucidate whether W171 of ppR interacts with retinal during the photoreaction and/or is involved in the hydrogen-bonding network as in bR, we formed W171-substituted mutants of ppR, W171A and W171T. Our low-temperature spectroscopic study has revealed that the substitution of W171 to Ala or Thr resulted in the stabilization of M- and O-intermediates. The stability of M and absorption spectral changes during the M-decay were different depending on the substituted residue. These findings suggest that W171 in ppR interacts with retinal and the degree of the interaction depends on the substituted residues, which might be rate determining in the M-decay. In addition, the involvement of W171 in the hydrogen-bonding network is suggested by the O-decay. We also found that glycerol slowed the decay of M and not of O.
法老嗜盐菌视紫红质（ppR），也称为法老嗜盐菌感官视紫红质II、NpSRII，是嗜盐栖热放线菌光ophobic反应的光感受器。细菌视紫红质（bR）的色氨酸182（W182）靠近发色团视黄醛，有人认为它在光反应过程中与视黄醛相互作用，并且还参与视黄醛周围的氢键网络。bR的W182在ppR中保守为色氨酸171（W171）。为了阐明ppR的W171在光反应过程中是否与视黄醛相互作用和/或是否像bR一样参与氢键网络，我们构建了ppR的W171取代突变体W171A和W171T。我们的低温光谱研究表明，将W171替换为丙氨酸或苏氨酸会导致M中间体和O中间体的稳定。M中间体的稳定性以及M中间体衰变过程中的吸收光谱变化取决于取代的残基。这些发现表明，ppR中的W171与视黄醛相互作用，相互作用的程度取决于取代的残基，这可能是M中间体衰变的速率决定因素。此外，O中间体衰变表明W171参与了氢键网络。我们还发现甘油减缓了M中间体的衰变，但没有减缓O中间体的衰变。
