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pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. In halobacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. In the present work, the truncated transducer, t-Htr, was used which interacts with ppR [Sudo et al. (2001) Photochem. Photobiol. 74, 489–494]. Two water-soluble reagents, hydroxylamine and azide, reacted both with the transducer-free ppR and with the complex ppR/t-Htr (the complex between ppR and its truncated transducer). In the dark, the bleaching rates caused by hydroxylamine were not significantly changed between transducer-free ppR and ppR/t-Htr, or that of the free ppR was a little slower. Illumination accelerated the bleach rates, which is consistent with our previous conclusion that the reaction occurs selectively at the M-intermediate, but the rate of the complex was about 7.4-fold slower than that of the transducer-free ppR. Azide accelerated the M-decay, and its reaction rate of ppR/t-Htr was about 4.6-fold slower than free ppR. These findings suggest that the transducer binding decreases the water accessibility around the chromophore at the M-intermediate. Its implication is discussed.

Halorhodopsin, isolated from Halobacterium salinarium cells incubated with tritiated palmitic acid, co-elutes with labeled palmitate in phenylsepharose CL-4B chromatography. Halorhodopsin-bound 3H-palmitate is not readily displaced by prolonged exposure to a large excess of detergents and by re-chromatography of radiolabeled halorhodopsin on phenylsepharose. On other hand, the association of labeled palmitate with purified halorhodopsin is not resistant to denaturation induced either by isopropanol/hexane or by SDS gel electrophoresis. We have tested the hypothesis that tightly associated palmitate is bound to halorhodopsin through a thioester bond, which is unstable in denaturing conditions. Using GC/MS, we have analysed the reaction products of native halorhodopsin with specific thioester reagents, thiols and NaBH4, which are inactive on free fatty acids. The results of this analytical approach indicate that there is no thioester bond between halorhodopsin and palmitic acid and that palmitic acid is associated with halorhodopsin as a free fatty acid.

Recent fast atom bombardment-mass spectrometry (FABMS) studies (Tsujimoto, K., Yorimitsu, S., Takahashi, T. and Ohashi, M. (1989) J. Chem. Commun. 668-670; Frederickson, H.L., De Leeuw, J.W., Tas, A.C., Van der Greef, J., LaVos, G.F. and Boon, J.J. (1989) Biomed. Environ. Mass. Spectrom. 18, 96-105; Kloppel, K.D. and Fredrickson, H.L. (1991) J. Chromatogr. 562, 369-376) have indicated that the structure of the major phospholipid of Halobacterium salinarium (formerly Halobacterium cutirubrum) is not 2,3-diphytanyl-sn-glycerol-1-phospho-3'-sn-glycerol-1'- phosphate (PGP), but the monomethylated derivative, 2,3-diphytanyl-sn-glycerol-1-phospho-3'-sn-glycerol-1'-methylphosphate (PGP-Me). We have now confirmed the structure of the major phospholipid of extremely halophilic archaebacteria as being this methylated structure (PGP-Me) by 1H- and 13C-NMR, FABMS and TLC of the native phospholipid and its product of mild acid hydrolysis PGP. The methylated structure (PGP-Me), rather than PGP itself, is also the major phospholipid in species of other genera of extreme halophiles examined so far, such as, Haloferax, Haloarcula, Halococcus, Natronobacterium and Natronococcus.