No abstract

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO(2) electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR(M) (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl(-). When Cl(-) was replaced with SO(4)(2-), the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl(-) binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl(-)-independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppR(M) state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.

A gene encoding putative retinal protein was cloned from Haloterrigena turkmenica (JCM9743). The deduced amino acid sequence was most closely related to that of deltarhodopsin, which functions as a light-driven H+ pump and was identified in a novel strain Haloterrigena sp. arg-4 (K. Ihara, T. Uemura, I. Katagiri, T. Kitajima-Ihara, Y. Sugiyama, Y. Kimura, Y. Mukohata, Evolution of the archaeal rhodopsins: Evolution rate changes by gene duplication and functional differentiation, J. Mol. Biol. 285 (1999) 163–174. GenBank Accession No. AB009620). Thus, we called the present protein H. turkmenica deltarhodopsin (HtdR) in this report. Differing from the Halobacterium salinarum bacteriorhodopsin (bR), functional expression of HtdR was achieved in Escherichia coli membrane with a high yield of 10–15 mg protein/L culture. The photocycle of purified HtdR was similar to that of bR. The photo-induced electrogenic proton pumping activity of HtdR was verified. We co-expressed both HtdR and EmrE, a proton-coupled multi-drug efflux transporter in E. coli, and the cells successfully extruded ethidium, a substrate of EmrE, on illumination

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 approximately 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.

No abstract