Artificial pigments of halorhodopsin and their chloride pumping activities.
Halorhodopsin (HR), the light-driven chloride pump of Halobacterium halobium, was bleached with hydroxylamine and regenerated with all-trans-retinal under several different conditions. The largest recovery of the pigment was found with apoprotein obtained from detergent-free HR [HR(BB)]. To compare the chloride-pumping mechanism of HR with that of bacteriorhodopsin (BR; the light-driven proton pump of the same bacteria), HR pigment analogues were reconstituted with the bleached HR (BB) and retinal analogues. The corresponding BR pigment analogues have previously been shown to have little or no proton-pumping activity, except for retinal2 (3,4-dehydroretinal). Pigment analogues with 13-demethylretinal or retinal2 showed an "opsin shift" similar to that of the all-trans-retinal pigment of both HR and BR. Opsin shifts of the pigments of 9-12-phenylretinal and 3,7-dimethyl-2,4,6,8-decatetraenal and haloopsin are slightly different from those of the corresponding BR pigment analogues, presumably reflecting differences of the chromophoric structures in HR and BR. In addition to the spectral properties, the effect of chloride ion on deprotonation of the Schiff base was measured. These pigment analogues showed the "chloride effect" (a shift of the pK value for deprotonation of the Schiff base), but a smaller one than that seen in HR. For a measurement of the chloride-pumping activity, each retinal analogue was added to a culture of L07 cells (BOP-, HOP+, Ret-), and the activity was measured with the cell suspension. Only cultures with retinal or retinal2 showed chloride-pumping activity, as is true for proton pumping by BR. This suggests that a similar retinal-protein interaction is necessary for both ion pumps.
Iwasa
T
T
Department of Life Science, Himeji Institute of Technology, Hyogo, Japan.
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry*; D006217:Halobacterium / Q000737:chemistry; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D016623:Ion Pumps; D007536:Isomerism; D012172:Retinaldehyde / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*
D002712:Chlorides; D025602:Halorhodopsins; D016623:Ion Pumps; D001436:Bacteriorhodopsins; D012172:Retinaldehyde