ABSORPTION AND PHOTOCHEMISTRY OF SENSORY RHODOPSIN .1. PH EFFECTS

Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH-probe to test whether there is a light-induced proton release to the bulk phase during the photochemical reaction cycle of sensory rhodopsin-I (SR-I). We conclude that the retinylidene Schiff-base proton is retained by SR-I-containing envelope vesicles during the SR-I photocycle under the conditions described here. Bacteriorhodopsin containing vesicles were used as a control to show that light-induced proton release can be observed under identical data acquisition parameters as those used for SR-I-containing vesicles. In addition, the effects of extravesicular pH on the absorption maximum (lambda(max)) and the SR-I photocycle were studied. SR-I properties are insensitive to pH in the range approximately 3 to approximately 8 with lambda(max) remaining at 587 nm. The lambda(max) shifts to 565 nm below pH 3.0 and to 552 nm at pH 10.8 with an apparent pK(a) of 8.5. Flash-induced absorbance changes of SR-I are described under neutral, alkaline and acidic conditions. The neutral, alkaline and acid SR-I forms each undergo similar photoreactions producing long-lived (>500 ms decay half-time) blue-shifted intermediates. The UV/near-UV absorption of the photoproducts from neutral and alkaline SR-I indicate a deprotonated Schiff base, whereas acid SR-I produces a species with lambda(max) > 460 nm indicative of a protonated Schiff base.