New photointermediates in the two photon signaling pathway of sensory rhodopsin-I

Sensory rhodopsin-I (SRT) functions as a color discriminating receptor in h alobacterial phototaxis. SRI exists in the membrane as a molecular complex with a signal transducer protein. Excitation of its thermally stable form, SRI587, generates a long-lived photointermediate of its photocycle, S-373, and an attractant phototactic response. S-373 decays thermally in a few sec onds into SRI587. However, when S-373 is excited by UV-blue light, it photo converts into SRI587 in less than a second, generating a repellent phototac tic response. Only one intermediate of this back-photoreaction, S-510(b), i s known. We studied the back-photoreaction in both native SRI and its trans ducer free form fSRI by measuring laser flash induced absorption changes of S-373 photoproducts from 100 ns to 1 s in the 350-750 nm range. Using glob al exponential fitting, we determined the spectra and kinetics of the photo intermediates. S-373 and fS(373) when pumped with 355 nm laser light genera te in less than 100 ns two intermediate species: a previously undetected sp ecies that absorbs maximally at about 410 nm, S-410(b), and the previously described S-510(b). These two intermediates appear to be in a rapid equilib rium, which probably entails protonation change of the Schiff base chromoph ore. At pH 6 this system relaxes to SRI587 via another intermediate absorbi ng maximally around 550 nm, which thermally decays back to the ground state . The same intermediates are seen in the presence and absence of transducer ; however, the kinetics are affected by binding of the transducer.