Monitoring The Activation Of Rhodopsin By The Transient Fluorescence Of Fluorescently Labeled Helix 8

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The transient changes of the fluorescence of bovine rhodopsin in ROS membranes selectively labeled with Alexa594 at cysteine 316 in helix 8 were followed in time from 1 μs to 10 s after flash excitation of the photoreceptor. A large light-induced transient fluorescence increase was observed with time constants in the ms- range at pH6. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state MII (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6), the sequence of events is: Schiff base deprotonation, structural change, proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.