Kinetics of the Light-Induced Proton Translocation Associated with the pH-Dependent Formation of the Metarhodopsin I/II Equilibrium of Bovine Rhodopsin

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Abstract
The kinetics of the formation of the metaII (MII) state of bovine rhodopsin was investigated by time-resolved electrical and absorption measurements with rod outer segment (ROS) fragments. Photoexcitation leads to proton transfer in the direction from the cytosolic to the intradiscal side of the membrane, probably from the Schiff base to the acceptor glutamate 113. Two components of comparable amplitude are required to describe the charge movement with exponential times of 1.1 (45%) and 3.0 ms (55%) (pH 7.8, 22 degreesC, 150 mM KCl). The corresponding activation energies are 86 and 123 kJ/mol, respectively (150 mM KCl). The time constants and amplitudes depend strongly on pH. Between pH 7.1 and 3.8 the kinetics becomes much faster, with the faster and slower components accelerating by factors of about 8 and 2, respectively. Complementary single-flash absorption experiments at 380 nm and 10 degreesC show that the formation of MII also occurs with two components with similar time constants and pH dependence. This suggests that both signals monitor the same molecular events. The pH dependence of the two apparent time constants and amplitudes of the optical data can be described well over the pH range 4-7.5 by two coupled equilibria between MI and two isochromic MII species MIIa and MIIb: MI MIIa(380) MIIb(380), with k0 proportional to the proton concentration. This model implies that deprotonation of the Schiff base and proton uptake are tightly coupled in ROS membranes. Models with k2 proportional to the proton concentration cannot describe the data. Photoreversal of MII by blue flashes (420 nm) leads to proton transfer in a direction opposite to that of the signal associated with MII formation. In this transition the Schiff base is reprotonated, most likely from glutamate 113. At pH 7.3, 150 mM KCl, 22 degreesC, this electrical charge reversal has an exponential time constant of about 30 ms and is about 10 times slower than the forward charge motion.