Kinetics of the light-induced proton translocation associated with the pH-dependent formation of the metarhodopsin I/II equilibrium of bovine rhodopsin

The kinetics of the formation of the meta(II) (M-II) 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 degrees C, 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 degrees C show that the formation of Mn 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 M-I and two isochromic M-II species M-IIa and M-IIb: M-I reversible arrow(k1)(k0) M-IIa(380) reversible arrow(k3)(k2) M-IIb(380), with k(0) 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 k(2) proportional to the proton concentration cannot describe the data. Photoreversal of M-II by blue flashes (420 nm) leads to proton transfer in a direction opposite to that of the signal associated with M-II formation. In this transition the Schiff base is reprotonated, most likely from glutamate 113, At pH 7.3, 150 mM KCl, 22 degrees C, this electrical charge reversal has an exponential time constant of about 30 ms and is about 10 times slower than the forward charge motion.