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 rhodop sin was investigated by time-resolved electrical and absorption measurement s with rod outer segment (ROS) fragments. Photoexcitation leads to proton t ransfer 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 move ment with exponential times of 1.1 (45%) and 3.0 ms (55%) (pH 7.8, 22 degre es C, 150 mM KCl). The corresponding activation energies are 86 and 123 kJ/ mol, respectively (150 mM KCl). The time constants and amplitudes depend st rongly 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 compone nts with similar time constants and pH dependence, This suggests that both signals monitor the same molecular events. The pH dependence of the two app arent time constants and amplitudes of the optical data can be described we ll over the pH range 4-7.5 by two coupled equilibria between M-I and two is ochromic M-II species M-IIa and M-IIb: M-I reversible arrow(k1)(k0) M-IIa(3 80) reversible arrow(k3)(k2) M-IIb(380), with k(0) proportional to the prot on concentration. This model implies that deprotonation of the Schiff base and proton uptake are tightly coupled in ROS membranes. Models with k(2) pr oportional to the proton concentration cannot describe the data. Photorever sal of M-II by blue flashes (420 nm) leads to proton transfer in a directio n opposite to that of the signal associated with M-II formation. In this tr ansition the Schiff base is reprotonated, most likely from glutamate 113, A t 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.