Kinetic and thermodynamic study of the bacteriorhodopsin photocycle over awide pH range

The photocycle of bacteriorhodopsin and its thermodynamic parameters were s tudied in the pH range of 4.5-9. Measurements were performed at five differ ent wavelengths (410, 500, 570, 610, and 650 nm), in the time interval 300 ns to 0.5 s, at six temperatures between 5 and 30 degrees C. Data were fitt ed to different photocycle models. The sequential model with reversible rea ctions gave a good fit, and the linear character of the Eyring plots was fu lfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linea r behavior. When a parallel model with reversible reactions, which has twic e as many free parameters as the sequential model, was considered, the qual ity of the fit did not improve and the Eyring plots were not linear. The se quential model was used to determine the thermodynamic activation parameter s (activation enthalpy, entropy, and free energy) of the transitions and th e free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transiti ons M-1 to M-2 and N to O disclosed a large entropy increase; which could b e interpreted as a loosening of the protein structure. The pH dependence of the energy levels explains the disappearance of intermediate O at high pH. A hypothesis is proposed to interpret the relation between the observed pK (a) of the photocycle energetics and the role of several amino acids in the protein.