Kinetic and Thermodynamic Study of the Bacteriorhodopsin Photocycle over a Wide pH Range

Abstract

The photocycle of bacteriorhodopsin and its thermodynamic parameters were studied in the pH range of 4.5-9. Measurements were performed at five different 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 degreesC. Data were fitted to different photocycle models. The sequential model with reversible reactions gave a good fit, and the linear character of the Eyring plots was fulfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linear behavior. When a parallel model with reversible reactions, which has twice as many free parameters as the sequential model, was considered, the quality of the fit did not improve and the Eyring plots were not linear. The sequential model was used to determine the thermodynamic activation parameters (activation enthalpy, entropy, and free energy) of the transitions and the free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transitions M1 to M2 and N to O disclosed a large entropy increase, which could be 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 pKa of the photocycle energetics and the role of several amino acids in the protein.

Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin

“…3 A and B). These spectra closely matched corresponding spectra of Archaerhodopsin 1 (32) and BR (33). The pH-dependent transient absorption (Fig.…”

Structure, dynamics and reactions of protein hydration water

“…The activation energy barrier found for this pathway is in agreement with experimental evidence (Ludman et al 1998), and thus provides evidence for internal water molecules playing a direct role in the primary proton transfer process in BR. Locating water molecules that play a direct role in protein function is a major challenge for crystallographers and computer simulations alike.…”

“…10 s (Ludman et al 1998). In this step, a proton is transferred from the retinal Schiff base NH group to Asp85 over a distance of ca.…”

Mechanism of a proton pump analyzed with computer simulations

“…Nevertheless, flexibility of the protein is essential for proton transfer [30,32,33]. In the presence of a flexible protein environment, computations with a pair of reactant and product states shown to be compatible with active proton pumping indicated an energy barrier of *12 kcal/mol, and the reaction was almost isoenergetic [30] (the enthalpic barrier estimated from experiments for the first proton-transfer step is *13 kcal/mol [34]). When only the active site groups (retinal, K216, D85, T89, D212, and w402) were allowed to move, the proton transfer barrier and the reaction energy were *23 kcal/mol and *11 kcal/mol, respectively [32,33].…”