Volume and Enthalpy Changes upon Photoexcitation of Bovine Rhodopsin Derived from Optoacoustic Studies by Using an Equilibrium between Bathorhodopsin and Blue‐Shifted Intermediate

Abstract

AbstractA complex multistep model including an equilibrium was used for the first time to analyze data obtained by laser‐induced optoacoustic spectroscopy (LIOAS) with bovine rhodopsin in washed membranes (T = 5–32°C). The thermal equilibrium was between bathorhodopsin, batho, and the blue‐shifted intermediate (BSI), i.e., batho ⇄ BSI. This analysis considers a 14‐ns intermediate detected by LIOAS at 25°C (with similar decay activation parameters as those reported for BSI in solubilized rhodopsin). The energy content of lumirhodopsin (lumi) and the structural volume difference between lumi and rhodopsin were essentially the same (Elumi. = 80 kJ/mol and ΔVlumi–rho = 40 mL/mol) for both a model based on a simple batho → BSI → lumi sequence and one including the batho ⇄; BSI equilibrium. However, the structural volume changes for the individual steps batho → BSI and BSI → lumi were markedly changed by the introduction of the equilibrium. With the rate constant for the forward reaction batho → BSI for solubilized rhodopsin determined by Hug et al. (Hug, S.J.; Lewis, W.J.; Einterz, C.M.; Thorgeirsson, T.E.; Kliger, D.S. Biochemistry, 1990, 29, 1475–1485) the structural volume changes obtained with the equilibrium model are ΔVBSI‐batho = 79 mL/mol and ΔVlumi‐BSI = −44 mL/mol. The resulting batho → BSI expansion is larger than with the sequential model, and ΔVlumi‐BSI is now a contraction instead of an expansion. A small expansion of 5 mL/mol is derived for the formation of batho with both models. The estimated energy content of BSI is clearly higher than the value of lumi. The structural volume changes between batho, BSI, and lumi derived from the equilibrium model correlate better than those from the simple sequential model with the chromophore transition dipole moment changes determined in the literature using linear dichroism measurements. The structural volume changes are discussed in terms of the variations upon transformation of the chromophore–protein interactions.