Energetics of primary processes in visual excitation: photocalorimetry of rhodopsin in rod outer segment membranes

Abstract

A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.

Rhodopsin photoenergetics: lumirhodopsin and the complete energy profile

“…Nevertheless, the overall energetics are consistent with a general mechanism involving rapid and efficient retinal photoisomerization as a first step [ 12,131, whilst at the same time unwanted thermal activation is suppressed by the large ground-state barrier [3]. Stored energy is subsequently released by conformational relaxation in stages to the metarhodopsin I state [7], following which the retinal-opsin linkage is probably hydrolysed [2,14] prior to release of the chromophore from the protein. These latter stages are undoubtedly more complex than pictured here, involving transient intermediates which cannot be resolved by calorimetry.…”

“…By combining this datum with the enthalpies of other intermediates determined [2,3] and with kinetic activation energy barriers observed in bovine rhodopsin under similar conditions [7-lo], it is now possible to draw a potential energy diagram covering the entire sequence of major events in the bleaching of rhodopsin (lig.2). This cannot, by itself, define the molecular mechanism of the process, but it does for the first time provide stringent thermochemical limits within which models may be developed.…”

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

“…Speculation on the Role of Arg-177/Asp-190 in Stabilizing Rhodopsin-Previous chemical models of the retinal-binding and release pathways have speculated that the protonated retinal Schiff base linkage can spontaneously hydrolyze and thus is in dynamic equilibrium with retinal covalently bound to rhodopsin (75,78). One interpretation of our data may be that the ion pair stabilizes the retinal plug structure.…”

Hydration of retinal and the nature of metarhodopsin II

“…After exposure to light, rhodopsin passes through a series of spectroscopically distinct intermediate steps culminating in the release of the free retinal aldehyde, as the all-trans isomer, from the apoprotein opsin [ 11. The precise stage at which hydrolysis of the Schiff base occurs is not known but, using a variety of circumstantial evidence, we earlier proposed that this most likely takes place between the intermediates known as metarhodopsins I and II, implying that metarhodopsin II consists of the aldehyde form of retinal non-covalently bound to the active site of opsin [2]. However, a subsequent resonance Raman study of metarhodopsin II failed to show the anticipated aldehydic C=O stretching band at this stage [3], leading the authors to support the interpretation of metarhodopsin II as an unprotonated retinalopsin Schiff base complex [4], despite the absence of a C=N band in the spectrum.…”