More evidence that light isomerises the chromophore of purple membrane protein

Abstract

LIGHT absorption by the purple membrane protein (bacterio-rhodopsin) of Halobacterium halobium leads to pumping of protons across the cell membrane¹; the resulting proton gradient can in turn be used to make ATP^2,3. As in photosynthesis, the energy for this proton pumping must be ultimately derived from the energy of the absorbed photon which is converted into chemical free energy in the formation of the primary photochemical product, bathobacteriorhodopsin or K (refs 4 and 5) (see Fig. 1). On warming, K decays to the photocycle intermediate L and then to M⁴, which, after warming to higher temperatures, returns to purple membrane protein (PM). The chromophore of the native pigment is all-trans retinal attached to the protein by way of a protonated Schiff base^6,7. The question arises : What is the action of light on the chromophore which results in K formation? In earlier papers^5,16, we have provided a partial answer to the nature of K by marshalling strong evidence that, as in visual pigments⁸, the primary effect of light is to isomerise the chromophore. Pettei et al.⁹ have recently shown using a chromophore extraction method that by the time of M formation, at least part and perhaps all of the chromophores have been isomerised to the 13-cis conformation. Resonance Raman studies of the chromophore of the pigment and its M intermediate also suggest that the chromophore is isomerised from the all-trans to the 13-cis conformation by the time of M formation⁷. We show here that when M or L is irradiated at −196 °C, their primary photoproducts—M′ or L′—can reform the original pigment when warmed to −90 °C. This property of these new photoproducts can most readily be explained if light has photoisomerised the chromophore of the intermediates back to the conformation originally present in the pigment. Thus, by the L and M intermediates in the photocycle, light has altered the geometry of the chromophore from the all-trans to another geometrical conformation.