Photoacoustic Studies of the Photocycles of Bacteriorhodopsin at Low Temperatures

Abstract

Halobacterium halobium is extremely halophilic and requires a high concentration of NaCl (CNaCl > 2M) for growth and maintenance of structure. It occurs in salt lakes (e.g. Dead Sea, salt ponds) and survives even in crystalline salt. Under conditions of limited oxygen supply, 1ight-energy-converting membrane patches, the so-called purple membranes, are synthesized within the outer membrane of the bacteria. The purple membrane contains practically only one protein, called bacteriorhodopsin (BR) (1), which is arranged in a two-dimensional lattice, whose structure is known within a resolution of 7Å (2). BR is a chromoprotein and the pigment of the purple membrane. The purple membrane can be isolated by lysis of the cells and following purification by different centrifugation steps. The chromophoric group of BR is retinal, alternatively either in the all-trans or 13-cis configuration (3). Other retinal isomers were not found in vivo. BR which contains all-trans retinal is called trans BR BRtrans568, BR which contains 13-cis retinal is called 13-cis BR BR13-cis548. The two BR isomers are interconvertible by means of several pathways (Fig. 1) (4). One pathway does not require light (‘dark adaptation’); the others are initiated by light. Each isomer forms its distinct primary photoproduct. In the case of trans BR, the photoproduct returns via dark reactions through a series of transients to its initial isomer, trans BR (trans BR cycle). The light-energy-converting process, i.e. the active transport of protons from one side of the purple membrane to the other, can be correlated to the trans BR cycle. The function of the 13-cis BR cycle is not yet known. In the case of 13-cis BR, the dark pathway is split: most of the molecules return to their initial isomer, 13-cis BR (13-cis BR cycle), but some, on an alter native path, go to trans BR. Both pathways are connected by light reaction pathways, which are occupied by photo-excitation of transients. The environmental light conditions determine the isomeric composition of BR. In the dark-adapted state, BR contains a mixture of 13-cis BR and trans BR in a ratio of 1:1. On illumination of a dark-adapted sample with moderate light (e.g. 1mW/cm2), nearly all BR molecules are found in the trans BR state within a few seconds. The photoproducts of each BR isomer can be identified by means of low temperature absorption spectroscopy. To overcome light scattering due to cracks forming in aqueous suspensions at low temperatures, the BR samples have to be suspended in a glycerol/water mixture (w:w = 2:1) for optical absorption measurements. Only photoacoustic spectroscopy enables us to examine the photoproducts and intermediates of the two BR photocycles in buffered aqueous suspensions at low temperatures. We thus obtained photoacoustic spectra of nearly the same quality as optical absorption spectra.