When grown under low oxygen tension in the light, Halobacterium halobium produces distinct patches in its plasma membrane which can be isolated by differential and sucrose density gradient centrifugation after lysis of the cells by dialysis against distilled water (Oesterhelt and Stoeckenius, 1974). These membrane fragments, which

No abstract

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.

Bacteriorhodopsin is a rhodopsin-like protein found in the cell membrane of Halobacterium halobium. It Halobacterium halobium contains in its cell membrane a rhodopsin-like protein, bacteriorhodopsin, which has a broad absorption band around 570 nm. The absorbed light energy is used by the pigment to translocate protons across the cell membrane (2, 3). Bacteriorhodopsin, like the visual pigments of animals, contains retinal linked as a Schiff base to a lysine residue of the opsin (2, 4, 5). We have studied mammalian rhodopsin with tunable laser resonance Raman spectroscopy (6). The results, together with biochemical and other spectroscopic data on several forms of rhodopsin, indicate that the Schiff base linkage of retinal to opsin is protonated (for recent reviews see refs. 7-9). Mendelsohn (1) used bacteriorhodopsin in similar studies and concluded that the retinal protein linkage in this pigment is unprotonated. This suggested an interesting difference between bacteriorhodopsin and mammalian rhodopsin. We have now extended our study to bacteriorhodopsin using a tunable laser and working at low temperature.Mendelsohn (1) assumed that bacteriorhodopsin is photochemically stable. Our recent data indicate (10, 11) that this is not so. In blue-green light the pigment undergoes cyclic conformational changes through a series of intermediates, including a complex with an absorption maximum at 412 nm (3,10,11 MATERIALS AND METHODSRaman spectra were obtained with a Spex 1401 double monochromator equipped with a home-built stepping motor and a RCA 31034 photomultiplier. A multichannel analyzer was used to record the counts at each wavelength setting of the monochromator. The spectra were averaged and plotted through a PDP-1 1 minicomputer. A Coherent Radiation model 53B argon ion laser equipped with UV mirrors, a model 53B krypton laser, and a Coherent Radiation model 431 tunable dye laser provided the exciting light. Some spectra were obtained with a Liconix model 401 He-Cd laser, which lases at 441.6 nm.The bacteriorhodopsin was used in the form of the purple membrane. This is a preparation of isolated membrane fragments from Halobacterium halobium R1 (12,13) The low-temperature experiments were performed in melting point capillaries surrounded by a glass jacket and cooled with a stream of nitrogen gas. The temperature was moni- 4462$ To whom reprint requests should be addressed.

Flash spectroscopy data were obtained for purple membrane fragments at pH 5, 7, and 9 for seven temperatures from 5 degrees to 35 degrees C, at the magic angle for actinic versus measuring beam polarizations, at fifteen wavelengths from 380 to 700 nm, and for about five decades of time from 1 microsecond to completion of the photocycle. Signal-to-noise ratios are as high as 500. Systematic errors involving beam geometries, light scattering, absorption flattening, photoselection, temperature fluctuations, partial dark adaptation of the sample, unwanted actinic effects, and cooperativity were eliminated, compensated for, or are shown to be irrelevant for the conclusions. Using nonlinear least squares techniques, all data at one temperature and one pH were fitted to sums of exponential decays, which is the form required if the system obeys conventional first-order kinetics. The rate constants obtained have well behaved Arrhenius plots. Analysis of the residual errors of the fitting shows that seven exponentials are required to fit the data to the accuracy of the noise level.