SPONTANEOUS AGGREGATION OF BACTERIORHODOPSIN IN BROWN MEMBRANE

Abstract— Halobacterium halobium cells grown in nicotine-containing medium synthesize bacterio-opsin (bO) but little bacteriorhod-opsin (bR) because nicotine inhibits retinal synthesis. In nicotine-grown cells, bacterio-opsin is found in a specific cell membrane fraction, the brown membrane (b.m.), which consists mainly of small round sheets. Freeze-fracture of isolated brown membrane reveals a dense particle population on its cytoplasmic fracture face, with few particles on the external face. There is no apparent order in the particle distribution and the fracture faces are practically indistinguishable from those of red membrane (r.m.). The absorbance spectrum of b.m. shows peaks at 550 and 410nm, the circular dichroism (CD) spectrum a positive band around 540nm and positive and negative bands around 410nm with a cross-over at 412nm. The photoreaction cycle of bR in b.m. has the same intermediates found in purple membrane (p.m.), but the apparent kinetics resemble those of bR monomers. Addition of 13-cis-, or all-trans-retinal to b.m. induces a rapid 5- to 10-fold increase in the visible absorbance band, and the absorbance maximum shifts to 560nm in the dark. Kinetic analysis of the absorbance increase shows three first order kinetic constants with t_½= 0.5 min, 6 min and 100 min, with most of the increase contributed by the fastest component. The CD bilobal pattern typical for purple membrane appears together with the strong negative band at 320nm. Ellipticity change at 590nm is nearly as rapid as the absorbance increase; within 5 min, 85% of the negative band is formed. Electron microscopy after addition of retinal reveals structurally distinct domains in the b.m. sheets which have the characteristic appearance of p.m. However, X-ray reflections are more diffuse compared to p.m. Flash spectroscopy of reconstituted b.m. detects the same photocycle intermediates as in b.m. or p.m., but with apparent kinetics closer to those of purple membrane than before reconstitution. These results show that bO in the b.m. binds retinal to form bR which spontaneously aggregates into a lattice. However, the reconstituted bR lattice is not as well-ordered as in p.m. and some bR is still present in the form of monomers and/or small aggregates.     

THE RADIOLYTIC REDUCTION OF THE SCHIFF BASE IN BACTERIORHODOPSIN

Abstract —Nanosecond electron beam pulses cause radiolytic reduction of the retinal Schiff base in bacteriorhodopsin. The effects of different scavengers show that both H atoms and the formate radical anion can act as the reducing species. Model system studies on the radiolysis of retinal indicate transient formation of a retinal radical anion and suggest an analogous two-step reaction in bR. Delocalization of the radical in the polyene system and reaction with surrounding groups would account for the relatively low yield of reduced Schiff base and the appearance of intra- and intermolecular crosslinks in irradiated bacteriorhodopsin.