Heterogeneity Effects in the Binding of All-Trans Retinal to Bacterio-opsin

The special trimeric structure of bacteriorhodopsin (bR) in the purple membrane of Halobacterium salinarum, and especially, the still controversial question as to whether the three protein components are structurally and functionally identical, have been subject to considerable work. In the present work, the problem is approached by studying the reconstitution reaction of the bR apo-protein with all-trans retinal, paying special attention to the effects of the apo-protein/retinal (P:R) ratio. The basic observation is that at high P:R values, the reconstitution reaction proceeds via two distinct, fast and slow, pathways associated with two different pre-pigment precursors absorbing at 430 nm (P430) and 400 nm (P400), respectively. These two reactions, exhibiting 2:1 (P430/P400) amplitude ratios, are markedly affected by the P:R value. The principal feature is the acceleration of the P400 → bR transition at low P:R ratios. The data are interpreted in terms of a scheme in which the added retinal first occupies two protein retinal traps, R1 and R2, from which it is transferred to two spectroscopically distinct binding sites corresponding to the two pre-pigments, P430 and P400, respectively. Two noncovalently bound retinal molecules occupy two P430 sites of the bR trimer, while one (P400) occupies the third. Binding is completed by generating the retinal−protein covalent bond. Analogous experiments were also carried out with an aromatic bR chromophore and with the D85N bR mutant. The accumulated data clearly point out the heterogeneity of the binding reaction intermediates, in which two are clearly distinct from the third. However, CD spectroscopy strongly suggests that even the two P430 sites are not structurally identical. The heterogeneity of the P intermediates in the binding reaction can be accounted for, either by being induced by cooperativity or by an intrinsic heterogeneity that is already present in the apoprotein. The question as to whether the final reconstituted pigment, as well as native bR, are nonhomogeneous should be the subject of future studies.