COVALENT LINK BETWEEN THE CHROMOPHORE AND THE PROTEIN BACKBONE OF BACTERIORHODOPSIN IS NOT REQUIRED FOR FORMING A PHOTOCHEMICALLY ACTIVE PIGMENT ANALOGOUS TO THE WILD-TYPE

Bacteriorhodopsin pigments lacking the retinal-lys-216 covalent bond w ere prepared by reconstituting the K216G mutant protein with retinal a lkylamine Schiff bases. The procedure follows the approach of Zhukovsk y et al. [Zhukovsky, E., Robinson, P., and Oprian, D. (1991) Science 2 51, 558-560] in the case of visual (rhodopsin) pigments. Reconstitutio n leads to a mixture of three pigments. One of them, bR(K216G)/566a, a bsorbs (pH = 6.9) at 566 nm. Its absorption is pH-dependent, exhibitin g a purple to blue transition. The pigment's laser-induced photocycle patterns are similar to those of wild-type all-trans-6R. A second comp onent, bR(K216G)/566b, exhibits an independent photocycle reminiscent of that of wild-type 13-cis-bR. A third pigment component, bR(K216G)/6 30, absorbs around 630 nm. Experiments in the presence of a pH dye ind icator show that illumination of bR(K216G)/566 produces a detectable p roton gradient. It is cancluded that a covalent bond between the retin al chromophore and the protein backbone is not a prerequisite for the basic structure and photochemical features of bR or for its proton pum p activity.