Chemical modification of purple membranes: role of arginine and carboxylic acid residues in bacteriorhodopsin

Light energy conversion by bacteriorhodopsin involves vectorial translocation of a proton across the purple membrane of Halobacterium halobium. Following photon activation of the retinal chromophore, a photoreaction cycle commences. During the first 30-50 ps of this photocycle, the light-adapted 570 nm chromophore is converted into the M4r2 transient species, whose Schiff base nitrogen has been found by resonance Raman spectroscopy [l] to be a deprotonated species. Reprotonation of MeI2 occurs during the 5-7 ms required for completion of the photocycle. These results suggest that deprotonation and reprotonation of the retinal Schiff base are essential for proton translocation across the membrane. Since the primary sequence of bacteriorhodopsin has been established [2,3], it is possible now to gain more information on the role of specific amino acid residues in proton translocation. Chemical modification of amino acids is a particularly promising approach to the problem because bacteriorhodopsin is the only protein component in the purple membrane and because the activity of these preparations is stable [4,5]. We report here the effect of treating purple membranes with two reagents which modify arginine residues and three carbodiimide reagents which modify carboxylic acid groups. In both cases, proton release in the early stages of the photocycle is slightly affected, but marked inhibition of the reprotonation phase occurs, leading to large increases in the amount of the M4r2 species seen in the photostationary state. The