Surface charge changes in purple membranes and the photoreaction cycle of bacteriorhodopsin

The surface potential of purple membrane fragments, determined from the distribution of the aqueous free and the membrane-bound positively charged, paramagnetic, amphiphilic probe 4-(dodecyldimethylammonium)-1-oxyl-2,2,6,6-tetramethylpiperidine bromide varied almost 60 mV as a function of ionic strength and 50 mV as a function of pH of the medium. Light-induced changes in surface potential followed the changes observed in the M412 intermediate of the photocycle of bacteriorhodopsin as a function of pH, temperature, and response to antibiotics beauvericin and valinomycin. The number of induced charges per M412 appearing at the surface of purple membranes decreased from about 0.75 to 0.45 as the surface potential became more negative. The stoichiometry would be twice as large if the charge changes were localized exclusively on one side of the purple membrane. Laser flash-induced kinetics of the rise and decay of surface charge changes were slightly slower than the kinetics of the rise and decay of M412 which is associated with the reversible deprotonation of the retinal Schiff base nitrogen in the chromophore. It is suggested that the light-induced charge changes monitor a dissociable amino acid residue which may be a step in the movement of protons across the purple membrane.