PROTONATION DYNAMICS OF THE EXTRACELLULAR AND CYTOPLASMIC SURFACE OF BACTERIORHODOPSIN IN THE PURPLE MEMBRANE

The dynamics of proton binding to the extracellular and the cytoplasmi c surfaces of the purple membrane were measured by laser-induced proto n pulses, Purple membranes, selectively labeled by fluorescein at Lys- 129 of bacteriorhodopsin, were pulsed by protons released in the aqueo us bulk from excited py ranine (8-hydroxy-1,3,6-pyrenetrisulfonate) an d the reaction of protons with the indicators was measured, Kinetic an alysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk pr otons, The extracellular surface of the purple membrane contains one a nionic proton binding site per protein molecule with pK = 5.1. This si te is within a Coulomb cage radius (approximate to 15 Angstrom) from L ys-129, The cytoplasmic surface of the purple membrane bears 4-5 proto nable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site, The reaction of the proton with this clust er is at a very fast rate (3 . 10(10) M(-1). s(-1)). The proximity bet ween the elements is sufficiently high that even in 100 mM NaCl they s till function as a cluster, Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmi c surface, and two to three of them assume positions that almost bar t heir reaction with bulk protons, The protonation dynamics determined a t the surface of the purple membrane is of relevance both for the vect orial proton transport mechanism of bacteriorhodopsin and for energy c oupling, not only in halobacteria, but also in complex chemiosmotic sy stems such as mitochondrial and thylakoid membranes.