Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane.
The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.
Nachliel
E
E
Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel.
Gutman
M
M
Kiryati
S
S
Dencher
N A
NA
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002021:Buffers; D003593:Cytoplasm / Q000378:metabolism; D004058:Diffusion; D005110:Extracellular Space; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D006217:Halobacterium / Q000737:chemistry / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D011522:Protons; D018717:Purple Membrane / Q000737:chemistry / Q000648:ultrastructure; D013499:Surface Properties
D002021:Buffers; D005452:Fluoresceins; D011522:Protons; D001436:Bacteriorhodopsins; D008239:Lysine; D019793:Fluorescein