Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.
Light-induced H+ release and reuptake as well as surface potential changes inherent in the bacterio-rhodopsin reaction cycle were measured between 10 degrees C and 50 degrees C. Signals of optical pH indicators covalently bound to Lys-129 at the extracellular surface of bacteriorhodopsin were compared with absorbance changes of probes residing in the aqueous bulk phase. Only surface-bound indicators monitor the kinetics of H+ ejection from bacteriorhodopsin and allow the correlation of the photocycle with the pumping cycle. During the L550----M412 transition the H+ appears at the extracellular surface of bacteriorhodopsin. Surface potential changes detected by bound fluorescein or by the potentiometric probe 4-[2-(di-n-butylamino)-6-naphthyl]vinyl-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS) occur in milliseconds concomitantly with the formation and decay of the N intermediate. pH indicators residing in the aqueous bulk phase reflect the transfer of H+ from the membrane surface into the bulk but do not probe the early events of H+ pumping. The observed retardation of H+ at the membrane surface for several hundred microseconds is of relevance for energy conversion of biological membranes powered by electrochemical H+ gradients.
Heberle
J
J
Hahn-Meitner-Institut, BENSC-N1, W-1000 Berlin 39, Germany.
Dencher
N A
NA
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000502:physiology*; D001692:Biological Transport; D002462:Cell Membrane / Q000502:physiology; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D019617:Halobacterium salinarum; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008564:Membrane Potentials; D010788:Photosynthesis; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D001436:Bacteriorhodopsins; D019793:Fluorescein