Late events in the photocycle of bacteriorhodopsin mutant L93A.
In the photocycle of bacteriorhodopsin (bR) from Halobacterium salinarum mutant L93A, the O-intermediate accumulates and the cycling time is increased approximately 200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which-with exception of the absorbance changes-had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800 ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N<-->O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum.
Tóth-Boconádi
R
R
Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary.
Keszthelyi
L
L
Stoeckenius
W
W
D000222:Adaptation, Physiological / Q000502:physiology / Q000528:radiation effects; D001436:Bacteriorhodopsins / Q000502:physiology* / Q000528:radiation effects*; D002478:Cells, Cultured; D003624:Darkness; D004307:Dose-Response Relationship, Radiation; D019617:Halobacterium salinarum / Q000235:genetics / Q000528:radiation effects*; D007834:Lasers; D008564:Membrane Potentials / Q000502:physiology* / Q000528:radiation effects*; D009154:Mutation; D010507:Periodicity; D010775:Photic Stimulation / Q000379:methods; D018462:Photobiology / Q000379:methods; D011522:Protons; D018717:Purple Membrane / Q000502:physiology* / Q000528:radiation effects*
D011522:Protons; D001436:Bacteriorhodopsins