Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium.
Single-turnover kinetics of the bacteriorhodopsin photocycle and proton-pumping capabilities of whole cells were studied. It was found that the Delta mu (tilde)H+ of the cell had a profound influence on the kinetics and components of the cycle. For example, comparing the photocycle in whole cells to that seen in PM preparations, we found that (1) the single-turnover time of the cycle was increased approximately 10-fold, (2) the mole fraction of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the time constant for M-slow increased significantly. The level of Delta mu(tilde)H+ was dependent on respiration, ATP formation and breakdown, and the magnitude of a pre-existing K+ diffusion gradient. The size of the Delta mu(tilde)H+ could be manipulated by additions of HCN, nigericin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levels of Delta mu(tilde)H+, further changes in the photocycle were seen. (4) Two slower components of M-decay appeared as major components. (5) The apparent conversion of the M-fast to the O intermediate disappeared. (6) A partial reversal of an early photocycle step occurred. The photocycle of intact cells could be changed to that seen in purple membrane suspensions by the energy-uncoupler CCCP or by lysis of the cells. In fresh whole cells, light-induced proton pumping was not seen until the K+ diffusion potential was dissipated and proton accumulation facilitated by use of a K+-H+ exchanger (nigericin), respiration was inhibited by HCN, and ATP synthesis and breakdown were inhibited by DCCD. In stored cells, the pre-existing K+ diffusion gradient was diminished through slow diffusion, and only DCCD and HCN were required to elicit proton extrusion.
Joshi
M K
MK
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
Bose
S
S
Hendler
R W
RW
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010101:Oxygen Consumption; D010777:Photochemistry; D017440:Photoperiod; D011309:Preservation, Biological; D017494:Proton Pumps / Q000737:chemistry* / Q000378:metabolism*; D013053:Spectrophotometry; D013997:Time Factors
D017494:Proton Pumps; D001436:Bacteriorhodopsins