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.
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
M K
MK
Joshi
S
S
Bose
R W
RW
Hendler
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