Regulation of the Bacteriorhodopsin Photocycle and Proton Pumping in Whole Cells of Halobacterium salinarium
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- 作者：Manoj K. Joshi ; Salil Bose ; and Richard W. Hendler
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：July 6, 1999
- 年：1999
- 卷：38
- 期：27
- 页码：8786 - 8793
- 全文大小：84K
- 年卷期：v.38,no.27(July 6, 1999)
- ISSN：1520-4995
文摘
Single-turnover kinetics of the bacteriorhodopsin photocycle and proton-pumping capabilitiesof whole cells were studied. It was found that the 
H+ of the cell had a profound influence on thekinetics and components of the cycle. For example, comparing the photocycle in whole cells to that seenin PM preparations, we found that (1) the single-turnover time of the cycle was increased ~10-fold, (2)the mole fraction of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the time constant forM-slow increased significantly. The level of H+ was dependent on respiration, ATP formation andbreakdown, and the magnitude of a pre-existing K+ diffusion gradient. The size of the H+ could bemanipulated by additions of HCN, nigericin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levelsof H+, further changes in the photocycle were seen. (4) Two slower components of M-decay appearedas 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 changedto that seen in purple membrane suspensions by the energy-uncoupler CCCP or by lysis of the cells. Infresh whole cells, light-induced proton pumping was not seen until the K+ diffusion potential was dissipatedand proton accumulation facilitated by use of a K+-H+ exchanger (nigericin), respiration was inhibitedby HCN, and ATP synthesis and breakdown were inhibited by DCCD. In stored cells, the pre-existingK+ diffusion gradient was diminished through slow diffusion, and only DCCD and HCN were requiredto elicit proton extrusion.
H+ of the cell had a profound influence on thekinetics and components of the cycle. For example, comparing the photocycle in whole cells to that seenin PM preparations, we found that (1) the single-turnover time of the cycle was increased ~10-fold, (2)the mole fraction of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the time constant forM-slow increased significantly. The level of H+ was dependent on respiration, ATP formation andbreakdown, and the magnitude of a pre-existing K+ diffusion gradient. The size of the H+ could bemanipulated by additions of HCN, nigericin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levelsof H+, further changes in the photocycle were seen. (4) Two slower components of M-decay appearedas 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 changedto that seen in purple membrane suspensions by the energy-uncoupler CCCP or by lysis of the cells. Infresh whole cells, light-induced proton pumping was not seen until the K+ diffusion potential was dissipatedand proton accumulation facilitated by use of a K+-H+ exchanger (nigericin), respiration was inhibitedby HCN, and ATP synthesis and breakdown were inhibited by DCCD. In stored cells, the pre-existingK+ diffusion gradient was diminished through slow diffusion, and only DCCD and HCN were requiredto elicit proton extrusion.