Regulation of the bacteriorhodopsin photocycle and proton pumping in wholecells of Halobacterium salinarium

Single-turnover kinetics of the bacteriorhodopsin photocycle and proton-pum ping capabilities of whole cells were studied. It was found that the Delta< (mu)over 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-turnov er time of the cycle was increased similar to 10-fold, (2) the mole fractio n of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the t ime constant for M-slow increased significantly, The level of Delta<(mu)ove r tilde>(+)(H) was dependent on respiration, ATP formation and breakdown, a nd the magnitude of a pre-existing K+ diffusion gradient. The size of the D elta<(mu)over tilde>(+)(H) could be manipulated by additions of HCN, nigeri cin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levels of Delta<(mu )over tilde>(+)(H), further changes in the photocycle were seen. (4) Two sl ower components of M-decay appeared as major components. (5) The apparent c onversion of the M-fast to the O intermediate disappeared. (6) A partial re versal 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-induc ed proton pumping was nor seen until the K+ diffusion potential was dissipa ted and proton accumulation facilitated by use of a K+-H+ exchanger (nigeri cin), respiration was inhibited by HCN, and ATP synthesis and breakdown wer e inhibited by DCCD. In stored cells, the pre-existing K+ diffusion gradien t was diminished through slow diffusion, and only DCCD and HCN were require d to elicit proton extrusion.