In order to identify the electrogenic steps in the bacteriorhodopsin (BR) photocycle, we employed the direct electrometrical method (DEM) of Drachev et al. to measure kinetics of voltage formation in fixed oriented sheets of purple membrane (PM) in combination with kinetics obtained optically and analyzed by singular value decomposition, using suspensions of PM. The approach based on parallel experiments using DEM and optical measurements to identify electrogenic steps for different energy-transducing enzymes has been used in several laboratories. We found, however, that the kinetics obtained in the DEM system were about 6-fold slower than those obtained optically, and have devoted the past year to discovering the reason for this discrepancy. The obvious possibilities that the slowdown was due to the presence of residual organic solvent or non-biological lipids used to fix and orient the PM in the DEM experiments were eliminated by suitable controls. We then tested two other possible explanations: 1.) In the DEM system, which consists of membrane-isolated closed compartments, the formation of membrane potential retards the kinetics of its formation (back-pressure effect), 2.) The difference in kinetics is only apparent and not real because the slow electrogenic steps are optically silent. Consistent with explanation #1 was the observation that as uncoupler (CCCP or valinomycin) decreased the amount of voltage formed in the DEM system, the kinetics were speeded to equal those obtained optically with the PM suspensions. However, this observation could also be consistent with explanation #2 if the uncoupler decreased the RC time constant of the electric circuit which represents the fixed PM in the DEM system and thereby speeded the electric response of the system. In the analogy, BR is a current generator and the membranes of the PM and fixed Teflon support for the PM provide both resistance and capacitance. We derived an equation to model the electric circuit analogy and found that decreasing the RC constant of the circuit in steps did lead to a family of curves resembling those obtained experimentally using the DEM system. There is, however, a major difference in the response to gradual additions of uncoupler according to the two explanations under consideration. In #1, there should be a slow continuous decrease of the kinetic constants for each individual step in the photocycle. In #2, there should be no change in any individual time constant until the RC constant becomes lower than the constant for the slowest photocycle transition. Then, the slow electrogenic step should abruptly become a voltage-dissipation step and the lower-valued RC constant become an electrogenic step. We found that the experimental titration data were consistent with explanation #1 and not #2. Additional data were obtained that support this conclusion but are not presented in this brief account. The DEM system is closely related to the situation existing in intact bacteria where the photocycle is also regulated by membrane potential. In control experiments, using intact bacteria, we show that the photocycle is also speeded by uncoupler addition. We also found that membrane potential regulates the relative amounts of the fast and slow M-intermediates of the photocycle in a manner that resembles the known regulation by the intensity of the actinic laser flash. These results, in addition to supporting the importance of thermodynamic back-pressue regulation, show that the approach using parallel experiments to measure voltage formation and optical transitions in order to assign electrogenicity must be interpreted with caution. - bioenergetics, proton-pump, membrane potential, kinetics of voltage-formation
| Hendler, R W; Drachev, L A; Bose, S et al. (2000) On the kinetics of voltage formation in purple membranes of Halobacterium salinarium. Eur J Biochem 267:5879-90 |
| Joshi, M K; Bose, S; Hendler, R W (1999) Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium. Biochemistry 38:8786-93 |
