EFFICIENCIES OF ENERGY TRANSDUCTION BY BACTERIORHODOPSIN

细菌视紫红质的能量转换效率

• 批准号: 6290374
• 负责人: richard w hendler
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290374
• 关键词: bacteriorhodopsins; bioenergetics; cytochrome oxidase; fiber optics; liposomes; membrane reconstitution /synthesis; method development; oxidative phosphorylation; phospholipids; photochemistry

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

为了鉴定细菌紫红素（BR）光循环中的电源步骤，我们采用了Drachev等人的直接电解方法（DEM）。用PM的悬浮液（测量紫色膜（PM）的固定定向片片的电压形成动力学，并使用PM的悬浮液（通过奇异值分解）结合使用，并通过奇异值分解。该方法基于使用DEM和光学测量值的平行实验来鉴定不同能量转换酶的电源步骤，已在几个实验室中使用。但是，我们发现，在DEM系统中获得的动力学比光学上获得的动力学慢了6倍，并致力于过去一年来发现这种差异的原因。通过合适的对照消除了放缓的可能性，这是由于存在用于修复DEM实验中PM的残留有机溶剂或非生物脂质引起的。然后，我们测试了另外两个可能的解释：1。）在DEM系统中，由膜分离的闭合室组成，膜电位的形成阻碍了其形成的动力学（后压效应），2。）动力学的差异仅是显而易见的，并且不是真实的，因为缓慢的电子步骤在缓慢的电子上是静音的。与解释＃1一致的是，观察到，作为解耦合器（CCCP或valinomycin）降低了DEM系统中形成的电压量，动力学被加快了以与PM悬浮液相等的光学相等。但是，如果解耦合器降低了代表DEM系统中固定PM的电路的RC时间常数，从而加快了系统的电响应，则该观察结果也可能与2＃2一致。在类比中，BR是电流发生器，PM的膜和固定的Teflon支持PM提供了电阻和电容。我们得出了一个方程式来对电路类比进行建模，并发现在步骤中降低电路的RC常数确实导致了类似于使用DEM系统实验获得的曲线家族。但是，根据所考虑的两个解释，对解偶子逐渐添加的响应有主要差异。在＃1中，对于光循环中每个单个步骤的动力学常数应缓慢连续减小。在＃2中，任何个体时间常数都不会发生变化，直到RC常数低于最慢的光循环转变的常数。然后，缓慢的电源步骤应突然成为电压散落步骤，而低价值的RC常数成为电源步骤。我们发现实验滴定数据与解释＃1一致，而不是＃2。获得了支持此结论但没有在此简短帐户中介绍的其他数据。 DEM系统与完整细菌中的情况密切相关，在该细菌中，光圈也受膜电位调节。在对照实验中，使用完整的细菌，我们表明光循环也通过添加了脱钩。我们还发现，膜电位调节光循环的快速和慢速中间体的相对量，以类似于已知调节的方式，即通过阳光激光闪光的强度。这些结果除了支持热力学背压调节的重要性外，还表明，使用平行实验来测量形成电压和光学转变以分配产生性的方法必须谨慎解释。 - 生物能学，质子泵，膜电位，电压形成动力学