Moltke S, Krebs M P, Mollaaghababa R, Khorana H G, Heyn M P
Department of Physics, Freie Universität Berlin, Germany.
Biophys J. 1995 Nov;69(5):2074-83. doi: 10.1016/S0006-3495(95)80078-0.
The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->Asn and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, both in the presence of Cl- (150 mM) and in its absence (75 mM SO4(2-)). Under both conditions the color of the pigment is blue (lambda max = 615 nm). The time course of the photovoltage kinetics is similar to that of the acid-blue form of wild-type, except that an additional transient charge motion occurs with time constants of 60 microseconds and 1.3 ms, indicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is suggested that this is the group XH, which is responsible for proton release in wild-type. At pH 1, the photovoltage signal of D85N changes upon the addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore, the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pKa of the Schiff base, there is a mixture of "M-like" and "N-like" states. Net proton transport in the same direction as in wild-type is restored in D85N from this N-like state. With the mutant Asp212-->Asn (D212N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire pH range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage experiments indicate that net proton pumping is restored. However, this Cl(-)-induced activation of net charge-transport in D212N is only partial. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chloride-dependent features as those accompanying the acid-blue to acid-purple transition of the wild-type.
在0至11的pH范围内，研究了细菌视紫红质突变体Asp212→Asn和Asp85→Asn在580nm激发后的光电压动力学。对于突变体Asp85→Asn（D85N），在pH 7时，无论存在Cl-（150mM）还是不存在Cl-（75mM SO4(2-)），均未观察到净电荷转移，信号相同。在这两种条件下，色素的颜色均为蓝色（λmax = 615nm）。光电压动力学的时间进程与野生型酸蓝形式相似，只是存在额外的瞬态电荷运动，时间常数分别为60微秒和1.3毫秒，表明膜外表面一个未知基团发生了瞬态去质子化和再质子化。推测这就是负责野生型中质子释放的XH基团。在pH 1时，加入Cl-后，D85N的光电压信号从野生型酸蓝状态的特征信号变为酸紫状态的特征信号。因此，85位基团的质子化对于氯离子结合是必要的，但并不充分。在pH 11时，远高于席夫碱的pKa，存在“M样”和“N样”状态的混合物。从这种N样状态开始，D85N恢复了与野生型相同方向的净质子转运。对于突变体Asp212→Asn（D212N），时间分辨光电压测量表明，在没有卤离子的情况下，信号与野生型酸蓝形式相似，并且在0至11的整个pH范围内均未发生净电荷转移。在3.8至7.2的pH范围内加入Cl-后，色素颜色恢复为紫色，光电压实验表明净质子泵浦得以恢复。然而，D212N中这种Cl-诱导的净电荷传输激活只是部分的。在这个pH范围之外，即使存在氯离子也未观察到净电荷传输，并且光电压显示出与野生型酸蓝到酸紫转变时相同的氯离子依赖性特征。
