Max-Planck-Institute of Biophysics, Department of Biophysical Chemistry, Frankfurt am Main, Germany.
PLoS One. 2013 Sep 3;8(9):e73338. doi: 10.1371/journal.pone.0073338. eCollection 2013.
The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum is tightly regulated by the [H(+)] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L) or stabilized (D96N, D96G) M intermediate in response to green light (to probe H(+) pumping) and blue laser flashes (to probe accumulation/decay of M). These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H(+) pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H(+) pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri) shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations. Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H(+) gradient generation.
来自盐沼盐杆菌的光驱动质子泵菌视紫红质（BR）受[H(+)]梯度和跨膜电位的严格调控。BR 表现出光电特性，因为在光循环期间的光谱变化在动力学上受到电压的控制，这使得 BR 适合于光学存储或处理设备。具有延长的蓝移 M 中间体寿命的 BR 突变体将是有利的，但这种突变体的光电特性仍然难以捉摸。使用在非洲爪蟾卵母细胞中的表达和双电极电压钳位，我们分析了动力学不稳定的（F171C、F219L）或稳定的（D96N、D96G）M 中间体的 BR 突变体的光电流对绿光（探测 H(+)泵送）和蓝激光闪光（探测 M 的积累/衰减）的响应。这些突变体具有不同的 M 中间体寿命。与 BR-WT 一样，这与 H(+)泵送的电压依赖性严格相关。BR-F171C 和 BR-F219L 显示出与 BR-WT 相似的光电流。然而，BR-F171C 显示出较弱的质子泵送电压依赖性。对于这两种突变体，在绿光照射期间和之后施加蓝激光闪光显示出 M 积累减少和 M 中间体寿命缩短。相比之下，BR-D96G 和 BR-D96N 表现出小的光电流，具有非线性电流-电压曲线，在叠氮化物存在下强烈增加。蓝激光闪光显示出大量的 M 积累和延长的 M 中间体寿命，这解释了 H(+)泵送速率的强烈降低。超极化电势增强了这些效果。BR-D96G/F171C/F219L 中的 M 稳定和不稳定突变的组合（BR-tri）表明，破坏主要质子供体 Asp-96 对 BR 作为质子泵是致命的。从机制上讲，M 不稳定突变不能补偿 Asp-96 的破坏。因此，BR-tri 和 BR-D96G 的光电流相似。然而，BR-tri 甚至在没有光激活绿光的情况下也显示出负的蓝激光闪光诱导电流，这表明 BR-tri 中的席夫碱去质子化在黑暗中存在，与以前的光谱研究一致。因此，包括三重突变在内的 M 稳定突变严重干扰电化学 H(+)梯度的产生。
