Druckmann S, Friedman N, Lanyi J K, Needleman R, Ottolenghi M, Sheves M
Department of Physical Chemistry, The Hebrew University of Jerusalem, Israel.
Photochem Photobiol. 1992;56(6):1041-7. doi: 10.1111/j.1751-1097.1992.tb09727.x.
The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin is investigated both for the native pigment and its D96N mutant. The experimental setup is based on creating the M intermediate by a first pulse, followed by a (blue) laser pulse which drives the back photoreaction of M. Experiments are carried out varying the delay between the two pulses, as well as the temperature over the -25 degrees C-20 degrees C range. It is found that the kinetic patterns of the M back photoreaction change with time after the generation of this intermediate. The data provide independent evidence for the suggestion of a photocycle mechanism based on two distinct M intermediates. They are thus in keeping with the consecutive model of Varo and Lanyi (Biochemistry 30, 5016-5022; 1991), although they cannot exclude other models such as those based on branched or parallel cycles. More generally, we offer a "photochemical" approach to discriminating between intermediate stages in the photocycle which does not depend on spectroscopic and/or kinetic data. While markedly affecting the rate of the M --> N transition in the photocycle, the rate of the thermal step in back photoreaction of M, at both room and low temperatures, is not significantly affected by the D96N mutation. It is proposed that while Asp 96 is the Schiff-base protonating moiety in the M --> N transition, another residue (most probably Asp 85) reprotonates the Schiff base following light absorption by M.
针对天然视紫红质及其D96N突变体，研究了细菌视紫红质光循环中M中间体的反向光反应。实验装置基于先用第一个脉冲产生M中间体，然后用一个（蓝色）激光脉冲驱动M的反向光反应。实验在改变两个脉冲之间的延迟以及在-25℃至20℃范围内改变温度的条件下进行。结果发现，M反向光反应的动力学模式在该中间体产生后随时间变化。这些数据为基于两种不同M中间体的光循环机制的建议提供了独立证据。因此，它们与瓦罗和兰伊的连续模型（《生物化学》30, 5016 - 5022；1991）一致，尽管它们不能排除其他模型，如基于分支或平行循环的模型。更一般地说，我们提供了一种“光化学”方法来区分光循环中的中间阶段，该方法不依赖于光谱和/或动力学数据。虽然D96N突变显著影响光循环中M→N转变的速率，但在室温和低温下，M反向光反应中热步骤的速率受其影响不显著。有人提出，虽然天冬氨酸96是M→N转变中席夫碱质子化部分，但在M吸收光后，另一个残基（很可能是天冬氨酸85）使席夫碱重新质子化。
