Zimányi L, Cao Y, Needleman R, Ottolenghi M, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine 92717.
Biochemistry. 1993 Aug 3;32(30):7669-78. doi: 10.1021/bi00081a010.
The time courses of chromophore reactions and proton uptake in the second half of the photocycle of the proton pump bacteriorhodopsin (BR) were examined. At pH > 8.5, the kinetics are simplified by the fact that only the M and N intermediates accumulate. The relaxation kinetics after perturbation of M with a second, blue flash confirm that M<==>N equilibration is the only significant cause of the biphasic M decay. With this feature, the analysis of time-resolved difference spectra yields a scheme which contains two sequential N states connected by a nearly unidirectional reaction. The proton uptake from the bulk, as measured with the pH-indicator dye pyranine, occurs during the decay of the first N rather than the recovery of BR. The results thus suggest the model M2(-1)<==>N(-1) + H+ (from the bulk)<==>N(0)-->BR, where the superscripts indicate the protonation state of the protein relative to BR. M2(-1)-->N(-1) is reprotonation of the Schiff base from D96, N(-1) + H+ (from the bulk)-->N(0) is uptake of proton from the cytoplasmic side, and N(0)-->BR represents 13-cis to all-trans reisomerization of the retinal and other relaxations which regenerate the initial state. R227, a residue near D96, affects the deprotonation of D96 more than the subsequent proton uptake. According to the changed [M2(-1)]/[N(-1)] equilibrium in the R227Q protein, interaction between R227 and D96 is responsible for about 1 pH unit of the decrease in the pKa of D96 during the reprotonation of the Schiff base. According to the pH dependencies of the rate constants in the N(-1)<==>N(0) equilibrium in wild-type and R227Q, interaction with R227 lowers the pKa for proton uptake from the bulk by 0.5 pH unit, to 11. We conclude from the proton uptake kinetics that at physiological pH free energy is converted to proton electrochemical potential in this pump not only as protons are released on the extracellular side [Zimányi, L., Váró. G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] but also as protons are taken up on the cytoplasmic side.
我们研究了质子泵细菌视紫红质（BR）光循环后半段发色团反应和质子摄取的时间进程。在pH > 8.5时，动力学因仅积累M和N中间体而简化。用第二次蓝色闪光扰动M后的弛豫动力学证实，M⇌N平衡是M双相衰减的唯一重要原因。基于此特征，对时间分辨差光谱的分析得出了一个包含两个通过近乎单向反应相连的连续N态的方案。用pH指示剂染料吡喃荧光素测量的来自主体溶液的质子摄取发生在第一个N态的衰减过程中，而不是BR的恢复过程中。因此，结果表明存在这样一个模型：M2(-1)⇌N(-1) + H⁺（来自主体溶液）⇌N(0)→BR，其中上标表示蛋白质相对于BR的质子化状态。M2(-1)→N(-1)是来自D96的席夫碱的再质子化，N(-1) + H⁺（来自主体溶液）→N(0)是从细胞质侧摄取质子，N(0)→BR代表视黄醛从13 - 顺式到全反式的异构化以及其他使初始状态再生的弛豫过程。R227是靠近D96的一个残基，它对D96去质子化的影响大于对随后质子摄取的影响。根据R227Q蛋白中[M2(-1)]/[N(-1)]平衡的变化，R227与D96之间的相互作用导致席夫碱再质子化过程中D96的pKa下降约1个pH单位。根据野生型和R227Q中N(-1)⇌N(0)平衡速率常数的pH依赖性，与R227的相互作用使从主体溶液摄取质子的pKa降低0.5个pH单位，至11。从质子摄取动力学我们得出结论，在生理pH下，在这个泵中自由能不仅在质子释放到细胞外侧时[齐马尼伊，L.，瓦罗，G.，张，M.，倪，B.，尼德曼，R.，& 兰伊，J. K.（1992年）《生物化学》31，8535 - 8543]转化为质子电化学势，而且在质子摄取到细胞质侧时也如此。
