THE ROLE OF BACK-REACTIONS AND PROTON UPTAKE DURING THE N-]O TRANSITION IN BACTERIORHODOPSINS PHOTOCYCLE - A KINETIC RESONANCE RAMAN-STUDY
THE ROLE OF BACK-REACTIONS AND PROTON UPTAKE DURING THE N-]O TRANSITION IN BACTERIORHODOPSINS PHOTOCYCLE - A KINETIC RESONANCE RAMAN-STUDY
复制标题
DOI：
10.1021/bi00483a005
复制
发表时间：
1990-08-07
期刊：
影响因子：
2.9
通讯作者：
MATHIES, RA
中科院分区：
文献类型：
--
作者：
AMES, JB;MATHIES, RA
用时间分辨共振拉曼光谱分析了pH为5，6，7，8和8.6时菌紫质光循环的动力学。通过使用502-nm（L550、N550、BR 568）、458-nm（M412）和752-nm（O 640）激发跟踪其共振拉曼强度，测定各种中间体的浓度作为时间的函数。从每个中间体的泵+探测数据的光谱贡献定量分离的最小二乘分解。然后通过使用分子守恒约束将这些相对浓度转换为绝对浓度。这使得各种动力学模型的明确细化，以找到准确描述数据的最简单的模型。动力学数据，包括L550和M412的双相衰减，最好通过包括逆反应的顺序方案再现（BR → BR）。L. tautm. M. fwdarw. N. O. BR）。此外，动力学的L. tautm。 M和N. O步骤被发现是pH依赖性的。连接L550和M412的正向和反向速率常数都随pH增加，证实了在碱性pH下催化席夫碱去质子化的早期建议。O_（640）速率常数与pH无关，但随pH的增大而线性减小。这表明蛋白质必须在N550 →期间拾取质子。O 640转变，并且该过程在pH 7以上成为速率决定。因此，在N550和O 640之间一定有一个中间体，我们记为N+550。一个分子图形模型，将这些意见纳入质子泵的机制。
The kinetics of bacteriorhodopsin''s photocycle have been analyzed at pH 5, 6, 7, 8, and 8.6 by using time-resolved resonance Raman spectroscopy. The concentrations of the various intermediates as a function of time were determined by following their resonance Raman intensities using 502-nm (L550, N550, BR568), 458-nm (M412), and 752-nm (O640) excitation. The spectral contributions to the pump + probe data from each intermediate were quantitatively separated by least-squares decomposition. These relative concentrations were then converted to absolute concentrations by using a conservation of molecules constraint. This enabled the unambiguous refinement of a variety of kinetic model to find the simplest one that accurately describes the data. The kinetic data, including the biphasic decay of L550 and M412, are best reproduced by a sequential scheme including back-reactions (BR .fwdarw. L .tautm. M .fwdarw. N .fwdarw. O .fwdarw. BR). In addition, the kinetics of the L .tautm. M and N .fwdarw. O steps are found to be pH-dependent. Both the forward and reverse rate constants connecting L550 and M412 increase with pH, confirming earlier proposals of catalyzed Schiff base deprotonation at alkaline pH. Below pH 7, the N550 .fwdarw. O640 rate constant is independent of pH, but it decreases linearly with pH above 7. This indicates that the protein must pick up a proton during the N550 .fwdarw. O640 transition and that this process becomes rate determining above pH 7. There must, therefore, be an intermediate between N550 and O640 which we denote as N+550. A molecular graphics model is presented which incorporates these observations into a mechanism for proton pumping.