Spencer D B, Dewey T G
Department of Chemistry, University of Denver, Colorado 80208.
Biochemistry. 1990 Mar 27;29(12):3140-5. doi: 10.1021/bi00464a034.
Phase lifetime spectroscopy is used to investigate the kinetics of the 520- and 640-nm intermediates in the halorhodopsin photocycle. These intermediates decay on the millisecond time scale and are strongly implicated in the chloride transport steps. The temperature dependence of the 520 and 640 relaxations was measured for chloride and nitrate buffers at pH 6, 7, and 8 and for iodide buffer at pH 6. The 640 relaxations have small activation energies but large entropy barriers. The two relaxation times observed for the 640 intermediate were interpreted by using a mechanism in which two 640 species exist in equilibrium. The second 640 species is not along the main decay path for the photocycle. A quantitative analysis of the data allowed rate constants and activation parameters to be calculated for the elementary steps of this isomerization process. These parameters are similar for both chloride and nitrate buffers but differ somewhat in iodide. The derived calculated rate constants were consistent with the relaxation times observed for the 520 intermediate. These results indicate that the 520 and two 640 intermediates have very similar free energies as well as similar free energies of activation for the various interconversion processes.
相寿命光谱法用于研究嗜盐菌视紫红质光循环中520纳米和640纳米中间体的动力学。这些中间体在毫秒时间尺度上衰变，并且与氯离子转运步骤密切相关。在pH值为6、7和8的氯离子和硝酸根缓冲液以及pH值为6的碘离子缓冲液中，测量了520纳米和640纳米弛豫的温度依赖性。640纳米弛豫具有较小的活化能但较大的熵垒。通过使用一种机制来解释在640纳米中间体中观察到的两个弛豫时间，在该机制中，两个640物种处于平衡状态。第二个640物种不在光循环的主要衰变路径上。对数据的定量分析使得能够计算出该异构化过程基本步骤的速率常数和活化参数。这些参数对于氯离子和硝酸根缓冲液是相似的，但在碘离子中略有不同。推导计算出的速率常数与在520纳米中间体中观察到的弛豫时间一致。这些结果表明，对于各种相互转化过程，520纳米和两个640纳米中间体具有非常相似的自由能以及相似的活化自由能。
