Activation parameters for the halorhodopsin photocycle: a phase lifetime spectroscopic study of the 520- and 640-nanometer intermediates

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.