MEASUREMENT AND GLOBAL ANALYSIS OF THE ABSORBENCY CHANGES IN THE PHOTOCYCLE OF THE PHOTOACTIVE YELLOW PROTEIN FROM ECTOTHIORHODOSPIRA-HALOPHILA

The photocycle of the photoactive yellow protein (PYP) from Ectothiorh odospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated s pectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T. E., E. Yakali, M. A. Cusano vich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophy s. J. 59:988-991). The first, red-shifted intermediate decays biexpone ntially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-s hifted intermediate. The last step of the photocycle is the biexponent ial (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the gr ound state of the protein. Reconstruction of the absolute spectra of t hese photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon(max) at about 50% and 40% relative to the epsilon(max) of the ground state. T he quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.