The phototransformation process in phytochrome. I. Ultrafast fluorescence component and kinetic models for the initial P r → P fr transformation steps in native phytochrome

Abstract

The fluorescence kinetics of native (124 kDa) phytochrome from oat has been studied by time-resolved spectroscopy in the picosecond to nanosecond time range. The excitation wavelength and the buffer conditions were varied and data were recorded over the whole emission wavelength range. The data were analyzed by gobal analysis methods and kinetic modelling. In addition to three lifetime components of about 45 ps, 180 ps and longer than 1 ns reported earlier, we now resolved an additional fast lifetime component in the range of 4 to 16 ps with dominant amplitude. The lifetimes and amplitudes of the three fastest components were not significantly dependent on the buffer used nor on the exchange of protons against deuterons. In particular, our data exclude a sign change in the amplitude of the ultrafast component from negative (rise term) in the non-deuterated buffer to a positive value (decay term) in the deuterated one. Thus, our data are in contrast to recently reported findings by Hermann et al. (Photochem. Photobiol. (1990) 52). The kinetic model consistent with the data involves a description in terms of two excited states which are in thermal equilibrium with each other. We suggest that these states represent the initially excited state of P r phytochrome and a conformationally relaxed, but still electronically excited, intermediate state of the chromophore which is reached along the path of the P r to P fr phototransformation. The rate constants of the interconversion of the excited states, their species-associated spectra, and the free energy differences of these states are determined from the fluorescence kinetics. The data confirm our previous results that there occurs no kinetic deuterium isotope effect on the lifetime of the excited P r. However, the rate constants of the primary reactions are to some extent dependent on the nature of the medium.