ULTRAFAST PUMP-PROBE SPECTROSCOPY OF NATIVE ETIOLATED OAT PHYTOCHROME

Absorption difference profiles were obtained at wavelengths from 640 t o 700 nm with 1-2-ps resolution in a study of primary photoprocesses i n the Pr --> Pfr transformation in native oat phytochrome. These exper iments were performed using low-intensity laser pulses at high repetit ion rate; fast sample recycling ensured that essentially all phytochro me species were excited from the Pr ground state. The Pr-stimulated e mission decay at wavelengths >670 nm exhibits major components with li fetimes of approximately 16 and 50-60 ps. Formation of the asymptotic 695-nm lumi-R absorption spectrum rapidly follows stimulated emission decay. Photoexcitation of one or both of the lumi-R intermediates inst antaneously recreates fluorescing Pr phytochrome, which is spectrosco pically and kinetically indistinguishable from that generated by direc t illumination of ground-state Pr. This is consistent with assignment of lumi-R as a species in which the chromophore has isomerized from th e Z,Z,Z to the Z,Z,E conformation. Anisotropy studies indicate that th e orientations of the Pr and lumi-R absorption transition moments are nearly parallel, since little anisotropy decay occurs during the 500-p s time window of these experiments.