A PHYTOCHROME STUDY USING 2-LASER 2-COLOR FLASH-PHOTOLYSIS - I-700 IS A MANDATORY INTERMEDIATE IN THE P(R)-]P(FR) PHOTOTRANSFORMATION

The phototransformation of native (124 kDa) oat phytochrome, P(r) --> P(fr) has been studied at 10-degrees-C by two-laser/two-color flash photolysis. The overall P(r) --> P(fr) reaction yield did not vary with temperature within the range 4-21-degrees-C. Following the excitation of P(r) with a single 15 ns laser flash at 650 nm, the formation of P(fr) was quantitatively measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. The second laser flash causes at 1.0 s after the initial laser flash a depletion of the intermediate I700 as well as a reduction of the P(fr) absorption at 730 nm. The depletion of I700 correlates quantitatively with the reduction of P(fr) formation. The absorption spectra of I700 and of the following intermediate, I(bl), were calculated assuming that the amount of P(r), which is photoconverted by a single laser flash, equals the amount of P(fr) formed.