Nature of phototransformation of phytochrome As probed by intrinsic tryptophan residues.

The phototransformation of the photomorphogenic photoreceptor phytochrome was probed by the intrinsic luminescence of the tryptophan (Trp) residues. The red light absorbing form of phytochrome (Pr) showed a decreased tryptophan phosphorescence intensity, compared to that of the far-red light absorbing form of phytochrome (Pfr), and a delayed fluorescence from the chromophore upon excitation of the tryptophan residues with 290-nm light. The tryptophan phosphorescence in both Pr and Pfr showed decreased lifetimes (0.29 and 1.84 s, respectively) compared to that of the free tryptophan (6.00 s). In addition, the decay kinetics of the delayed fluorescence in Pr showed a short-lifetime component (0.24 s), which is similar to the tryptophan phosphorescence lifetime value. This is due to an efficient triplet-singlet (3Trp-1Pr) energy transfer in the Pr from. The increases in the tryptophan phosphorescence quantum yield and lifetime in the Pfr form have been interpreted on the basis of chromophore reorientation on the protein surface as a result of the Pr lead to Pfr phototransformation. The Stern-Volmer plot of the quenching data further confirms preferential exposure of the tryptophan residues in the Pfr form (46% "exposed' tryptophan residues in the Pr form as compared to 72% in the Pfr form). These results provide strong support for the hydrophobic model of Pfr [Hahn, T. R., & Song, P. S. (1981) Biochemistry 20, 2602-2609).