A differential molecualr topography of the Pr and Pfr forms of native oat phytochrome as probed by fluoresence quenching.

Tryptophan (Trp) surface topography of the red- and far-red-absorbing forms of phytochrome (Pr, Pfr) ofAvena sativa L. has been investigated by analyzing quenching of the two components of Trp fluorescence decay, in order to understand the differences in the two forms at the molecular level. Stern-Volmer kinetic analysis of the quenching data for two cationic surface quenchers, Cs(+) and Tl(+), showed strong quenching of the short component of the Pr fluorescence (Stern-Volmer constants,K sv , 27.2 and 21.4 M(-1), respectively) relative to that of Pfr fluorescenceK sv , 10.4 and 12.3 M(-1), respectively). The long component of the Trp fluorescence was quenched differentially by Cs(+) and Tl(+), withK sv of 9.0 and 19.8 M(-1), respectively, for the Pr fluorescence andK sv of 13.7 and 8.7 M(-1), respectively, for the Pfr fluorescence. The results indicate that the phytochrome Trp residues with short fluorescence lifetime are more accessible to the cationic surface quenchers than those with long fluorescence lifetime. The data, taken together with our earlier study (Singh et al. 1988, Biochim, Biophys. Acta936, 395-405), indicate that most, if not all the ten Trp residues of phytochrome, are fluorescent and exist in distinct groups differing in their topography and microenvironment, and the peptide segment containing Trp-774 and Trp-778 within the 55-kilodalton C-terminal domain of phytochrome also undergoes a subtle alteration in its surface topography during Pr→Pfr phototransformation.