Physicochemical differences between the red- and the far-red-absorbing forms of phytochrome.

We investigated some of the chemical and physical differences between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of immunoaffinity-purified, undegraded oat (Avena sativa L., ev. Garry) phytochrome. Both Pr and Pfr had identical surface charges as measured by isoelectric focusing and identical secondary structure as judged from their circular dichroic spectra. Modification of specific amino acid residues, however, revealed some chemical differences between Pr and Pfr. Compared to Pr, Pfr had one more His and Cys residue per monomer modified immediately. His residues on Pfr were modified more rapidly than were those on Pr, as opposed to Cys modification which, after the initial burst, occurred more rapidly on Pr than on Pfr. Both His- and Cys-modified phytochromes were fully photoreversible. Both Pr and Pfr had the same number of immediately modified carboxyl functions, but those on Pr reacted slightly more rapidly than those on Pfr. Carboxyl-modified phytochrome was denatured by the acid pH used for modification so that its photoreversibility could not be measured. Modification of Tyr on Pr and Pfr resulted in two residues per monomer being modified very rapidly, with those on Pfr reacting even faster than those on Pr. Subsequent slower modification of remaining Tyr residues, however, occurred more rapidly on Pr than on Pfr. Phytochrome photoreversibility declined as a linear function of the number of Tyr modified and was lost completely when two Tyr per monomer had been modified. These data are consistent with the hypothesis of a small conformational change in phytochrome upon photoconversion and also fit a conformation-stabilization mechanism in which photo-conversion from Pr to Pfr stabilizes phytochrome in one particular conformation.