In vitroformation of a photoreversible adduct of phycocyanobilin and tobacco apophytochrome B

Abstract

The light‐stable tobacco phytochrome apoprotein (PHYB) expressed in yeast can be assembled with phycocyanobilin to give a photoreversible adduct. The spectral properties of the reconstituted PHYB‐phycocyanobilin species were determined by absorbacen and difference absorbance spectroscopies. The holoprotein exhibits absorbance maxima at 408 nm and 712 nm for the far‐red‐light‐absorbing (Pfr) form and 356 nm and 658 nm for the red‐light‐absorbing (Pr) form. The ligation of the chromophores to the dimeric PHYB apoprotein resulted in a PHYB‐phycocyanobilin adduct with the spectral properties of the Pr form. Kinetic analyses of the in vitro reconstitution for PHYB apoprotein under saturating concentrations of phycocyanobilin revealed a pseudo first‐order rate constant of 2.8 × 10⁻²s⁻¹. The similarity with the reported rate constant for the reconstitution of light‐labile phytochrome (PHYA) from oat [Li, L. & Lagarias, J. C. (1992) Phytochrome assembly, J. Biol. Chem. 267, 19 204–19 210] suggests that the mechanisms of chromophore attachment are probably very similar for PHYA and PHYB.