Chromophore incorporation, Pr, to Pfr kinetics, and Pfr thermal reversion of recombinant N-terminal fragments of phytochrome A and B chromoproteins

N-Terminal apoprotein fragments of oat phytochrome A (phyA) of 65 kDa (amino acids 1-595) and potato phyB of 66 kDa (1-596) were heterologously expressed in Escherichia coli and in the yeasts Saccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (P Phi B; native chromophore) and phycocyanobilin (PCB), The phyA65 apoprotein from yeast showed a monoexponential assembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showed only a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore more slowly than the phyA65s, with biexponential kinetics. With all apoproteins, P Phi B was incorporated faster than PCB, The thermal stabilities of the P-fr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 P-fr is highly stable, whereas potato phyB66P(fr) is rapidly converted into P-r. Thus, neither the C-terminal domain nor homodimer formation regulates this property. Rather, it is a characteristic of the phytochrome indicating its origin from mono- or dicots. The P-r to P-fr kinetics of the N-terminal phyA65 and phyB66 are different. The primary photoproduct I-700 of phyA65-PCB decayed monoexponentially and the P Phi B analogue biexponentially, whereas the phyB66 I-700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of P-fr from P-r is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvement of the C-terminal domain in the relatively slow protein conformational changes.