Formation of a photoreversible phycocyanobilin-apophytochrome adduct in vitro.
Avena seedlings grown in the presence of the plant tetrapyrrole synthesis inhibitor 4-amino-5-hexynoic acid contain less than 10% of the spectrally detectable phytochrome levels found in untreated seedlings, but continue to accumulate phytochrome apoprotein (Elich, T. D., and Lagarias, J. C. (1988) Plant Physiol. 88, 747-751). Using such tetrapyrrole-deficient seedlings, we have previously reported that phycocyanobilin, the cleaved prosthetic group of C-phycocyanin, can be incorporated into phytochrome in vivo to yield spectrally active holoprotein (Elich, T. D., McDonagh, A. F., Palma, L. A., and Lagarias, J. C. (1988) J. Biol. Chem. 264, 183-189). Here we show that addition of phycocyanobilin to soluble extracts of inhibitor-treated seedlings results in a rapid increase in spectrally active phytochrome holoprotein. The newly formed photoactive species displays a blue-shifted absorbance difference spectrum similar to that observed in the previous in vivo studies. The increase in spectral activity is consistent with conversion of all of the preexisting phytochrome apoprotein to functionally active holoprotein. The formation of a covalent phycocyanobilin-apophytochrome adduct is shown by an increase in Zn2+-dependent bilin fluorescence of the phytochrome polypeptide. A photoreversible, covalent adduct with a similar optical spectrum also forms when immunopurified apophytochrome is incubated with phycocyanobilin. ATP, reduced pyridine nucleotides, or other cofactors are not required for adduct formation. When biliverdin IX alpha is substituted for phycocyanobilin, no spectrally active covalent adduct is produced. These results indicate that an A-ring ethylidene-containing bilatriene is required for post-translational covalent attachment of bilin to apophytochrome and that apophytochrome may be the bilin C-S lyase which catalyzes bilin attachment.
Elich
T D
TD
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Lagarias
J C
JC
D000213:Acyl Carrier Protein / Q000096:biosynthesis; D000614:Aminocaproates; D001059:Apoproteins / Q000096:biosynthesis; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D010777:Photochemistry; D052980:Phycobilins; D010798:Phycocyanin / Q000096:biosynthesis* / Q000302:isolation & purification; D010834:Phytochrome / Q000096:biosynthesis* / Q000302:isolation & purification; D010860:Pigments, Biological / Q000096:biosynthesis*; D010940:Plant Proteins / Q000096:biosynthesis*; D011233:Precipitin Tests; D011758:Pyrroles / Q000096:biosynthesis* / Q000302:isolation & purification; D012639:Seeds / Q000378:metabolism; D045725:Tetrapyrroles; D020888:Vigabatrin
D000213:Acyl Carrier Protein; D000614:Aminocaproates; D001059:Apoproteins; D029968:Escherichia coli Proteins; D052980:Phycobilins; D010860:Pigments, Biological; D010940:Plant Proteins; D011758:Pyrroles; D045725:Tetrapyrroles; C040157:acpP protein, E coli; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin; D054889:Fatty Acid Synthase, Type II; D020888:Vigabatrin