FTIR studies of phytochrome photoreactions reveal the C=O bands of the chromophore: consequences for its protonation states, conformation, and protein interaction.

The molecular changes of phytochrome during red --> far-red and reverse photoreactions have been monitored by static infrared difference spectroscopy using the recombinant 65 kDa N-terminal fragment assembled with a chromophore chemically modified at ring D or with a chromophore isotopically labeled with (18)O at the carbonyl group of ring A. This allows the identification of the C=O stretching vibrations of rings D and A. We exclude the formation of an iminoether in Pfr. The positions of both these modes show that the chromophore always remains protonated. The upshift of the C=O stretch of ring D in the first photoproducts is explained by a twisted methine bridge connecting rings C and D. The changes in the vibrational pattern during the red --> far-red conversion show that the backreaction is not just the reversal of the forward reaction. The infrared difference spectra of the fragment deviate very little from those of the full-length protein. The differences which are related to the lack of the C-terminal half of the protein constituting the signaling domain are possibly important for the understanding of the signaling mechanism.

Chromophore attachment to biliproteins: specificity of PecE/PecF, a lyase-isomerase for the photoactive 3(1)-cys-alpha 84-phycoviolobilin chromophore of phycoerythrocyanin.

PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-alpha 84 of the phycoerythrocyanin alpha-subunit (PecA), and (2) the Delta 4-->Delta 2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore [Zhao et al. (2000) FEBS Lett. 469, 9-13]. The alpha-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M. laminosus, with and without His-tags. The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-alpha 98/99 with thiol-directed reagents. The spontaneous addition leads to a 3(1)-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 3(1)-Cys-PVB adduct which after purification was identical to alpha-PEC. The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 3(1)-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive. Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F. The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.

Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon.

The structure of phycoviolobilin, the photoactive chromophore of alpha-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a Delta3, 3(1)-double bond of the phycobilin. The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-alpha-phycoerythrocyanin (PecA). In the absence of the components of alpha-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (lambda(max)=641 nm) and low photoactivity (<10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (lambda(max)=565 nm) and high photoreversible photochromism (100% type I) has been identified as integral alpha-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.