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Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full-length, truncated and cysteine point-mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV-visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red-green photochromism (k max = 650 and 535 nm) that is assigned to the reversible 15Z ⁄ E isomerization of a single phycocyanobilin-chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E-PCB was more active than the 650 nm state containing Z-PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB-GAF1 is photochromic, with the proposed 15E state (k max = 685 nm) reverting slowly thermally to the thermostable 15Z state (k max = 635 nm). PCB-GAF3 showed a novel red-orange photochromism; the unstable state (putative 15E, k max = 595 nm) reverts very rapidly (s 20 s) back to the thermostable Z state (k max = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E-GAF1 ⁄ E-GAF3 shows the highest rate of autophosphorylation activity, while E-GAF1 ⁄ Z-GAF3 has intermediate activity, and Z-GAF1 ⁄ Z-GAF3 is the least active state. Structured digital abstractl AphC phosphorylates AphC by protein kinase assay (View interaction) l RGS phosphorylates RGS by protein kinase assay (View interaction) Abbreviations AphC, protein encoded by aphC = all2699; CBR, cyanobacteriochrome; GAF, cGMP phosphodiesterase, adenylyl cyclase and FhlA protein domain (SMART acc. no. SM00065); KPB, potassium phosphate buffer; Nostoc, Anabaena (Nostoc) sp. PCC 7120; P XXX ⁄ P YYY , the two photoconvertible states of CBR or Phy designated by the absorption maxima, with the stable generally 15Z state (k max = XXX nm) preceding the light-activated generally 15E-configured state (k max = YYY nm); PAS, period circadian protein, Ah receptor nuclear translocator protein and single-minded protein domain (SMART acc. no. SM00091); PCB, phycocyanobilin; Phy, phytochrome; PVB, phycoviolobilin; PFB, phytochromobilin; RGS, red-green switchable protein encoded by rgs = slr1393; Synechocystis, Synechocystis sp. PCC 6803.

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the ␤-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the ␣-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.biliprotein biosynthesis ͉ light-harvesting ͉ photosynthesis ͉ phycobilisome P hycobilisomes, the extramembraneous light-harvesting antennas in cyanobacteria and red algae, use four different types of linear tetrapyrrole chromophores to harvest light in the green gap of chlorophyll absorption (1-6). These phycobilins are covalently bound to seven or more proteins, each carrying one to four binding sites. The chromophores are biosynthesized from the cyclic irontetrapyrrole, heme, by ring opening at C-5, followed by reduction and, sometimes, also by isomerization (7-9). In the last step, these phycobilins are covalently attached to cysteines of the apoprotein via a thioether bond to C-3 1 on ring A ( Fig. 1) and in some cases by an additional thioether bond to C-18 1 on ring D (6, 10-12). This step, the binding to the apoprotein, is presently only poorly understood; it involves a considerable number of binding sites and chromophores, as well as the proper regulation and coordination of events. ¶ An increasing number of lyases has recently been identified that catalyze the chromophore addition and are specific not only for the chromophore but also for the apoprotein and the binding site (12-16). Based on the capacity of several of the respective apoproteins to also bind the chromophores autocatalytically (17-21), a chaperone-like function has been suggested (12). It enhances and guides the autocatalytic binding, which is generally of low fidelity, possibly by conformational control of the chromophore (18). At the same time, this autocatalytic binding interferes with the lyase analyses (22). The situation is some...

Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-greenabsorbing phycourobilin ( max ϳ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its ␣-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters.To perform photosynthesis, the main energetic basis for life on earth, phototrophic organisms have to cope with large spatial and temporal variations of light conditions. A major evolutionary step in meeting this challenge was the development of light-harvesting complexes, the most variable part of the photosynthetic apparatus (1). By binding a large number of chromophores, these antennas can considerably enhance the photon absorption capacity of reaction centers that are responsible for the conversion of solar energy into chemical energy. Pigmented proteins associated with light-harvesting complexes also fill (at least partially) the large gap between the absorption bands of reaction center chlorophylls (e.g. ϳ440 and 680 nm for chlorophyll a found in most oxygenic organisms). Antennas also transport the excitons with minimal loss and transduce high energy excitons into the low energy ones required by the reaction centers (1, 2). They do not only vary among the different organisms but also with time within individual organisms, thereby providing the flexibility needed by the photosynthetic apparatus to work efficiently under varying ambient conditions. Cyanobacteria, which contribute a substantial fraction of global photosynthesis (...