Summary: Biliproteins are a widespread group of brilliantly coloured photoreceptors characterized by linear tetrapyrrolic chromophores, bilins, which are covalently bound to the apoproteins via relatively stable thioether bonds. Covalent binding stabilizes the chromoproteins and is mandatory for phycobilisome assembly; and, it is also important in biliprotein applications such as fluorescence labelling. Covalent binding has, on the other hand, also considerably hindered biliprotein research because autocatalytic chromophore additions are rare, and information on enzymatic addition by lyases was limited to a single example, an EF‐type lyase attaching phycocyanobilin to cysteine‐α84 of C‐phycocyanin. The discovery of new activities for the latter lyases, and of new types of lyases, have reinvigorated research activities in the subject. So far, work has mainly concentrated on cyanobacterial phycobiliproteins. Methodological advances in the process, however, as well as the finding of often large numbers of homologues, opens new possibilities for research on the subsequent assembly/disassembly of the phycobilisome in cyanobacteria and red algae, on the assembly and organization of the cryptophyte light‐harvesting system, on applications in basic research such as protein folding, and on the use of phycobiliproteins for labelling.

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 β-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 α-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.

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-green-absorbing phycourobilin (lambda(max) approximately 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 alpha-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.

Significance: Photosynthesis, the basis for life on earth, relies on proper balancing of the beneficial and destructive potentials of light. In cyanobacteria and red algae, which contribute substantially to photosynthesis, the core-membrane linker, L _CM , is critical to this process. Light energy harvested by large antenna complexes, phycobilisomes, is funneled to L _CM . Depending on light conditions, L _CM passes this energy productively to reaction centers that transform it into chemical energy or, on oversaturating conditions, to the photoprotecting orange carotenoid protein (OCP). The details of these functions in the complex-structured L _CM are poorly understood. The crystal structure and time-resolved data of the chromophore domain of L _CM provide a rationale for the functionally relevant energetic matching, and indicate a mechanism for switching between photoproductive and photoprotective functions.

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 (λ_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 (λ_max = 685 nm) reverting slowly thermally to the thermostable 15Z state (λ_max = 635 nm). PCB‐GAF3 showed a novel red–orange photochromism; the unstable state (putative 15E, λ_max = 595 nm) reverts very rapidly (τ∼ 20 s) back to the thermostable Z state (λ_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 abstract AphC phosphorylates AphC by protein kinase assay (View interaction) RGS phosphorylates RGS by protein kinase assay (View interaction)