Cyanochromes Are Blue/Green Light Photoreversible Photoreceptors Defined by a Stable Double Cysteine Linkage to a Phycoviolobilin-type Chromophore

Abstract

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C3 1 carbon of the ethylidene side chain and the C4 or C5 carbons in the A-B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb 3 Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals. Phytochromes (Phys)3 comprise a large and diverse superfamily of photoreceptors that regulate a wide range of physiological responses in plants, fungi, bacteria, and cyanobacteria (1-3). They are unique among photoreceptors by being able to photoconvert between two stable states, a red light-absorbing Pr form that is typically the dark-adapted and biologically inactive conformer and a far-red light-absorbing Pfr form that requires light for its production and is typically the biologically active conformer. By interconverting between Pr and Pfr, Phys act as light-regulated switches in controlling processes ranging from phototaxis and pigmentation in bacteria to seed germination, photomorphogenesis, and flowering time in higher plants.Light absorption by Phys is directed by a bilin (or linear tetrapyrrole) chromophore produced by the oxidative cleavage of heme. Although bacterial and fungal Phys use the immediate cleavage product biliverdin (BV), cyanobacterial and higher plant Phys use phycocyanobilin (PCB) and phytochromobilin, respectively, produced by enzymatic reduction of BV (1, 2). The bilin is then covalently bound autocatalytically to the photosensory unit of the apoprotein, which typically contains a sequence of Per/Arndt/Sim (PAS), cGMP phosphodiesterase/aden...

Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp. PCC 6803

“…2A). Irradiation of Pg with green light restored the Puv spectrum with a broad absorption maximum at 382 nm, results similar to those previously reported for recombinant UirS/Slr1212-GAF (18). Absorption and difference spectra of UirS-GAF expressed in Synechocystis cells were nearly identical to those of the E. coli-expressed version (Fig.…”

“…UirS belongs to a CBCR subfamily with two conserved Cys residues within its phycocyanobilin (PCB)-binding GAF domain (14), with other notable examples being Synechocystis SyPixJ/TaxD1/Sll0041 (5,15) and TePixJ/Tll0569 and Tlr0924 from Thermosynechococcus elongatus (14,16,17). Previous studies have shown that the region containing TM helices is responsible for ethylene binding in UirS (12), whereas its GAF domain can bind to the linear tetrapyrrole PCB to yield a violet/green light-switchable CBCR (18).…”

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

“…This energy transfer requires the two chromophores in close proximity and indicates that the holo-GAFs aggregate in solution. 14 In agreement, the solutions contain exclusively dimers as determined by gel filtration (Fig. S7 †).…”

“…6 Some CBCRs contain doubly linked PCB or phycoviolobilin (PVB) chromophores. 3,4,[12][13][14][15] Such GAF domains of Nostoc punctiforme ATCC 29133 and one of Cph2 of Synechocystis sp. PCC 6803 have been tested for PEB chromophorylation.…”

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

“…sanger.ac.uk) and were used for phylogenetic analysis. ATG1 and ATG13 protein sequences were aligned by ClustalX (Chenna et al, 2003) under the default settings and compared by MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003) using the proportion of invariable sites g rate model and the mixed amino acid model as described (Ulijasz et al, 2009). The program was run for 1,000,000 generations, and the first 25% (337,500 generations) was discarded as "burn in" after checking that the log likelihood values had reached a plateau.…”