Photophysical diversity of two novel cyanobacteriochromes with phycocyanobilin chromophores: photochemistry and dark reversion kinetics.
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.
College of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan, China.
Yu
Y
Chen
Juan
J
Zhang
Juan
J
Luo
Jun-Ming
JM
Tu
Xiao-Li
XL
Zeng
Jie
J
Xie
Ming
M
Zhou
Jing-Quan
JQ
Zhao
Hugo
H
Scheer
Kai-Hong
KH
Zhao
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000071677:Histidine Kinase; D007700:Kinetics; D008969:Molecular Sequence Data; D046937:Nostoc / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry; D020283:Photoreceptors, Microbial / Q000737:chemistry / Q000378:metabolism*; D052980:Phycobilins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010798:Phycocyanin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010834:Phytochrome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D052980:Phycobilins; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin; D011494:Protein Kinases; D000071677:Histidine Kinase