bilin ͉ biliprotein ͉ photochromic ͉ histidine kinase

Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle. The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB). The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm. The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1). Excitation at 615 nm leads to a slightly broadened distribution. The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps. In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime. Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed. This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) [double bond] C(16) double bond. The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction. This suggests that the initial reactions were established already before evolution of plant phytochromes began.

The phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 forms holoprotein adducts with close spectral similarity to plant phytochromes when autoassembled in vitro with bilin chromophores. Cph1 is a 85-kDa protein that acts as a light-regulated histidine kinase seemingly involved in`two-component' signalling. This paper describes the improvement of Cph1 purification, estimation of the extinction coefficient of holo-Cph1, spectral analyses of the assembly procedure and studies on quaternary structure. During assembly with the natural chromophore phycocyanobilin (PCB), a red-shifted intermediate is observed. A similar result was obtained when phycoerythrobilin was used as chromophore. As shown by SDS/PAGE and Zn 21 fluorescence, the covalent attachment of PCB is blocked by 1 mm iodoacetamide, a cysteinederivatizing agent. When PCB was incubated with blocked apo-Cph1, again a shoulder at longer wavelengths appeared. It is therefore proposed that the long-wavelength-absorbing form represents the protonated, noncovalently bound bilin.Biliverdin, which is neither protonated nor covalently attached, undergoes spectral changes in its blue-absorbing band upon incubation with apo-Cph1. On the basis of these data we therefore propose a three-step model for phytochrome autoassembly. Size-exclusion chromatography revealed different mobilities for the apoprotein, redabsorbing Cph1-PCB and far-red-absorbing Cph1-PCB. The major peaks of both holoprotein adducts had apparent molecular masses < 200 kDa, a result in agreement with the notion that autophosphorylation in sensory histidine kinases requires dimerization. When Cph1-PCB was further purified by preparative native electrophoresis, the mobility on size-exclusion chromatography was < 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.Keywords: biliverdin; extinction coefficient; iodoacetamide; phycocyanobilin; phycoerythrobilin.Plant phytochromes are photoreceptors with a bilin chromophore that control many developmental processes in both higher and lower plants. Light triggers the interconversion of phytochrome between two thermostable, spectrally different forms, the red-absorbing form (Pr) with l max around 660 nm, and the far-red absorbing form (Pfr) with l max around 730 nm. This unusual feature allowed early spectral detection in etiolated plants [1], which was followed by biochemical [2] and molecular [3] characterization long before other plant photoreceptors were identified. Plant phytochromes are protein dimers consisting of < 124 kDa subunits that carry a single bilin chromophore, either phytochromobilin [4] or phycocyanobilin [5], covalently linked via a thioether to a conserved cysteine residue in the N-terminal moiety. The photoconversion of Pr to Pfr involves a Z to E isomerization around the double bond between rings 3 and 4 of the tetrapyrrole chromophore [4].A number of genes with homology to the chromophorebinding domain of phytochromes were recently identified by geno...

Photoreceptor chromoproteins undergo light-induced conformational changes that result in a modulation of protein interaction and enzymatic activity. Bacterial phytochromes such as Cph1 from the cyanobacterium Synechocystis PCC 6803 are light-regulated histidine kinases in which the light signal is transferred from the N-terminal chromophore module to the C-terminal kinase module. In this study, purified recombinant Cph1 was subjected to limited proteolysis using trypsin and endoproteinase Glu-C (V8). Cleavage sites of chromopeptide fragments were determined by MALDI-TOF and micro-HPLC on-line with tandem mass spectrometry in an ion trap mass spectrometer. Trypsin produced three major chromopeptides, termed F1 (S56 to R520), F2 (T64 to R472), and F3 (L81 to R472). F1 was produced only in the far-red absorbing form Pfr within 15 min and remained stable up to >1 h; F2 and F3 were obtained in the red-light absorbing form Pr within ca. 5-10 min. When F1 was photoconverted to Pr in the presence of trypsin, this fragment degraded to F2 and F3 within 1-2 min. On size exclusion chromatography, F1 eluted as a dimer in the Pfr and as a monomer in the Pr form, whereas F2 and F3 behaved always as monomers, irrespective of the light conditions. These and other results are discussed in the context of light-dependent subunit interactions, in which amino acids 473-520 within the PHY domain are required for chromophore-module subunit interaction within the homodimer. V8 proteolysis yielded five major chromopeptides, F4 (T17 to N449), F5 (T17 to E335), F6 (T17 to E323), F7 (unknown sequence), and F8 (tentatively L121 to E323). F6 and F8 were formed in the Pr form, whereas F4, F5, and F7 were preferentially formed in the Pfr form. Three amino acids next to specific cleavage sites, R520, R472, and E323, were altered by site-directed mutagenesis. The mutants were analyzed by UV-vis spectroscopy, size exclusion chromatography, and autophosphorylation. Histidine kinase activity was low in R472A, R520P, and R520A; in all mutants, the ratio of phosphorylation intensity between Pr and Pfr was reduced. Thus, light regulation of autophosphorylation is negatively affected in all mutants. In R472P, E323P, and E323D, the phosphorylation intensity of the Pfr form exceeded that of the wild-type control. This result shows that the histidine kinase activity of Cph1 is actively inhibited by photoconversion into Pfr.

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