PubMed · 9097603
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A Inazu, J Koizumi, H Mabuchi. 1997. [Functioning test for cholesterol turnover].. https://pubmed.ncbi.nlm.nih.gov/9097603/
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A phytochrome-encoding cDNA from the cyanobacterium Synechocystis has been heterologously expressed in Escherichia coli and reconstituted into functional chromoproteins by incubation with either phycocyanobilin (PCB) or phytochromobilin (PPhiB). These materials were studied by Raman spectroscopy and nanosecond flash photolysis. The Raman spectra suggest far-reaching similarities in chromophore configuration and conformation between the Pfr forms of Synechocystis phytochrome and the plant phytochromes (e.g. phyA from oat), but some differences, such as torsions around methine bridges and in hydrogen bonding interactions, in the Pr state. Synechocystis phytochrome (PCB) undergoes a multistep photoconversion reminiscent of the phyA Pr --> Pfr transformation but with different kinetics. The first process resolved is the decay of an intermediate with red-shifted absorption (relative to parent state) and a 25-micros lifetime. The next observable intermediate grows in with 300 (+/-25) micros and decays with 6-8 ms. The final state (Pfr) is formed biexponentially (450 ms, 1 s). When reconstituted with PPhiB, the first decay of this Synechocystis phytochrome is biexponential (5 and 25 micros). The growth of the second intermediate is slower (750 micros) than that in the PCB adduct whereas the decays of both species are similar. The formation of the Pfr form required fitting with three components (350 ms, 2.5 s, and 11 s). H/D Exchange in Synechocystis phytochrome (PCB) delays, by an isotope effect of 2.7, both growth (300 micros) and decay rates (6-8 ms) of the second intermediate. This effect is larger than values determined for phyA (ca. 1.2) and is characteristic of a rate-limiting proton transfer. The formation of the Pfr state of the PCB adduct of Synechocystis phytochrome shows a deuterium effect similar as phyA (ca. 1.2). Activation energies of the second intermediate in the range 0-18 degrees C are 44 (in H2O/buffer) and 48 kJ mol-1 (D2O), with essentially identical pre-exponential factors.
The complete sequence of the Synechocystis chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in Escherichia coli. In this paper we describe this recombinant Synechocystis phytochrome in more detail. Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related. An approximately 300 residue portion that is important for plant phytochrome function is missing from the Synechocystis sequence, immediately in front of the putative kinase region. The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography. Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation. Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the Synechocystis phytochrome. According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively. Both tend to form dimers in vitro and aggregate under low salt conditions. Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography.
The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.