Raman spectroscopic and light-induced kinetic characterization of a recombinant phytochrome of the cyanobacterium Synechocystis.

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.