Raman Spectroscopic and Light-Induced Kinetic Characterization of a Recombinant Phytochrome of the Cyanobacterium Synechocystis

Abstract

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 (PΦB). 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 P_fr 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 P_r state. Synechocystis phytochrome (PCB) undergoes a multistep photoconversion reminiscent of the phyA P_r → P_fr 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-μs lifetime. The next observable intermediate grows in with 300 (±25) μs and decays with 6−8 ms. The final state (P_fr) is formed biexponentially (450 ms, 1 s). When reconstituted with PΦB, the first decay of this Synechocystis phytochrome is biexponential (5 and 25 μs). The growth of the second intermediate is slower (750 μs) than that in the PCB adduct whereas the decays of both species are similar. The formation of the P_fr 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 μs) 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 P_fr 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 °C are 44 (in H₂O/buffer) and 48 kJ mol^-1 (D₂O), with essentially identical pre-exponential factors.