Photoconversion Mechanism of a Green/Red Photosensory Cyanobacteriochrome AnPixJ: Time-Resolved Optical Spectroscopy and FTIR Analysis of the AnPixJ-GAF2 Domain

The photoconversion mechanism of a green/red sensory cyanobacteriochrome AnPixJ was studied. The phycocyanobilin-binding second GAF domain of AnPixJ of Anabaena sp. PCC 7120 was expressed in Escherichia coli cells. The His-tagged AnPixJ-GAF2 domain exhibited photoconversion between the green- and red-absorbing forms, APg(543) and APr(648), respectively. We detected four intermediate states in the photocycle between them, as follows: APr648 -> red light -> APr(648)* -> (with a rise time constant tau(r) of < 100 ns) R1(650-80) (with a decay time constant tau(d) of < 1 mu s) -> R2(610) (tau(d) = 920 mu s) -> APg(543) -> green light -> APg(543)* -> (tau(r) < 50 ns) G1(570) (tau(d) = 190 mu s) -> G2(630) (tau(d) = 1.01 ms) -> APr(648). These intermediates were named for their absorption peak wavelengths, which were estimated on the basis of the time-resolved difference spectra and global analysis of the time courses. The absorption spectrum of APr648 resembles that of the Pr form of the phytochrome, while all the other states showed peaks at 530-650 nm and had wider bandwidths with smaller peak amplitudes. The fastest decay phases of fluorescence from APr(648)* and APg(543)* gave lifetimes of 200 and 42 ps, respectively, suggesting fast primary reactions. The APg(543)-minus-APr(648) difference FTIR spectrum in an H2O medium was significantly different from those reported for the Pfr/Pr difference spectra in phytochromes. Most of the peaks in the difference spectrum were shifted in the D2O medium, suggesting the high accessibility to the aqueous phase. The interactions of the phycocyanobilin chromophore with the surrounding amino acid residues, which are fairly different from those in the GAF domain of phytochromes, realize the unique green/red photocycle of AnPixJ.