The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination.
Ppr from the purple phototrophic bacterium, Rhodospirillum centenum (also known as Rhodocista centenaria), is a hybrid of photoactive yellow protein (PYP), bacteriophytochrome (Bph), and histidine kinase (HK) domains. The holo-Ppr (containing both chromophores) exhibits characteristic absorption maxima at 435 nm due to the PYP domain and at 400, 642, and 701 nm due to the Bph domain. Illumination of the Ppr with white light causes a bleach of both PYP and Bph absorbance; weak blue light primarily bleaches the PYP, and red light activates only the Bph. When excited by blue light, the PYP domain in Ppr recovers with biphasic kinetics at 445 nm (32% with a lifetime of 3.8 min and the remainder with a lifetime of 46 min); white light primarily results in fast recovery, whereas the 130-residue PYP construct shows only the faster kinetics in both blue and white light. Furthermore, there is a slight red shift of the ground state Bph when the PYP is activated; thus, both spectroscopy and kinetics suggest interdomain communication. When Ppr is illuminated with red light, the recovery of the Bph domain to the dark state is significantly slower than that of PYP and is biphasic (57% of the 701 nm decay has a lifetime of 17 min and the remainder a lifetime of 50 min). However, when illuminated with white light or red followed by blue light, the Bph domain in Ppr recovers to the dark-adapted state in a triphasic fashion, where the fastest phase is similar to that of the fast phase of the PYP domain (in white light, 25% of the 701 nm recovery has a lifetime of approximately 1 min) and the slower phases are like the recovery after red light alone. Apo-holo-Ppr (with the biliverdin chromophore only) recovers with biphasic kinetics similar to those of the slower phases of holo-Ppr when activated by either red or white light. We conclude that the photoactivated PYP domain in Ppr accelerates recovery of the activated Bph domain. Phytochromes can be reversibly switched between Pr and Pfr forms by red and far-red light, but the consequence of a bleaching phytochrome is that it cannot be photoreversed by far-red light. We thus postulate that the function of the PYP domain in Ppr is to act as a blue light switch to reverse the effects of red light on the Bph.
Kyndt
John A
JA
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Fitch
John C
JC
Meyer
Terry E
TE
Cusanovich
Michael A
MA
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C120281:Ppr protein, Rhodospirillum centenum