FTIR Study of the Photoinduced Processes of Plant Phytochrome Phya using Isotope-Labeled Bilins and Density Functional Theory Calculations

Abstract

Fourier transform infrared spectroscopy was used to analyze the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global (13)C-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE-->ZZZ), which involves only small protein structural changes.

Light-induced chromophore activity and signal transduction in phytochromes observed by 13 C and 15 N magic-angle spinning NMR

“…The signal doubling observed for carbons 1 and 2 in the Pr state disappears in the Pfr state. These observations suggest an increase of mechanical tension occurring in the Pfr state, as is also observed by 15 N MAS NMR (19) and vibrational spectroscopy (13). Such tension would also explain the change at the 3 1 carbon atom that links the chromophore via a thioether covalently to Cys-259.…”

“…3 shows an 1 H-15 N heteronuclear correlation experiment that indicates good agreement with data obtained in the solution state (27). Based on the 15 N assignments of refs. 19 and 24 (Table S2) Fig.…”

“…However, the exact geometry of the chromophore in the Pfr state and the role of the chromophore-binding pocket in the phototransformation have yet to be established. Moreover, slight rotations around single bonds that are hardly reflected by the crystal structures may cause a more distorted chromophore, a scenario that is supported by a recent investigation using vibrational spectroscopy and density functional theory calculations (15).…”

“…A 1 H-13 C heteronuclear 2D spectrum of the region of the methine carbons of u-[ 13 C, 15 N]-PCB in Cph1⌬2 is shown in Fig. S5.…”

OWL1: An Arabidopsis J-Domain Protein Involved in Perception of Very Low Light Fluences

“…phyA, like all phytochromes, is synthesized in the cytoplasm, where it accumulates to high levels in darkness. Upon light activation, which leads to conformational changes of the chromophore transduced to the protein backbone (Pfr form), phyA moves from the cytoplasm to the nucleus where it localizes to nuclear foci (Kircher et al, 2002;Schwinte et al, 2008). The phyA photoreceptor itself is modulated by light on multiple levels.…”