Characterization of the covalent and noncovalent adducts of Agp1 phytochrome assembled with biliverdin and phycocyanobilin by circular dichroism and flash photolysis.
The functional role of the covalent attachment of the bilin chromophores biliverdin (BV) and phycocyanobilin (PCB) was investigated for phytochrome Agp1 from Agrobacterium tumefaciens using circular dichroism (CD) and transient absorption spectroscopy. Covalent and noncovalent adducts with these chromophores were prepared by using wild-type (WT) Agp1 (covalent BV and noncovalent PCB binding), mutant C20A in which the covalent BV binding site is eliminated, and mutant V249C in which the covalent PCB binding site is introduced. While the CD spectra of the P(r) forms of all these photochromic adducts are qualitatively the same, the CD spectrum of the P(fr) form of the covalent PCB adduct is unique in having a positive rotational strength in the Q-band which we assign to the Z-E isomerization of the C-D methine bridge. In the three other adducts, the Q-band CD in the P(fr) state is almost zero, suggesting that upon photoconversion a negative contribution from an out-of-plane rotation of the A ring of the chromophore compensates for the positive contribution from ring D. The contribution from ring A is absent or strongly reduced in the shorter pi-conjugation system of the covalent PCB adduct. The results from CD spectroscopy are consistent with a uniform geometry of the bilin chromophore in the covalent and noncovalent adducts. Transient absorption spectroscopy showed that the spectral changes and the kinetics of the P(r) to P(fr) photoconversion are not substantially affected by the covalent attachment of BV and PCB. The kinetics in the BV and PCB adducts mainly differ in the formation of P(fr) that is accelerated by 2 orders of magnitude in the PCB adducts, whereas the sequence of spectral transitions and the associated proton transfer processes are quite similar. We conclude that the P(r) to P(fr) photoconversion in the BV and PCB adducts of Agp1 involves the same relaxation processes and is thus governed by specific protein-cofactor interactions rather than by the chemical structure of the chromophore or the mode of attachment. The strongly reduced photostability of the noncovalent BV adduct suggests that covalent attachment in native Agp1 phytochrome prevents irreversible photobleaching and stabilizes the chromophore. The N-terminal peptide segment including amino acids 2-19 is essential for covalent attachment of the chromophore but dispensable for the spectral and kinetic properties of Agp1.
Borucki
Berthold
B
Biophysics Group, Department of Physics, Freie Universitat Berlin, Arnimallee 14, 14195 Berlin, Germany. borucki@physik.fu-berlin.de
Seibeck
Sven
S
Heyn
Maarten P
MP
Lamparter
Tilman
T
D016960:Agrobacterium tumefaciens / Q000737:chemistry; D001664:Biliverdine / Q000737:chemistry*; D002942:Circular Dichroism; D007700:Kinetics; D010777:Photochemistry; D052980:Phycobilins / Q000737:chemistry*; D010798:Phycocyanin / Q000737:chemistry*; D010834:Phytochrome / Q000737:chemistry*; D011078:Polychlorinated Biphenyls / Q000737:chemistry; D011522:Protons
D052980:Phycobilins; D011522:Protons; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin; D011078:Polychlorinated Biphenyls; D001664:Biliverdine