Regulation of the Ppr histidine kinase by light-induced interactions between its photoactive yellow protein and bacteriophytochrome domains.

Ppr is a unique bacteriophytochrome that bleaches rather than forming a far-red-shifted Pfr state upon red light activation. Ppr is also unusual in that it has a blue light photoreceptor domain, PYP, which is N-terminally fused to the bacteriophytochrome domain (Bph). When both photoreceptors are activated by light, the fast phase of Bph recovery (1 min lifetime) corresponds to the formation of an intramolecular long-lived complex between the activated PYP domain and the Bph domain (lifetime of 2-3 days).

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.

Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1.

The bacteriophytochrome Agp1 was reconstituted with a locked 5Zs-biliverdin in which the C(4)=C(5) and C(5)-C(6) bonds of the methine bridge between rings A and B are fixed in the Z and syn configuration/conformation, respectively. In Agp1-5Zs the photoconversion proceeds via the Lumi-R intermediate to Meta-R(A), but the following millisecond-transition to Meta-R(C) is blocked. Consistently, no transient proton release was detected.

Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation.

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy.

Light-induced proton release of phytochrome is coupled to the transient deprotonation of the tetrapyrrole chromophore.

The Pr --> Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R(C) were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 micros and 3.