The P r 3 P fr 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 P r and ZZEssa in P fr , with all pyrrole nitrogens being protonated. Photoexcitation of P r is followed by (at least) three thermal relaxation components in the formation of P fr with time constants of 230 s and 3.1 and 260 ms. H 2 O/D 2 O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R C , respectively. Resonance Raman measurements of Meta-R C indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R C are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the P r 3 P fr photoconversion mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the P fr formation that switch between the active and the inactive state of the photoreceptor.Phytochromes are photoreceptors that utilize light as a source of information for controlling numerous biological processes (1, 2). The chromophore, a methine-bridged tetrapyrrole ( Fig. 1), acts as a photoswitch between two stable, spectrally distinct forms, denoted as P r and P fr according to the red and far-red absorption maxima, respectively. The P r /P fr interconversion is initiated by the rapid Z/E photoisomerization of the C-D methine bridge (3), followed by chromophore relaxations that are coupled to structural changes of the apoprotein (4). These structural changes are the trigger for signal transduction. Resonance Raman (RR) 2 and IR spectroscopy have provided valuable insight into light-induced chromophore and protein structural changes (e.g. see Refs. 5-10), but molecular and mechanistic details are not yet known and no crystal structure of a phytochrome has been reported so far.While phytochromes were originally thought to be restricted to plants, the discovery of these chromoproteins in cyanobacteria (11) and other bacteria points to the prokaryotic origin of this family of photoreceptors. In contrast to plant phytochromes, typical bacterial phytochromes are light-regulated histidine kinases. Despite the quite different regulatory functions (12, 13), plant and bacterial phytochromes exhibit structural and mechanistic similarities. The phytochromobilin chromophore of plant phytoc...

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. Titration experiments demonstrate a lowering of the pK a from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc 3 Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

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. The photoconversion of Agp1-5Zs is apparently arrested in a Meta-R A -like intermediate, since the subsequent syn to anti rotation around the C 5 -C 6 bond is prevented by the lock. The Meta-R A -like photoproduct was characterized by its distinctive CD spectrum suggesting a reorientation of ring D.

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.

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). Since this state is unusually long-lived as compared to other intermediates in the photocycle of both PYP and Bph, we interpret this as formation of a metastable complex between activated PYP and Bph domains that takes days to relax. In the metastable complex, the PYP domain is locked in its activated UV absorbing state and the Bph domain is in a slightly red-shifted state (from 701 to 702 nm), which is photochemically inactive to red or white light. The amount of metastable complex formed increases with the degree of prior activation of PYP, reaching a maximum of 50% when PYP is fully activated compared to 0% when no PYP is activated. The saturation of complex formation at 50% is believed to be due to light-induced heterogeneity within the Ppr dimer. UV irradiation (365 nm) of the metastable complex state photoreverses the activated PYP and the red-shifted Bph to the initial dark state within seconds. We therefore postulate that Ppr functions as a UV-red light sensor and describe the different Ppr states that can be obtained depending on the light quality. Both red and white light upregulate the autokinase activity, while it is downregulated in the dark. The physiological state of Ppr is most likely a mixture of three different states, dark, metastable complex, and red light-activated, with fractional populations whose amounts depend on the light quality of the environment and that regulate the extent of phosphorylation by the kinase.