Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15‫؍‬C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15‫؍‬C16 double bond is in either the Z or E configuration and the C14 -C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15‫؍‬C16 Z configuration and a C14 -C15 anti conformation in the Pr form and a C15‫؍‬C16 E configuration and a C14 -C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.Phytochromes are photoreceptors of plants, bacteria, and fungi with a bilin chromophore that absorb light in the red and far-red region (1). The natural chromophore differs between species: land plants use phytochromobilin (2) and cyanobacteria use phycocyanobilin (3), whereas other bacteria use biliverdin (BV) 1 (4). The natural chromophore attaches covalently to a cysteine residue by an autocatalytical process, although in vitro adducts with non-covalently bound chromophores may also be spectrally active (5, 6). Agrobacterium phytochrome Agp1, which is used in the present study, belongs to the BV binding phytochromes and attaches its chromophore to Cys-20 (5, 7) via the ring A vinyl side chain of BV (8). Upon assembly, the red absorption maximum ( max ) of the chromophore shifts toward a longer wavelength, accompanied by an absorbance increase, to form the red-absorbing Pr. Light absorption of Pr initiates photoconversion into the Pfr form, which results in a further ϳ50 -70-nm red shift of max . Light absorption of Pfr initiates the reverse photoconversion into Pr. For plant phytochromes it has been shown that the first step of the Pr to Pfr photoconversion is a Z to E isomerization of the chromophore around the C15ϭC16 double bond (9), which occurs in the picosecond time scale (10, 11). Isomerization is ...

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15‫؍‬C16 double bond is fixed in either the E or Z configuration and the C14 -C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4‫؍‬C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4‫؍‬C5 Z C5-C6 syn C15‫؍‬C16 Z C14 -C15 anti stereochemistry and that in the Pfr chromophore the C15‫؍‬C16 double bond has isomerized to the E configuration, whereas the C14 -C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.Almost all organisms have adopted mechanisms to sense light via photoreceptors and respond to different light conditions in manifold ways. Phytochromes are photoreceptors that are reversibly converted by light between two stable or long lived forms, the red-absorbing Pr 3 and the far-red-absorbing Pfr (1). Depending on the species, phytochromes bind one of three bilins as a chromophore: land plants utilize phytochromobilin (P⌽B) (2), cyanobacteria use phycocyanobilin (PCB) (3, 4), whereas all other organisms including proteobacteria and fungi use biliverdin (BV) (5-7). The chromophore is covalently attached to a cysteine residue during an autocatalytic process. Whereas plant and cyanobacterial phytochromes use a cysteine in the so-called GAF domain of the protein as chromophore attachment site (2), the chromophore-binding cysteine of BVbinding phytochromes is close to the N terminus of the protein (8 -10). NMR studies with plant phytochromes showed that the chromophore undergoes isomerization around the double bond connecting the rings C and D during photoconversion from Pr to Pfr (11). The configuration of this double bond is Z in the Pr and E in the Pfr form. Time-resolved spectroscopy techniques show that this Z-E isomerization, leading to the lumi-R photoproduct, takes place on a picosecond time scale (12, 13). Later intermediates, termed meta-Ra, meta-Rb, and meta-Rc, as well as the photoproduct Pfr are formed in the microsecond and millis...

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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.

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended -electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.