Biliverdin Binds Covalently to Agrobacterium Phytochrome Agp1 via Its Ring A Vinyl Side Chain

Abstract

The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction. In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein. Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20). By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment. Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner. Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249). It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket. In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein. However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin. This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain.Phytochromes are biliprotein photoreceptors that were discovered in plants (1) but were recently also found in bacteria (2-4), fungi (5), and slime molds (6). The photocycle of phytochromes has two long-lived forms, the red-absorbing form (Pr), 1

The Heme Oxygenase(s)-Phytochrome System of Pseudomonas aeruginosa

“…BV) was then associated with BphP, indicating that BV was removed from BphO and was attached to BphP. This attachment was identified to be covalent by zinc blotting (40) (data not shown) and is in agreement with other known bacterial phytochromes that form a covalent complex with BV (11,41). Experiments to identify real physical interaction between BphO and BphP using the GPC method after Hummel and Dreyer (42) with BphO⅐BV in the mobile phase did not reveal a stable complex of both proteins (data not shown).…”

“…From reports by other groups it is known that an A-ring endo vinyl-group of the BV is necessary for binding to BphP (41). Therefore, only the BV IX␦ isomer should yield a photoactive holo-BphP.…”

The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity

“…Addition of SDS to the biliverdin/ GST-PKC␦ sample followed by size-exclusion separation of GST-PKC␦ from low molecular weight components yielded a spectrum identical to that of SDS-treated GST-PKC␦, including the shifted ultraviolet absorbance peak, indicating that the inhibitor is removed by simple physical dissociation and is therefore not a consequence of covalent association of biliverdin with the PKC. This is in contrast to the observation of covalent binding of biliverdin to phytochrome proteins in Pseudomonas aeroginosa and Agrobacterium tumefaciens (44,47).…”

“…Test of Covalent Binding of Biliverdin to PKC␦-Association between PKC␦ and biliverdin was examined essentially as described by Lamparter et al (44). Biliverdin was dissolved in 0.1 M NaOH and diluted to 2 M in PBS, pH 7.4; a 500-l sample was used to measure the absorption spectrum between 260 and 760 nm.…”

The Structure of Phytochrome: A Picture Is Worth a Thousand Spectra

“…Heme oxygenases convert heme to biliverdin IXa (BV), which is the direct precursor of the chromophores of bacteriophytochromes (Bhoo et al, 2001;Giraud et al, 2002Giraud et al, , 2005Lamparter et al, 2003Lamparter et al, , 2004Tasler et al, 2005) and probably also those of fungal phytochromes (Blumenstein et al, 2005;Froehlich et al, 2005). In cyanobacteria and algae, BV is converted to phycocyanobilin (PCB) via a four-electron reduction mediated by ferredoxin-dependent bilin reductases of the PcyA subfamily ).…”