The Biliverdin Chromophore Binds Covalently to a Conserved Cysteine Residue in the N-Terminus of Agrobacterium Phytochrome Agp1

Abstract

Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

“…Notably, canonical GAF and PHY domains were identified in the PSM of both protein sequences, but the N-terminal PAS domain sequence was predicted only in Tp-DPH ( Figure 1A; Supplemental Data Set 1). In spite of this difference, both DPHs possess a Cys residue in the N-terminal extremity, at the same position as the Cys residue that mediates chromophore binding in BphPs ( Figure 1A; Supplemental Figure 1) (Lamparter et al, 2004;Wagner et al, 2005). This conservation suggests that Biliverdin IXa (BV) might constitute the hemederived chromophore of DPHs.…”

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 ).…”

“…In all phytochromes, the bilin chromophore precursor is covalently attached to the protein via a thioether linkage between a Cys residue and the bilin A-ring ( Figure 1A). Plant and cyanobacterial phytochromes utilize a conserved Cys residue in the P3 domain (Lagarias and Rapoport, 1980;Hü bschmann et al, 2001), whereas bacteriophytochromes (and probably fungal phytochromes) instead utilize a different Cys in the P2 region (Lamparter et al, 2004;Karniol et al, 2005;Tasler et al, 2005) (Figure 1C). Unlike the phycobiliproteins, which utilize C-S lyases for covalent attachment of their bilin chromophores (Schluchter and Glazer, 1999), plant phytochromes do not require enzymes or cofactors to assist the covalent assembly reaction (Terry et al, 1993).…”

“…Interestingly, introduction of a P3 Cys into bacteriophytochromes supports assembly with PCB (Davis et al, 1999;Lamparter et al, 2004;Quest and Gä rtner, 2004), arguing for structural conservation of the chromophore binding pocket.…”

Apo-bacteriophytochromes modulate bacterial photosynthesis in response to low light

“…In vitro studies have characterized the reversible photoconversion of RpBphP2 and RpBphP3 between a red light (700 nm)-absorbing form, which is also the form that the proteins assume in the dark, and a far-red (750 nm)-absorbing or near-red (650 nm)-absorbing form, respectively (9)(10)(11)(12). BphPs bind the chromophore biliverdin IXα (BV), a linear tetrapyrrole that is incorporated autocatalytically into the N-terminal photosensory domain (13,14). BV is synthesized by heme oxygenase from heme, a reaction that requires oxygen (15).…”