Phytochromes are a widespread family of photosensory proteins first discovered in plants, which measure the ratio of red to far-red light to control many aspects of growth and development. Phytochromes interconvert between red-absorbing Pr and far-redabsorbing Pfr states via photoisomerization of a covalently-bound linear tetrapyrrole (bilin) chromophore located in a conserved photosensory core. From recent crystal structures of this core region, it has been inferred that the chromophore structures of Pr and Pfr are conserved in most phytochromes. Using circular dichroism spectroscopy and ab initio calculations, we establish that the Pfr states of the biliverdin-containing bacteriophytochromes DrBphP and PaBphP are structurally dissimilar from those of the phytobilincontaining cyanobacterial phytochrome Cph1. This conclusion is further supported by chromophore substitution experiments using semisynthetic bilin monoamides, which indicate that the propionate side chains perform different functional roles in the 2 classes of phytochromes. We propose that different directions of bilin D-ring rotation account for these distinct classes of red/far-red photochemistry.bilin amides ͉ biliprotein ͉ circular dichroism ͉ photoisomerization P hytochromes comprise a class of red/far-red biliprotein photoreceptors that regulate photomorphogenesis, shade avoidance, and development in higher plants (1, 2). Also found in bacteria and fungi, phytochromes characteristically photoconvert between red-absorbing P r and far-red-absorbing P fr states (3,4). Conversion between these 2 states involves Z/E photoisomerization of the 15/16 double bond of their linear tetrapyrrole (bilin) chromophores. Photosensory signaling by phytochromes relies on a conserved core comprising PAS (PER, ARNT, SIM), GAF (cGMP phosphodiesterase, adenylate cyclase, FhlA), and PHY (phytochrome-specific GAF-related) domains (5), with the bilin bound within a conserved pocket of the GAF domain (1, 6). The precise structure of the bilin chromophore, the nature of its covalent linkage to cysteine (Cys) side chains in the protein, and the location of those Cys residues vary among phytochromes (7), and 2 distinct subclasses can be distinguished on these grounds.Phytobilin-containing phytochromes, which include the plant (Phys) and cyanobacterial (Cph1) phytochromes, are found exclusively in oxygenic photosynthetic organisms. The chromophore precursor for these phytochromes is either phytochromobilin or phycocyanobilin (PCB), both of which possess reduced ethylidene-containing A-rings. For these phytochromes, covalent linkage forms between a GAF-domain Cys and the ␣-carbon of the A-ring ethylidene (Fig. S1 A). The much more widespread biliverdin-containing phytochromes (4), which include the bacteriophytochromes (BphPs) and fungal phytochromes, possess covalent linkages between a Cys residue upstream of the PAS domain and the ␤-carbon of the A-ring endo-vinyl group of biliverdin IX␣ (BV; Fig. S1B) (8). P r is commonly the thermally stable dark state for both classes of...

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Linear tetrapyrroles (bilins) perform important antioxidant and light harvesting functions in cells from bacteria to humans. To explore the role of the propionate moieties in bilin metabolism, we report the semisynthesis of mono- and di-amides of biliverdin IXα and those of its non-natural XIIIα isomer. Initially, these were examined as substrates of two types of NADPH-dependent biliverdin reductase, BVR and BvdR, and of the representative ferredoxin-dependent bilin reductase, phycocyanobilin:ferredoxin oxidoreductase (PcyA). Our studies indicate that the NADPH-dependent biliverdin reductases are less accommodating to amidation of the propionic acid sidechains of biliverdin IXα than PcyA, which does not require free carboxylic acid sidechains to yield its phytobilin product, phycocyanobilin. Bilin amides were also assembled with BV-type and phytobilin-type apophytochromes, demonstrating a role for the 8-propionate in formation of the spectroscopically native Pr dark states of these biliprotein photosensors. Neither ionizable propionate sidechain proved essential to primary photoisomerization for both classes of phytochromes, but an unsubstituted 12-propionate was required for full photointerconversion of phytobilin-type phytochrome Cph1. Taken together, these studies provide insight into the roles of the ionizable propionate sidechains in substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences between the photoconversions of BV-type and phytobilin-type phytochromes.

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