Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 Å resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its darkadapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an ''arm'' structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common and may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, crossdomain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.phytochrome ͉ photoreceptor

Signaling photoreceptors use the information contained in the absorption of a photon to modulate biological activity in plants and a wide range of organisms. The fundamental-and as yet imperfectly answered-question is, how is this achieved at the molecular level? We adopt the perspective of biophysicists interested in light-dependent signal transduction in nature and the three-dimensional structures that underpin signaling. Six classes of photoreceptors are known: light-oxygen-voltage (LOV) sensors, xanthopsins, phytochromes, blue-light sensors using flavin adenine dinucleotide (BLUF), cryptochromes, and rhodopsins. All are water-soluble proteins except rhodopsins, which are integral membrane proteins; all are based on a modular architecture except cryptochromes and rhodopsins; and each displays a distinct, light-dependent chemical process based on the photochemistry of their nonprotein chromophore, such as isomerization about a double bond (xanthopsins, phytochromes, and rhodopsins), formation or rupture of a covalent bond (LOV sensors), or electron transfer (BLUF sensors and cryptochromes).

Bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris work in tandem to modulate synthesis of the light-harvesting complex LH4 in response to light. Although RpBphP2 and RpBphP3 share the same domain structure with 52% sequence identity, they demonstrate distinct photoconversion behaviors. RpBphP2 exhibits the ''classical'' phytochrome behavior of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states, whereas RpBphP3 exhibits novel photoconversion between Pr and a nearred (Pnr) light-absorbing states. We have determined the crystal structure at 2.2-Å resolution of the chromophore binding domains of RpBphP3, covalently bound with chromophore biliverdin IX␣. By combining structural and sequence analyses with site-directed mutagenesis, we identify key residues that directly modulate the photochemical properties of RpBphP3 and RpBphP2. Remarkably, we identify a region spanning residues 207-212 in RpBphP3, in which a single mutation, L207Y, causes this unusual bacteriophytochrome to revert to the classical phenotype that undergoes reversible photoconversion between the Pr and Pfr states. The reverse mutation, Y193L, in the corresponding region in RpBphP2 significantly diminishes the formation of the Pfr state. We propose that residues 207-212 and the spatially adjacent conserved residues, Asp-216 and Tyr-272, interact with the chromophore and form part of the interface between the chromophore binding domains and the PHY domain that modulates photoconversion.biliverdin ͉ red-light photoreceptor P hytochromes are photoreceptors found in plants, cyanobacteria, fungi, and nonphotosynthetic bacteria that regulate a range of physiological responses such as chlorophyll synthesis, seed germination, floral induction, and phototaxis by using light in the red/far-red region of the spectrum (1). Upon absorption of a photon in the appropriate wavelength range, their linear tetrapyrrole chromophores (bilins) switch between two stable, spectrally distinct, red-and far-red-light absorbing forms, denoted Pr and Pfr, respectively. In most phytochromes Pr is the dark-adapted, ground state; in others, it is Pfr. The primary photochemical event for the Pr/Pfr photoconversion in plant phytochromes (Phys) and bacteriophytochromes (Bphs) is believed to involve rapid 15Z anti to 15E anti (15Za/15Ea) isomerization of the C15AC16 double bond between rings C and D of the bilin chromophore. Isomerization is followed by slower transitions via several spectrally distinct intermediates (2-4) that are presumably accompanied by structural changes in the chromophore and the surrounding protein.A pair of Bphs from the photosynthetic bacterium Rhodopseudomonas palustris, denoted RpBphP2 and RpBphP3, was characterized that in tandem modulates synthesis of the LH4 light-harvesting complex (5). RpBphP2 and RpBphP3 share the same biliverdin IX␣ (BV) chromophore and the same domain structure, in which three N-terminal photosensory domains, PAS (Per-ARNT-Sim), GAF (cGMP phosphodies...

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a ''flip-and-rotate'' model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket.biliverdin ͉ photoconversion ͉ red-light photoreceptor P hytochromes are red-light photoreceptors that undergo reversible photoconversion between a red-light-absorbing state (Pr) and a far-red-light-absorbing state (Pfr), and thereby they regulate a wide range of physiological responses in plants, fungi, and photosynthetic bacteria (1-5). Using linear tetrapyrroles as chromophores to detect light in the long-wavelength range of the visible spectrum, the photosensory core module (PCM) of bacteriophytochromes contains three domains (PAS, GAF, and PHY). The PAS and GAF domains constitute the chromophore-binding module (CBM); and the PHY domain is essential for efficient photoconversion (5). Upon absorbing a photon, 15Za/15Ea isomerization occurs about the C15AC16 double bond between rings C and D of the bilin chromophore, followed by thermal relaxation events in the chromophore and the protein matrix (6). Local conformational changes originating in the photosensory domains propagate to the C-terminal histidine kinase (HK) domain, where they modulate the kinase activity and thus convert a light signal into a chemical signal (5). Fundamental questions about the molecular mechanisms of photoconversion and signal transduction remain unanswered. What are the local and long-range conformational changes? What molecular events are involved? In what sequence do they occur?Extensive studies on a variety of phytochromes and bacteriophytochromes suggest that significant structural changes occur in both the chromophore and protein moieties during Pr/Pfr photoconversion, but details of these changes are still lacking (7-11). Two crystal structures of bacteriophytochromes with intact PCMs have been determined recently: that of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) (12) and that of cyanobacterial phyt...

Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria1. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses2. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states3-7. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We employed cryo-trapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeds. Light-induced conformational changes in PaBphP originate in ring D of the bilin chromophore, and E to Z isomerization about the C15=C16 double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C15 methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data suggest that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.