Profile for J Clark Lagarias

BACKGROUND: Over the past decade, a number of tools have emerged for the examination of homology relationships among protein sequences in a structural context. Most recent software implementations for such analysis are tied to specific molecular viewing programs, which can be problematic for collaborations involving multiple viewing environments. Incorporation into larger packages also adds complications for users interested in adding their own scoring schemes or in analyzing proteins incorporating unusual amino acid residues such as selenocysteine. RESULTS: We describe homolmapper, a command-line application for mapping information from a multiple protein sequence alignment onto a protein structure for analysis in the viewing software of the user's choice. Homolmapper is small (under 250 K for the application itself) and is written in Python to ensure portability. It is released for non-commercial use under a modified University of California BSD license. Homolmapper permits facile import of additional scoring schemes and can incorporate arbitrary additional amino acids to allow handling of residues such as selenocysteine or pyrrolysine. Homolmapper also provides tools for defining and analyzing subfamilies relative to a larger alignment, for mutual information analysis, and for rapidly visualizing the locations of mutations and multi-residue motifs. CONCLUSION: Homolmapper is a useful tool for analysis of homology relationships among proteins in a structural context. There is also extensive, example-driven documentation available. More information about homolmapper is available at http://www.mcb.ucdavis.edu/faculty-labs/lagarias/homolmapper_home/homolmapper%20web%20page.htm.

Phytochromes are a widespread family of red/far-red responsive photoreceptors first discovered in plants, where they constitute one of the three main classes of photomorphogenesis regulators. All phytochromes utilize covalently attached bilin chromophores that enable photoconversion between red-absorbing (P(r)) and far-red-absorbing (P(fr)) forms. Phytochromes are thus photoswitchable photosensors; canonical phytochromes have a conserved N-terminal photosensory core and a C-terminal regulatory region, which typically includes a histidine-kinase-related domain. The discovery of new bacterial and cyanobacterial members of the phytochrome family within the last decade has greatly aided biochemical and structural characterization of this family, with the first crystal structure of a bacteriophytochrome photosensory core appearing in 2005. This structure and other recent biochemical studies have provided exciting new insights into the structure of phytochrome, the photoconversion process that is central to light sensing, and the mechanism of signal transfer by this important family of photoreceptors.

Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of biliverdin IXalpha to phycocyanobilin-the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semireduced bilin radical intermediates that are profoundly stabilized by selected mutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for this stabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. PCC 6803 PcyA in the initial oxidized and one-electron reduced radical states. Unlike wild-type PcyA, both mutants possess a bilin-interacting axial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radical complex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located for D-ring vinyl reduction. On the basis of these structures and companion (15)N-(1)H long-range HMQC NMR analyses to assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.

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 diamides of biliverdin IXalpha and those of its non-natural XIIIalpha 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 side chains of biliverdin IXalpha than PcyA, which does not require free carboxylic acid side chains 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 the formation of the spectroscopically native P(r) dark states of these biliprotein photosensors. Neither ionizable propionate side chain proved to be 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 side chains in substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences between the photoconversions of BV-type and phytobilin-type phytochromes.

Photosensory proteins enable living things to detect the quantity and quality of the light environment and to transduce that physical signal into biochemical outputs which entrain their metabolism with the ambient light environment. Phytochromes, which photoconvert between red-absorbing P(r) and far-red-absorbing P(fr) states, are the most extensively studied of these interesting proteins. Critical regulators of a number of key adaptive processes in higher plants, including photomorphogenesis and shade avoidance, phytochromes are widespread in photosynthetic and nonphotosynthetic bacteria, and even in fungi. Cyanobacterial genomes also possess a plethora of more distant relatives of phytochromes known as cyanobacteriochromes (CBCRs). Biochemical characterization of representative CBCRs has demonstrated that this class of photosensors exhibits a broad range of wavelength sensitivities, spanning the entire visible spectrum. Distinct protein-bilin interactions are responsible for this astonishing array of wavelength sensitivities. Despite this spectral diversity, all members of the extended family of phytochrome photosensors appear to share a common photochemical mechanism for light sensing: photoisomerization of the 15/16 double bond of the bilin chromophore.

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase (FDBR) family. Unlike other FDBRs that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria and cryptophytes. To address the structural basis for PcyA's reduction regiospecificity, we report new high-resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803 - all of which lack exo-vinyl reduction activity. These include the BV complex of the Glu76Gln (E76Q) mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha (BV13), a synthetic substrate that lacks an exo-vinyl group. While the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu76 side-chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side-chain of Glu76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA.

Phytochromes are an important class of red/far-red responsive photoreceptors that act as light-activated biological switches, ultimately driving growth and development in plants, bacteria, and fungi. The composition of the red-absorbing ground-state has been widely debated due to the presence of a shoulder feature on the blue edge of electronic absorption spectra, which many have attributed to the presence of multiple ground-state conformers. Here we use resonance Raman intensity analysis to calculate the vibronic absorption profile of cyanobacterial phytochrome Cph1 and show that this shoulder feature is due simply to vibronic transitions from a single species, thus reflecting a homogeneous ground-state population.

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 P(r) and far-red-absorbing P(fr) 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 P(r) and P(fr) are conserved in most phytochromes. Using circular dichroism spectroscopy and ab initio calculations, we establish that the P(fr) states of the biliverdin-containing bacteriophytochromes DrBphP and PaBphP are structurally dissimilar from those of the phytobilin-containing 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.

The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two-step four-electron reduction of biliverdin IXalpha to phycocyanobilin, the precursor of biliprotein chromophores found in phycobilisomes. It is known that catalysis proceeds via paramagnetic radical intermediates, but the structure of these intermediates and the transfer pathways for the four protons involved are not known. In this study, high-field electron paramagnetic resonance (EPR) spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of two PcyA mutants D105N from the cyanobacteria Synechocystis sp. PCC6803 and Nostoc sp. PCC7120 are examined. Detailed analysis of Synechocystis D105N mutant spectra at 130 and 406 GHz reveals a biliverdin radical with a very narrow g tensor with principal values 2.00359(5), 2.00341(5), and 2.00218(5). Using density-functional theory (DFT) computations to explore the possible protonation states of the biliverdin radical, it is shown that this g tensor is consistent with a biliverdin radical where the carbonyl oxygen atoms on both the A and the D pyrrole rings are protonated. This experimentally confirms the reaction mechanism recently proposed (Tu, et al. Biochemistry 2007, 46, 1484).

Photochemical interconversion between the red-absorbing (P(r)) and the far-red-absorbing (P(fr)) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The P(r)-to-P(fr) transition commences with a rapid Z-to-E photoisomerization at the C(15)=C(16) methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C(15)-H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted C=C stretch and N-H in-plane rocking modes within 500 fs, reveal that initial distortion of the C(15)=C(16) bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to P(r) in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C(15)-H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C(15)=C(16) Z-to-E isomerization occurs on the excited-state surface.

Dominant gain-of-function alleles of Arabidopsis phytochrome B were recently shown to confer light-independent, constitutive photomorphogenic (cop) phenotypes to transgenic plants (Su and Lagarias, 2007). In the present study, comparative transcription profiling experiments were performed to assess whether the pattern of gene expression regulated by these alleles accurately reflects the process of photomorphogenesis in wild-type Arabidopsis. Whole-genome transcription profiles of dark-grown phyAphyB seedlings expressing the Y(276)H mutant of phyB (YHB) revealed that YHB reprograms about 13% of the Arabidopsis transcriptome in a light-independent manner. The YHB-regulated transcriptome proved qualitatively similar to but quantitatively greater than those of wild-type seedlings grown under 15 or 50 mumol m(-2) m(-1) continuous red light (Rc). Among the 2977 genes statistically significant two-fold (SSTF) regulated by YHB in the absence of light include those encoding components of the photosynthetic apparatus, tetrapyrrole/pigment biosynthetic pathways, and early light-responsive signaling factors. Approximately 80% of genes SSTF regulated by Rc were also YHB-regulated. Expression of a notable subset of 346 YHB-regulated genes proved to be strongly attenuated by Rc, indicating compensating regulation by phyC-E and/or other Rc-dependent processes. Since the majority of these 346 genes are regulated by the circadian clock, these results suggest that phyA- and phyB-independent light signaling pathway(s) strongly influence clock output. Together with the unique plastid morphology of dark-grown YHB seedlings, these analyses indicate that the YHB mutant induces constitutive photomorphogenesis via faithful reconstruction of phyB signaling pathways in a light-independent fashion.

Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/ E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.

The photoreversibility of plant phytochromes enables continuous surveillance of the ambient light environment. Through expression of profluorescent, photoinsensitive Tyr-to-His mutant alleles of Arabidopsis thaliana phytochrome B (PHYB(Y276H)) and Arabidopsis phytochrome A (PHYA(Y242H)) in transgenic Arabidopsis plants, we demonstrate that photoconversion is not a prerequisite for phytochrome signaling. PHYB(Y276H)-expressing plants exhibit chromophore-dependent constitutive photomorphogenesis, light-independent phyB(Y276H) nuclear localization, constitutive activation of genes normally repressed in darkness, and light-insensitive seed germination. Fluence rate analyses of transgenic plants expressing PHYB(Y276H), PHYA(Y242H), and other Y(GAF) mutant alleles of PHYB demonstrate that a range of altered light-signaling activities are associated with mutation of this residue. We conclude that the universally conserved GAF domain Tyr residue, with which the bilin chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction by plant phytochromes.

The X-ray crystal structure of the substrate-free form of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) from the cyanobacterium Nostoc sp. PCC7120 has been solved at 2.5 A resolution. A comparative analysis of this structure with those recently reported for substrate-bound and substrate-free forms of PcyA from the cyanobacterium Synechocystis sp. PCC6803 (Hagiwara et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 27-32; Hagiwara et al. (2006) FEBS Lett. 580, 3823-3828) provides a compelling picture of substrate-induced changes in the PcyA enzyme and the chemical basis of PcyA's catalytic activity. On the basis of these structures and the biochemical analysis of site-directed mutants of Nostoc PcyA, including mutants reported in recent studies (Tu et al. (2006) J. Biol. Chem. 281, 3127-3136) as well as mutants described in this study, a revised mechanism for the PcyA-mediated four-electron reduction of biliverdin IXalpha to 3E/3Z-phycocyanobilin via enzyme-bound bilin radical intermediates is proposed. The mechanistic insight of these studies, along with homology modeling, have provided new insight into the catalytic mechanisms of other members of the ferredoxin-dependent bilin reductase family that are widespread in oxygenic photosynthetic organisms.

Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1Delta, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1Delta apoprotein (apoCph1Delta) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1Delta altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1Delta and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein.

Phycocyanobilin:ferredoxin oxidoreductase is a member of the ferredoxin-dependent bilin reductase family and catalyzes two vinyl reductions of biliverdin IXalpha to produce phycocyanobilin, the pigment precursor of both phytochrome and phycobiliprotein chromophores in cyanobacteria. Atypically for ferredoxin-dependent enzymes, phycocyanobilin:ferredoxin oxidoreductase mediates direct electron transfers from reduced ferredoxin to its tetrapyrrole substrate without metal ion or organic cofactors. We previously showed that bound bilin radical intermediates could be detected by low temperature electron paramagnetic resonance and absorption spectroscopies (Tu, S., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C. (2004) J. Am. Chem. Soc. 126, 8682-8693). On the basis of these studies, a mechanism involving sequential electron-coupled proton transfers to protonated bilin substrates buried within the phycocyanobilin:ferredoxin oxidoreductase protein scaffold was proposed. The present investigation was undertaken to identify catalytic residues in phycocyanobilin:ferredoxin oxidoreductase from the cyanobacterium Nostoc sp. PCC7120 through site-specific chemical modification and mutagenesis of candidate proton-donating residues. These studies identified conserved histidine and aspartate residues essential for the catalytic activity of phycocyanobilin:ferredoxin oxidoreductase. Spectroscopic evidence for the formation of stable enzyme-bound biliverdin radicals for the H85Q and D102N mutants support their role as a "coupled" proton-donating pair during the reduction of the biliverdin exovinyl group.

The phytochrome family of red/far-red photoreceptors has been optimized to support photochemical isomerization of a bound bilin chromophore, a process that triggers a conformational change and modulates biochemical output from the surrounding protein scaffold. Recent studies have established that the efficiency of this photochemical process is profoundly altered by mutation of a conserved tyrosine residue (Tyr176) within the bilin-binding GAF domain of the cyanobacterial phytochrome Cph1 [Fischer, A. J., and Lagarias, J. C. (2004) Harnessing phytochrome's glowing potential, Proc. Natl. Acad. Sci. U.S.A. 101, 17334-17339]. Here, we show that the equivalent mutation in plant phytochromes behaves similarly, indicating that the function of this tyrosine in the primary photochemical mechanism is conserved. Saturation mutagenesis of Tyr176 in Cph1 establishes that no other residue can support comparably efficient photoisomerization. The spectroscopic consequences of Tyr176 mutations also reveal that Tyr176 regulates the conversion of the porphyrin-like conformation of the bilin precursor to a more extended conformation. The porphyrin-binding ability of the Tyr176Arg mutant protein indicates that Tyr176 also regulates the ligand-binding specificity of apophytochrome. On the basis of the hydrogen-bonding ability of Tyr176 substitutions that support the nonphotochemical C15-Z,syn to C15-Z,anti interconversion, we propose that Tyr176 orients the carboxyl side chain of a conserved acidic residue to stabilize protonation of the bilin chromophore. A homology model of the GAF domain of Cph1 predicts a C5-Z,syn, C10-Z,syn, C15-Z,anti configuration for the chromophore and implicates Glu189 as the proposed acidic residue stabilizing the extended conformation, an interpretation consistent with site-directed mutagenesis of this conserved acidic residue.

Directed evolution of a cyanobacterial phytochrome was undertaken to elucidate the structural basis of its light sensory activity by remodeling the chemical environment of its linear tetrapyrrole prosthetic group. In addition to identifying a small region of the apoprotein critical for maintaining phytochrome's native spectroscopic properties, our studies revealed a tyrosine-to-histidine mutation that transformed phytochrome into an intensely red fluorescent biliprotein. This tyrosine is conserved in all members of the phytochrome superfamily, implicating direct participation in the primary photoprocess of phytochromes. Fluorescent phytochrome mutants also hold great promise to expand the present repertoire of genetically encoded fluorescent proteins into the near infrared.

Cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the four electron reduction of biliverdin IXalpha (BV) to phycocyanobilin, a key step in the biosynthesis of the linear tetrapyrrole (bilin) prosthetic groups of cyanobacterial phytochromes and the light-harvesting phycobiliproteins. Using an anaerobic assay protocol, optically detected bilin-protein intermediates, produced during the PcyA catalytic cycle, were shown to correlate well with the appearance and decay of an isotropic g approximately 2 EPR signal measured at low temperature. Absorption spectral simulations of biliverdin XIIIalpha reduction support a mechanism involving direct electron transfers from ferredoxin to protonated bilin:PcyA complexes.

The covalently bound phytochromobilin (PphiB) prosthetic group is required for the diverse photoregulatory activities of all members of the phytochrome family in vascular plants, whereas by contrast, green algal and cyanobacterial phytochromes use the more reduced linear tetrapyrrole pigment phycocyanobilin (PCB). To assess the functional consequence of the substitution of PphiB with PCB in plants, the phytochrome chromophore-deficient hy2 mutant of Arabidopsis was transformed with a constitutively expressed pcyA gene that encodes the cyanobacterial enzyme, PCB:ferredoxin oxidoreductase. Spectroscopic analyses of extracts from etiolated seedlings revealed that PcyA expression restored photoactive phytochrome to WT levels, albeit with blue-shifted absorption maxima, while also restoring light lability to phytochrome A. Photobiological measurements indicated that PcyA expression rescued phytochrome-mediated red high-irradiance responses, low-fluence red/far-red (FR) photoreversible responses, and very-low-fluence responses, thus confirming that PCB can functionally substitute for PphiB for these photoregulatory activities. Although PcyA expression failed to rescue phytochrome A-mediated FR high-irradiance responsivity to that of WT, our studies indicate that the FR high-irradiance response is fully functional in pcyA-expressing plants but shifted to shorter wavelengths, indicating that PCB can functionally complement this phytochrome-mediated response in vascular plants.

The recent discovery of large numbers of phytochrome photoreceptor genes in both photosynthetic and non-photosynthetic prokaryotes has led to efforts to understand their physiological roles in environmental acclimation. One receptor in this class, RcaE, is involved in controlling complementary chromatic adaptation, a process that regulates the transcription of operons encoding light-harvesting proteins in cyanobacteria. Although all previously identified phytochrome responses are maximally sensitive to red and far red light, complementary chromatic adaptation is unique in that it is responsive to green and red light. Here, we present biochemical and genetic evidence demonstrating that RcaE is a photoreceptor and that it requires the cysteine at position 198 to ligate an open chain tetrapyrrole covalently in a manner analogous to chromophore attachment in plant phytochromes. Furthermore, although the wild-type rcaE gene can rescue red and green light photoresponses of an rcaE null mutant, a gene in which the codon for cysteine 198 is converted to an alanine codon rescues the red light but not the green light response. Thus, RcaE is a photoreceptor that is required for both green and red light responsiveness during complementary chromatic adaptation and is the first identified phytochrome class sensor that is involved in sensing and responding to green and red light rather than red and far red light.

Previous studies have established that the expression of mammalian biliverdin IXalpha reductase (BVR) in transgenic tobacco (Nicotiana tabacum cv. Maryland Mammoth) resulted in the loss of photoregulatory activity of all phytochromes together with a pronounced chlorophyll deficiency. This study was undertaken to assess the contribution of BVR-mediated alteration of tetrapyrrole metabolism to the observed phenotypes of BVR transgenic plants. BVR expression in dark-grown plants led to the reduced accumulation of protochlorophyllide and transcripts for the two committed enzymes for 5-aminolevulinic acid (ALA) synthesis despite the marked increased capacity for synthesis of ALA. Together with the observation that Mg-porphyrin accumulation in dark-grown seedlings treated with an iron chelator was unaffected by BVR expression, these results indicate that BVR diverts tetrapyrrole metabolism toward heme synthesis while also reducing heme levels to de-repress ALA synthesis. By contrast with dark-grown seedlings, light-grown BVR plants showed a marked inhibition of ALA synthesis compared with wild-type plants - a result that was correlated with the disappearance of the CHL I subunit of Mg-chelatase and an increase in heme oxygenase protein levels. As transcript levels of all tetrapyrrole biosynthetic genes tested were not strongly affected by BVR expression, these results implicate misregulated tetrapyrrole metabolism to be a major mechanism for BVR-dependent inhibition of chlorophyll biosynthesis in light-grown plants.

In cyanobacteria, the biosynthesis of the phycobiliprotein and phytochrome chromophore precursor phycocyanobilin is catalyzed by the ferredoxin-dependent enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA), which mediates an atypical four-electron reduction of biliverdin IXalpha. Here we describe the expression, affinity purification, and biochemical characterization of recombinant PcyA from Anabaena sp. PCC 7120. A monomeric protein with a native M(r) of 30,400 +/- 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its substrate biliverdin IXalpha. The enzyme exhibits a strong preference for plant type [2Fe-2S] ferredoxins; however, flavodoxin can also serve as an electron donor. HPLC analyses establish that catalysis proceeds via the two electron-reduced intermediate 18(1),18(2)-dihydrobiliverdin, indicating that exovinyl reduction precedes A-ring (endovinyl) reduction. Substrate specificity studies indicate that the arrangement of the A- and D-ring substituents alters the positioning of the bilin substrate within the enzyme, profoundly influencing the course of catalysis. Based on these observations and the apparent lack of a metal or small molecule cofactor, a radical mechanism for biliverdin IXalpha reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.