Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.

PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis.

Crystal structure of phytochromobilin synthase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phytochrome.

Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.

Bilin-Dependent Photoacclimation in Chlamydomonas reinhardtii.

In land plants, linear tetrapyrrole (bilin)-based phytochrome photosensors optimize photosynthetic light capture by mediating massive reprogramming of gene expression. But, surprisingly, many green algal genomes lack phytochrome genes. Studies of the heme oxygenase mutant (hmox1) of the green alga Chlamydomonas reinhardtii suggest that bilin biosynthesis in plastids is essential for proper regulation of a nuclear gene network implicated in oxygen detoxification during dark-to-light transitions. hmox1 cannot grow photoautotrophically and photoacclimates poorly to increased illumination. We show that these phenotypes are due to reduced accumulation of photosystem I (PSI) reaction centers, the PSI electron acceptors 5'-monohydroxyphylloquinone and phylloquinone, and the loss of PSI and photosystem II antennae complexes during photoacclimation. The hmox1 mutant resembles chlorophyll biosynthesis mutants phenotypically, but can be rescued by exogenous biliverdin IXα, the bilin produced by HMOX1. This rescue is independent of photosynthesis and is strongly dependent on blue light. RNA-seq comparisons of hmox1, genetically complemented hmox1, and chemically rescued hmox1 reveal that tetrapyrrole biosynthesis and known photoreceptor and photosynthesis-related genes are not impacted in the hmox1 mutant at the transcript level. We propose that a bilin-based, blue-light-sensing system within plastids evolved together with a bilin-based retrograde signaling pathway to ensure that a robust photosynthetic apparatus is sustained in light-grown Chlamydomonas.

Function and distribution of bilin biosynthesis enzymes in photosynthetic organisms.

Bilins are open-chain tetrapyrrole molecules essential for light-harvesting and/or sensing in many photosynthetic organisms. While they serve as chromophores in phytochrome-mediated light-sensing in plants, they additionally function in light-harvesting in cyanobacteria, red algae and cryptomonads. Associated to phycobiliproteins a variety of bile pigments is responsible for the specific light-absorbance properties of the organisms enabling efficient photosynthesis under different light conditions. The initial step of bilin biosynthesis is the cleavage of heme by heme oxygenases (HO) to afford the first linear molecule biliverdin. This reaction is ubiquitously found also in non-photosynthetic organisms. Biliverdin is then further reduced by site specific reductases most of them belonging to the interesting family of ferredoxin-dependent bilin reductases (FDBRs)-a new family of radical oxidoreductases. In recent years much progress has been made in the field of heme oxygenases but even more in the widespread family of FDBRs, revealing novel biochemical FDBR activities, new crystal structures and new ecological aspects, including the discovery of bilin biosynthesis genes in wild marine phage populations. The aim of this review is to summarize and discuss the recent progress in this field and to highlight the new and remaining questions.

Biosynthesis of open-chain tetrapyrroles in Prochlorococcus marinus.

Members of the genus Prochlorococcus belong to the most abundant phytoplankton on earth. In contrast to other cyanobacteria, Prochlorococcus is characterized by divinyl-chlorophyll containing light-harvesting complexes and the lack of phycobilisomes. Despite the lack of phycobilisomes, all sequenced genomes of Prochlorococcus possess genes that putatively encode enzymes involved in the biosynthesis of open-chain tetrapyrrole molecules. Here, biochemical evidence is presented indicating that high-light- and low-light-adapted Prochlorococcus ecotypes possess genes encoding functional enzymes for the biosynthesis of open-chain tetrapyrrole molecules. Experiments on recombinant protein as well as through complementation studies of a cyanobacterial insertion mutant revealed the functionality of the bilin reductases investigated.

Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli.

By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors.

Agrobacterium phytochrome as an enzyme for the production of ZZE bilins.

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.

Green colouration of cocoons in Antheraea yamamai (Lepidoptera: Saturniidae): light-induced production of blue bilin in the larval haemolymph.

When the larvae of a saturniid silkmoth, Antheraea yamamai, are maintained under high intensity light (5000 lux), they produce green cocoons whereas the cocoons produced under light of low intensity (e.g., 50 lux) or in darkness are yellow. The green colour of the cocoon is due to the presence of a blue bilin pigment in combination with yellow pigment, and light stimulates the accumulation of blue bilin. In the present study, we show that two blue bilins, with similar characteristics to the sarpedobilin in the green cocoon, can be induced in larval haemolymph both in vivo and in vitro. In both conditions, the amount of these bilins increased with increasing intensity or duration of light exposure. Induction also occurred at 0 degrees C. In contrast, the chromophore of the constitutive biliprotein of the haemolymph did not change depending on light conditions. Size fractionation of the haemolymph indicates that the precursor of the blue bilins induced by light is bound to a protein with a molecular mass of 5000 Da or more. Thus, in these insects, the blue bilin responsible for green colouration is facultative under photochemical stimulation.

Recombinant holophytochrome in Escherichia coli.

We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXalpha which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies.

Gallbladder mucin and cholesterol and pigment gallstone formation in hamsters.

We measured gallbladder mucin production by hamsters fed diets lithogenic for either cholesterol or pigment gallstones. In hamsters on the cholesterol stone diet, gallbladder production of 3H-glucosamine-labeled mucin was elevated two- and seven-fold after 1 and 3 weeks, respectively. After 1 week cholesterol crystals were seen in a mucus gel on the gallbladder surface. In hamsters on the pigment stone diet, gallbladder mucin production was significantly elevated after 1 and 3 weeks. The first precipitation of pigment crystals was in mucus in bile or on the gallbladder surface. Black pigment stones grew by agglomeration of pigment crystals enmeshed in mucus. In conclusion, gallbladder mucin production is increased before cholesterol or pigment stone formation, and the earliest deposition of crystals is in mucus in bile or on the gallbladder surface.

Oral calcium promotes pigment gallstone formation.

Dietary calcium supplementation has been recommended for prevention of osteoporosis and has become a standard component of most "health food" diets. Biliary calcium has been recognized to play a central role in the formation of pigment gallstones. We have recently demonstrated that 5 days of oral calcium supplementation significantly increases biliary calcium in the prairie dog (K. D. Lillemoe, T. H. Magnuson, G. E. Peoples, et al., Gastroenterology 94: A563, 1988). We hypothesized, therefore, that long-term oral calcium supplementation would promote pigment gallstone formation. Sixteen adult male prairie dogs were maintained on a standard nonlithogenic diet. Eight animals received calcium supplementation (2.5 x control levels) in their water, while the remaining eight animals served as controls. After 8 weeks, cholecystectomy was performed, and the common bile duct was cannulated. Bile was examined microscopically and analyzed for ionized calcium, bilirubin, glycoprotein, and biliary lipids. The cholesterol saturation index (CSI) was calculated. Pigment stones and calcium bilirubinate sludge were present in all animals receiving calcium supplementation. Only one control animal had evidence of pigment stones (P less than 0.001). Biochemical analysis of gallbladder bile demonstrated a significant increase in total bilirubin and bilirubin monoglucuronide (P less than 0.01) as well as bile glycoprotein content (P less than 0.05) after oral calcium supplementation. Gallbladder bile ionized calcium was also increased although not significantly. These data suggest that oral calcium supplementation promotes gallbladder sludge and pigment gallstone formation in the prairie dog. This observation raises concern that oral calcium supplementation, especially in the older female population, may enhance gallstone formation.

A two-molecule mechanism of haem degradation.

Coupled oxidation of octaethylhaemin and phenylhydrazine hydrochloride with 16,16O2 and 18,18O2 produced octaethyl[16O]verdohaemochrome and octaethyl[18O]-verdohaemochrome respectively. Reactions of these products with 16,16O2 in the presence of phenylhydrazine hydrochloride yielded octaethyl[16O, 16O]biliverdin and octaethyl[18O, 16O]biliverdin. The same reactions with 18,18O2 yielded octaethyl[16O, 18O]biliverdin and octaethyl[18O, 18O]biliverdin. Accordingly, the two oxygen atoms of biliverdin are incorporated from different O2 molecules in separate reactions, namely the formation of verdohaemochrome and the conversion of verdohaemochrome into biliverdin. These reactions account for a "two-molecule mechanism' of biliverdin formation from haem with verdohaemochrome participating as an intermediate product.

Formation and disposition of newly synthesized heme in adult rat hepatocytes in primary culture.

Studies with the intact liver have suggested that newly synthesized heme exists transiently in a small pool before its incorporation into tissue heme proteins. The same or a closely related pool may regulate synthesis of heme and serve as the precursor of "early peak" bilirubin. To delineate this postulated pool by a direct approach, we have utilized primary cultures of adult rat hepatocytes. Cultures pulse-labeled with delta-amino[3H]levulinic acid at various time points were fractionated into 105,000 X g supernatant and pellet. Labeled heme appeared within 1 to 2 min in the cytosol fraction, followed by transfer to the pellet. The kinetics of heme formation and transfer and of labeled bilirubin production were analyzed by computer simulation utilizing the least squares method. The experimental findings conformed best to a four-compartment model that includes a second cytosolic heme compartment exchanging with the initially labeled compartment but not serving as a direct precursor of bilirubin. Calculation of apparent rate coefficients indicated that, in cultured hepatocytes, 20% of newly formed heme is converted directly to bile pigment, whereas 80% is utilized for formation of cellular heme proteins (64% in the pellet, 16% in the second cytosol compartment). This experimental approach has provided direct evidence for a rapidly formed cytosolic heme fraction which appears to be identical with the previously postulated regulatory or "unassigned" heme pool of the liver.

Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium.

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.

Mechanism of bile-pigment synthesis in algae. 18O incorporation into phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium.

The origin of the lactam oxygen atoms of phycocyanobilin from Cyanidium caldarium was studied using 18O labelling. By inhibiting photosynthesis, a high 18O enrichment was maintained in the gas phase and the resulting incorporation of label showed that the lactam oxygen atoms were derived from two oxygen molecules. Slow exchange of these oxygen atoms with water was demonstrated directly by using H218O.

Comparative aspects of bile pigment formation and excretion.

Breakdown of haem which is of key importance in most organisms, involves oxidative CO-evolving cleavage of the macrocyclic ring with formation of biliverdin-IX. In two major pathways established so far formation of biliverdin-IX alpha is followed by (a) biliary secretion or (b) reduction to bilirubin-IX alpha, formation of more hydrophilic derivatives (usually glycosidic conjugates) and biliary secretion. The scattered comparative information available indicates marked species variation with regard to the methin-bridge carbon atom removed from haem and the metabolic site of cleavage, the nature of bilirubin conjugates and the developmental sequence of maturation of enzyme activities and transport proteins involved in the chain of events leading from breakdown of haem to the excretion of the final end products.