Báo cáo khoa học: The use of synthetic linear tetrapyrroles to probe the verdin sites of human biliverdin-IXa reductase and human biliverdin-IXb reductase potx

The use of synthetic linear tetrapyrroles to probe the verdin sites of human biliverdin-IXa reductase and human biliverdin-IXb reductase Edward M. Franklin 1 , Seamus Browne 1 , Anne M. Horan 1 , Katsuhiko Inomata 2 , Mostafa A. S. Hammam 3 , Hideki Kinoshita 2 , Tilman Lamparter 4 , Georgia Golfis 1 and Timothy J. Mantle 1 1 School of Biochemistry and Immunology, Trinity College, Dublin, Ireland 2 Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan 3 Department of Chemistry, School of Science, Nagoya University, Aichi, Japan 4 Institut fu ¨ r Botanik I, Universita ¨ t Karlsruhe (TH), Germany Introduction The reduction of biliverdin-IXa to bilirubin-IX a catal- ysed by biliverdin-IXa reductase (BVR-A) comprises an ancient reaction that has been conserved through- out evolution from cyanobacteria to man [1,2]. Until recently, the pathway in mammals has been considered functionally as the catabolic elimination of excess haem. This view has been challenged by recent observations that haem oxygenase plays a major cytoprotective role [3], and there is clear evidence that bilirubin-IXa is a physiologically significant antioxi- dant [4]. Central to this model is the enzyme biliverdin reductase, which is responsible for the maintenance of Keywords Biliverdin, dimethyl ester, ditaurate, inhibitor, jaundice, tolerance Correspondence E. M. Franklin, School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland Fax: +353 1677 2400 Tel: +353 1896 1612 E-mail: efrankli@tcd.ie (Received 23 April 2009, revised 9 June 2009, accepted 11 June 2009) doi:10.1111/j.1742-4658.2009.07148.x Many vertebrate species express two enzymes that are capable of catalysing the reduction of various isomers of biliverdin. Biliverdin-IXa reductase (BVR-A) is most active with its physiological substrate biliverdin-IXa, but can also reduce the three other biliverdin isomers IXb,IXd and IXc. Bili- verdin-IXb reductase (BVR-B) catalyses the reduction of only the IXb,IXd and IXc isomers of biliverdin. Therefore, the activity of BVR-A can be measured using biliverdin-IXa as a specific substrate. We now show that the dimethyl esters of biliverdin-IXb and biliverdin-IXd are substrates for BVR-B, but not for BVR-A. This provides a useful method for specifically assaying the activity of both BVR-A and BVR-B in crude mixtures, using biliverdin-IXa for BVR-A and the dimethyl ester of either biliverdin-IXb or biliverdin-IXd for BVR-B. Human BVR-A has been suggested as a pharmacological target for neonatal jaundice. Because of the absence of a crystal structure with biliverdin bound to BVR-A, we have investigated indirect ways of examining tetrapyrrole binding. In the present study, we report that a number of sterically locked conformers of 18-ethylbiliverdin- IXa are substrates for human BVR-A, and discuss the implications for the biliverdin binding site. The oxidation of bilirubin-IXa ditaurate to biliver- din-IXa ditaurate is also described. We show that biliverdin-IXa ditaurate is a substrate for human BVR-A and discuss the possibility of using a com- peting substrate, which is reduced to a water soluble and excretable rubin, as a prototypic inhibitor of BVR-A. Abbreviations 18EtBV, 18-ethylbiliverdin-IXa; BVR-A, biliverdin-IXa reductase; BVR-B, biliverdin-IXb reductase; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone; hBVR-A, human biliverdin-IXa reductase; hBVR-B, human biliverdin-IXb reductase. FEBS Journal 276 (2009) 4405–4413 ª 2009 The Authors Journal compilation ª 2009 FEBS 4405 cytoprotective levels of bilirubin-IXa [5]. This antioxi- dant mechanism has been suggested to involve cycling between biliverdin-IXa and bilirubin-IXa [5]. Intrigu- ing evidence has been obtained from animal transplan- tation studies [6–8] showing that the administration of biliverdin-IXa is cytoprotective for the heart, colon and liver. In a notable study, Yamashita et al. [8] demonstrated that short-term treatment (3 weeks) with biliverdin-IXa is sufficient to induce tolerance in a recipient to the donor heart for 120 days. The intro- duction of an allogeneic third heart at 120 days was rejected, whereas the introduction of a third syngeneic heart was accepted, clearly indicating tolerance [8]. The exact mechanism for achieving tolerance remains obscure, although it is clear that components of the haem catabolic pathway play a major role in successful organ tranplants as well as in physiological cytopro- tection [4,8]. BVR-A has also been identified as a pharmacologi- cal target for treating neonatal jaundice [9]. Mammals maintain relatively high levels of circulating bilirubin- IXa. This is excreted in the bile, predominantly as a conjugate with glucuronic acid. At birth, the delayed expression of glucuronyltransferase UGT1A1 results in increased levels of unconjugated bilirubin in the liver, which reflux into plasma. This results in transiently elevated levels of bilirubin-IXa, which manifests as neonatal jaundice. If the albumin binding capacity is exceeded, bilirubin-IXa can partition into the brain and cause irreversible damage or death. The design of pharmacological inhibitors for BVR- A would not only combat the hyperbilirubinemia asso- ciated with neonatal jaundice, but also might prove significant during organ transplantation by elevating the concentration of cytosolic biliverdin-IXa. The pau- city of information on the biliverdin binding site has hampered the development of inhibitors of BVR-A. With this in mind, and in the absence of a crystal structure of the ternary complex of BVR-A with pyri- dine nucleotide and biliverdin, we set out to further probe the nature of the biliverdin binding pocket in a significant extension to our earlier studies [2]. In the present study, sterically-locked conformers of 18-ethyl- biliverdin-IXa (18EtBV) are shown to be substrates for human BVR-A (hBVR-A), and the implications for the nature of the binding of biliverdin-IXa to BVR-A are discussed. We also show that biliverdin ditaurate is a substrate for BVR-A and discuss the possibility of using a com- peting substrate such as this to reduce the levels of the lipophilic and potentially toxic bilirubin-IXa by pro- ducing bilirubin ditaurate, which is water soluble and readily excreted. Additionally, by studying various biliverdin-IX iso- mers, we present a method that specifically allows the assay of both BVR-A and biliverdin-IXb reductase (BVR-B) enzymes in crude preparations. Catalytically, the major difference identified to date between the two human enzymes is that BVR-B catalyses the reduction of biliverdin-IXb, biliverdin-IXd and biliverdin-IXc, but not biliverdin-IXa [2,10]. BVR-A prefers biliverdin- IXa as substrate, but can reduce all three other isomers [10]. Thus, although it is possible to specifically measure the activity of BVR-A with biliverdin-IXa, it is not pos- sible to specifically measure the activity of BVR-B with any of the free acids of biliverdin (IXa,IXb,IXc or IXd). We now show that BVR-B can reduce biliverdin- IXb, biliverdin-IXd and biliverdin-IXc as their dimethyl esters in clear distinction to BVR-A. This permits the specific assay of the activity of BVR-A and BVR-B in crude cytosols. Increasingly, straightforward assays are required that are specific for measuring the activity of BVR-A and BVR-B. Protein levels of BVR-A are signi- ficantly elevated in chronic hypoxia in the rat lung [11] and after focal cerebral ischaemia in mice [12]. In addi- tion, lipopolysaccharide has been reported to increase rat kidney BVR-A by a post-transcriptional mechanism [13] and thymidine induces BVR-A in human K562 ery- throleukemic cells [14]. Furthermore, both BVR-A and BVR-B have recently been reported to form adducts with environmental toxins [15] and endogenous ligands [16]. BVR-B is adducted at equimolar stoichiometry by electrophilic metabolites of 1-nitronapthalene [15] and BVR-A by 15-deoxy-D12,14-prostaglandin J2 [16]. It is unclear whether the adducted proteins retain activity. Measurement of both the RNA and protein level of both BVR-A and BVR-B is straightforward; however, to date, there has been no method of specifically assay- ing the activity of both enzymes in crude preparations with biliverdin substrates. Results Non-IXa biliverdin dimethyl esters as substrates for human BVR-B (hBVR-B) All three ‘non-IXa’ biliverdin dimethyl esters were reduced to the corresponding bilirubins by BVR-B. The spectra of the dimethyl ester of biliverdin-IXb in the presence and absence of 37 lm BSA revealed no significant change in the pigment spectrum (Fig. 1A). The addition of hBVR-B (18.5 lg) revealed a time- dependent decrease at 660 nm and an increase at 460 nm (Fig. 1B). Similar results were obtained with the dimethyl ester of biliverdin-IXd (data not shown). We did not have sufficient material for detailed studies Probing the verdin site of BVR-A and -B E. M. Franklin et al. 4406 FEBS Journal 276 (2009) 4405–4413 ª 2009 The Authors Journal compilation ª 2009 FEBS with the dimethyl ester of biliverdin-IXc. Initial rate kinetics show that the dimethyl esters of the IXb and IXd isomers all demonstrate substrate inhibition with apparent substrate inhibitory K i values in the lm range (data not shown). Probing the biliverdin binding pocket of BVR-A using synthetic biliverdin isomers The substrate specificity of hBVR-A was examined using a range of synthetic biliverdins locked in various conformations. These biliverdin derivatives have their C and D rings sterically locked by cycliz- ing with an additional two- or three-carbon chain [22] (and are termed 15Z-syn 18EtBV, 15Z-anti 18EtBV, 15E-syn 18EtBV and 15E-anti 18EtBV; Fig. 2). All of the fixed isomers were substrates for human BVR-A (Fig. 4). The spectra of all of the fixed isomers exhibited the characteristic immediate A B Fig. 1. Spectra of the hBVR-B catalysed reaction of the dimethyl ester of biliverdin-IXb. (A) Biliverdin-IXb dimethyl ester (7 l M)in 100 m M potassium phosphate (pH 7.5) containing 1% (v ⁄ v) metha- nol from the stock solution of ester (blue); addition of 37 l M BSA from a stock solution of 50 mgÆmL )1 produced the red trace and subsequent addition of 50 l M NADPH produced the green trace. After addition of enzyme, the reaction was allowed to go to completion (orange trace). (B) The BVR-B reaction was initiated by the addition of hBVR-B (18.5 lg) and the spectra recorded over a period of 20 min. Fig. 2. The structures of the open chain and locked tetrapyrroles. The structures shown are 15Z-syn 18EtBV, 15Z-anti 18EtBV, 15E-syn 18EtBV and 15E-anti 18EtBV. E. M. Franklin et al. Probing the verdin site of BVR-A and -B FEBS Journal 276 (2009) 4405–4413 ª 2009 The Authors Journal compilation ª 2009 FEBS 4407 red shift (for the ‘390’ and ‘660’ peaks) on the addi- tion of BSA and this was particularly marked for the 15E-syn 18EtBV isomer (Fig. 3C). On addition of human BVR-A (1.4 lg), all the locked isomers showed an increase in absorbance in the 460 nm region coupled with a decrease at 650 nm, which was consistent with reduction to the corresponding bilirubins (Fig. 4A–D). When the initial rate was measured (DA 460 ) at a range of concentrations of the various locked isomers, the characteristic substrate inhibition profile was evident (Fig. 5A–C). Although we have not isolated or characterized the putative bilirubin products from the locked isomer reactions, the spectral changes that we observed are entirely consistent with enzyme-catalysed formation of the corresponding bilirubin. The possibility of a C-10 adduct (other than hydride from NADPH) is highly unlikely. Thiol compounds will adduct to biliverdin to give a yellow product; however, because neither NADPH nor BVR-A alone were capable of initiating an increase in A 460 , adduct formation of this type can be ruled out. The fact that the linear increase with time at A 460 required NADPH, BVR-A and the locked biliverdin isomer is entirely consistent with enzyme-catalysed hydride transfer from NADPH to the various locked isomers. The demonstration that locked isomers of biliverdin can be substrates for BVR-A has been reported previously for the rat liver enzyme [26], although the physiologically less rele- vant IXc isomer and its locked variants were used in that study. Competitive substrates as inhibitors As an alternative strategy to biliverdin reductase inhibitors for human BVR-A, we were interested in the possibility that competing substrates may provide a means of inhibiting BVR-A in vivo. In the case of the natural isomer, biliverdin-IXa, the reduction product bilirubin-IXa is lipophilic and requires sub- sequent conjugation with glucuronic acid for efficient elimination. However, replacing the propionate side chains of biliverdin-IXa by sulfonate analogues should produce a water soluble bilirubin that would not need conjugation prior to excretion. Figure 6 shows the initial rate kinetics for human BVR-A with biliverdin-IXa ditaurate. The substrate inhibi- tion with this sulfonate is less potent than that seen with biliverdin-IXa. Biliverdin-IXa sulfonate is also a good substrate (data not shown) and, similar to bili- verdin-IXa ditaurate, the substrate inhibition is not as potent as that seen with biliverdin-IXa. A B C D Fig. 3. Spectra of the locked conformers of 18EtBV. The spectra of the fixed isomers are shown in (A) 15Z-syn 18EtBV, (B) 15Z-anti 18EtBV, (C) 15E-syn 18EtBV and (D) 15E-anti 18EtBV in 100 m M Tris ⁄ HCl (pH 8). The immediate effect of the addition of 37 lM BSA is also shown. Probing the verdin site of BVR-A and -B E. M. Franklin et al. 4408 FEBS Journal 276 (2009) 4405–4413 ª 2009 The Authors Journal compilation ª 2009 FEBS Discussion The induction of haem oxygenase has been implicated in a number of models for cytoprotection, and subse- quently, there has been an increasing interest in the enzymes of tetrapyrrole metabolism. The two down- stream products of haem oxygenase activity, the linear tetrapyrroles biliverdin-IXa and bilirubin-IXa, have been identified as agents that improve the efficacy of organ transplantation [8,27]. BVR-A has been identi- fied as a pharmacological target for treating neonatal jaundice [9]. Increasingly, there is a requirement for straightforward assays that are specific for measuring the activity of BVR-A and BVR-B in crude prepara- tions. Until now, no method of specifically assaying the activity of both enzymes in crude preparations with biliverdin substrates has been proposed. As described previously, the free acid of biliverdin- IXa is not a substrate for hBVR-B [2], and the dimethyl ester behaves similarly. The bridging propio- nate side chains of biliverdin-IXa preclude access to the substrate pocket of hBVR-B in a productive mode and the tetrapyrrole rotates 90° compared to meso- biliverdin-IVa [28]. The rotated configuration observed with biliverdin-IXa bound to BVR-B is not consistent with hydride transfer [28]. It is likely that a similar binding orientation is adopted when the dimethyl ester of biliverdin-IXa binds to hBVR-B. Biliverdin-IXa dimethyl ester clearly does bind because it inhibits hBVR-B activity with an apparent K i in the micro- molar range. The dimethyl ester of biliverdin-IXa is not a substrate for BVR-A (data not shown), which is in agreement with previous findings [29]. Clearly, the activity of BVR-A can be measured specifically using biliverdin-IXa as a substrate [2] and, in the present study, we report that it is now possible to specifically measure the activity of BVR-B using the dimethyl ester of biliverdin-IXb or biliverdin-IXd. It is not possible to use the free acids of the IXb,IXd and IXc biliver- din isomers as specific substrates because they are reduced by BVR-A as well as BVR-B [10]. The substrate specificity of hBVR-A was also analy- sed using a range of synthetic biliverdins locked in various conformations to gain further insight on the biliverdin binding site of human BVR-A. It is highly A B C D Fig. 4. Spectra of the BVR-A catalysed reactions of the locked con- formers of biliverdin-IXa. The spectra of the fixed isomers are shown in (A) 15Z-syn 18EtBV, (B) 15Z-anti 18EtBV, (C) 15E-syn 18EtBV and (D) 15E-anti 18EtBV in 100 m M Tris ⁄ HCl (pH 8). Zero time has 120 l M NADPH added and the subsequent scans, over 10 and 12 min (as indicated), show the hBVR-A dependent reduction of the pigment and the associated oxidation of NADPH. E. M. Franklin et al. Probing the verdin site of BVR-A and -B FEBS Journal 276 (2009) 4405–4413 ª 2009 The Authors Journal compilation ª 2009 FEBS 4409 likely that BVR-A and BVR-B follow the same reac- tion mechanism with hydride transfer to a carbocation intermediate [30]. The angle of attack is likely to be the same such that the reaction centre for both BVR-B and BVR-A is likely to involve the B-face hydrogen atom attached to C4 of the nicotinamide ring and the two bridging pyrroles (rings B and C) with the linking C10 methene bridge as the electrophilic centre. Over- lapping conformationally stable forms of the sterically- locked conformers of 18EtBV, where the two C-10 bridging pyrroles (B and C) are ‘fixed’, reveals that the outer pyrrole rings (A and D) can adopt a variety of conformations in the verdin binding site of BVR-A. There are two models that require consideration to define biliverdin binding in light of the present obser- vations. The first model assumes that all four pyrroles are bound in the active site, which must be able to accommodate various conformations of the tetrapyr- role. If we assume a helical ‘lock washer’ conformation for the ‘nonlocked’ physiological substrate biliverdin- IXa, then, although the B and C rings may deviate slightly from planarity, the deviation for the outer A and D rings is sufficient to suggest that they may inter- act with the ‘roof’ and ‘floor’ of a hypothetical ‘verdin binding site’, with the nicotinamide ring forming part of the floor. If the bound conformation is helical, there would be a significant distance between the ‘roof’ and the ‘floor’, allowing a variety of conformational states to be accommodated, as appears to be the case for the locked isomers shown to be substrates in the present study. Such a model would also allow the binding of both P- and M-helical configurations described by Hayes & Mantle [31] for the wild-type and Y102A A B C Fig. 5. Initial rate kinetics of the BVR-A catalysed reactions of the locked conformers of biliverdin-IXa. The initial rate kinetics of the fixed isomers are shown in (A) 15Z-syn 18EtBV, (B) 15Z-anti 18EtBV, (C) 15E-anti 18EtBV in 100 m M Tris ⁄ HCl (pH 8) with 120 l M NADPH. The initial rates were measured by recording DA 460 at the range of concentrations indicated for the various locked isomers. The data are fitted to partial substrate inhibition [25]. The limited availability of the locked isomers curtailed the range of substrate concentrations studied. Fig. 6. Initial rate kinetics of BVR-A with biliverdin-IXa ditaurate. The reaction was carried out at 30 °C and was initiated by the addi- tion of enzyme. Probing the verdin site of BVR-A and -B E. M. Franklin et al. 4410 FEBS Journal 276 (2009) 4405–4413 ª 2009 The Authors Journal compilation ª 2009 FEBS mutant forms, respectively, of the cyanobacterial enzyme BVR-A from Synechocystis PCC6803. The second model requires that only the central B and C pyrroles are bound with the two outer pyrroles in solvent and therefore not constrained. This would allow the various locked conformers to be substrates but would not be consistent with the CD spectra dem- onstrating that helical conformations bind to human (E. Franklin and J. M. Hayes, unpublished work) and cyanobacterial [31] forms of BVR-A. The second model is clearly applicable for BVR-B, where the inner pyrroles are in good density [28], although the outer pyrroles show low occupancy and are modelled in the solvent. The second model does not allow protein binding to stabilize either P- or M-helical forms, which we clearly see in the case of both human and cyano- bacterial forms of BVR-A. Clearly, we require the crystal structures of ternary complexes with biliverdin-IX a to define the biliverdin binding site. It is likely that conformational changes may be needed to facilitate biliverdin binding once the enzyme-pyridine nucleotide complex has formed. This information will allow the development of potential BVR-A inhibitors. In this regard, we have shown that biliverdin-IXa sulfonate is a substrate for human BVR-A, although its synthesis is not straightforward. The rubin product is water soluble and readily excreted, whereas bilirubin-IXa requires conjugation with glucuronic acid prior to excretion. The concept of a competing substrate as a BVR-A inhibitor, where the product is readily excretable, is of some interest. In the present study, we have also shown that the synthe- sis of biliverdin ditaurate is relatively straighforward and that it is a substrate for human BVR-A. Our preli- minary investigations indicate that an in vivo evalua- tion of this compound is warranted to determine whether it can competitively reduce the levels of the potentially toxic bilirubin-Ixa, whereas the bilirubin ditaurate is predicted to retain the ability to be elimi- nated efficiently from circulation. The supplementation of competitive substrates that are reduced to a soluble bilirubin product might also boost the degree of physi- ological cytoprotection afforded by the cycling of bilirubin described by Baranano et al. [5]. Experimental procedures Preparation of biliverdin-IXd dimethyl ester and biliverdin-IXb dimethyl ester The dimethyl esters of biliverdin-IXb, biliverdin-IXc and biliverdin-IXd were synthesized by coupled oxidation of haem, producing the linear free acids. Esterification of the resulting free acids with BF 3 ⁄ MeOH and separation of all three biliverdin dimethyl esters by TLC was performed as described [17]. The band corresponding to the various dimethyl esters was scraped off, and the pigment extracted into acetone. Isomers with the greatest degree of separation on TLC were used in the present study. The IXd dimethyl ester runs as a distinctly separate band, whereas the upper- most fraction of the IXb dimethyl ester band was isolatable in a homogenous form. The NMR spectra of the dimethyl ester of the IXd and IXb isomers were in accordance with a previous study [17], and the absorption spectra in methanol were as reported previously [18]. The isomers also showed the red shift and doubling of extinction coefficient on the addition of HCl [18]. The dimethyl ester of biliverdin-IXa was synthesized by oxidation of the free acid of bilirubin- IXa to biliverdin-IXa using 2,3-dichloro-5,6-dicyanobenzo- quinone (DDQ) [19] and subsequent esterification with BF 3 ⁄ MeOH [17]. The synthesis of the sterically locked bilins has been described previously [20,21] and the recorded absorbance spectra are reported [22]. Biliverdin ditaurate was synthesized by oxidation of bilirubin-IXa ditaurate using DDQ. Briefly, bilirubin-IXa ditaurate (53 mg) was dissolved in 20 mL of sterile distilled water in a round bottomed flask, to which 30 mg of DDQ was added and mixed. The solution was allowed to react for 5 min and then silica gel (3 g) was added to the flask. The biliverdin ditaurate was dried onto silica gel by rotary evap- oration. A silica gel 60G column of 20 mL bed volume was equilibrated in ethyl acetate (100 mL). The dried biliverdin ditaurate ⁄ silica gel material was loaded onto the column. The column was washed with 300 mL of ethyl acetate to remove the reduced quinone. The column was then washed with 150 mL of ethyl acetate ⁄ methanol (4 : 1, v ⁄ v) to elute the unreacted quinone and bilirubin ditaurate. Finally, the biliverdin ditaurate was eluted with 100% methanol and dried to a powder by rotary evaporation. This material was homogenous by TLC and the NMR spectrum of the final product was consistent with oxidation at C10. The extinc- tion coefficient at 660 nm at pH 6.8 is 11.3 mm )1 Æcm )1 . Biliverdin-IXa sulfonate was a generous gift from Professor David Lightner (University of Reno, NV, USA). Enzyme assays Recombinant hBVR-A and hBVR-B were purified and assayed as described previously [2]. The biliverdin dimethyl esters were solubilized in methanol and additions from this stock solution were made to the assay mix. The final con- centration of methanol in the assay mix never exceeded 1%, which was shown not to affect enzyme activity. The assay mix also contained 37 lm BSA to aid solubilization of the dimethyl ester. The addition of BSA lowers the free concentration of the various verdins and their dimethyl esters. For this reason, we have not reported the K i or K m values, although we show the data plotted against the total E. M. Franklin et al. Probing the verdin site of BVR-A and -B FEBS Journal 276 (2009) 4405–4413 ª 2009 The Authors Journal compilation ª 2009 FEBS 4411 verdin concentration for comparative purposes with the lit- erature. This issue has been discussed previously [23], along with kinetic studies on BVR-A conducted in the absence of biliverdin-binding proteins [24] and the difficulties in cor- recting for biliverdin binding when it is known to occur [25]. When sterically-locked conformers of 18EtBV were used as substrates, the assay mix contained 120 lm NADPH in 100 mm Tris ⁄ HCl (pH 8). Spectra of the vari- ous locked bilins were measured in 100 mm Tris ⁄ HCl (pH 8) using a Cary 300 UV-Vis spectrophotometer (Varian Inc., Palo Alto, CA, USA). The effect of 37 lm BSA and also the subsequent addition of 120 lm NADPH on the spectra were recorded. BVR-A (1.4 lg) was then added, and spectra were recorded at intervals over a period of 20 min. Acknowledgement This work was supported by a grant from Science Foundation Ireland. References 1 Schluchter WM & Glazer AN (1997) Characterization of cyanobacterial biliverdin reductase. 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