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Clark J Lagarias - One of the best experts on this subject based on the ideXlab platform.
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Biliverdin amides reveal roles for propionate side chains in bilin reductase recognition and in holophytochrome assembly and photoconversion
Biochemistry, 2010Co-Authors: Lixia Shang, Nathan C Rockwell, Shelley S Martin, Clark J LagariasAbstract: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 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 side chains of Biliverdin IXα 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 Pr dark states of these biliprotein photos...
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structure of the Biliverdin radical intermediate in phycocyanobilin ferredoxin oxidoreductase identified by high field epr and dft
Journal of the American Chemical Society, 2009Co-Authors: Stefan Stoll, Clark J Lagarias, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C Kohler, Andrew Ozarowski, Andrew J Fisher, David R BrittAbstract: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).
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structure of the Biliverdin radical intermediate in phycocyanobilin ferredoxin oxidoreductase identified by high field epr and dft
Journal of the American Chemical Society, 2009Co-Authors: Stefan Stoll, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C Kohler, Andrew Ozarowski, Andrew J Fisher, Clark J LagariasAbstract:The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two-step four-electron reduction of Biliverdin IXα 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 s...
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Biliverdin reduction by cyanobacterial phycocyanobilin ferredoxin oxidoreductase pcya proceeds via linear tetrapyrrole radical intermediates
Journal of the American Chemical Society, 2004Co-Authors: Shihlong Tu, Alexander Gunn, Michael D Toney, David R Britt, Clark J LagariasAbstract:Cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the four electron reduction of Biliverdin IXα (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 ≈ 2 EPR signal measured at low temperature. Absorption spectral simulations of Biliverdin XIIIα reduction support a mechanism involving direct electron transfers from ferredoxin to protonated bilin:PcyA complexes.
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phycocyanobilin ferredoxin oxidoreductase ofanabaenasp pcc 7120 biochemical and spectroscopic characterization
Journal of Biological Chemistry, 2003Co-Authors: Nicole Frankenberg, Clark J LagariasAbstract:Abstract 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 IXα. 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 IXα. 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 181,182-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 IXα reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.
Timothy J. Mantle - One of the best experts on this subject based on the ideXlab platform.
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In silico and crystallographic studies identify key structural features of Biliverdin IXβ reductase inhibitors having nanomolar potency.
The Journal of biological chemistry, 2018Co-Authors: Natasha M. Nesbitt, Xiliang Zheng, José A. Manso, Wan-yi Yen, Lisa E. Malone, Jorge Ripoll-rozada, Pedro Pereira, Timothy J. Mantle, Jin WangAbstract:Heme cytotoxicity is minimized by a two-step catabolic reaction that generates Biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant Biliverdin reductases (IXα (BLVRA) and IXβ (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP+-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of Biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.
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the use of synthetic linear tetrapyrroles to probe the verdin sites of human Biliverdin ixα reductase and human Biliverdin ixβ reductase
FEBS Journal, 2009Co-Authors: Edward Franklin, Tilman Lamparter, Katsuhiko Inomata, Seamus Browne, Anne M Horan, Mostafa A S Hammam, Hideki Kinoshita, Georgia Golfis, Timothy J. MantleAbstract:Many vertebrate species express two enzymes that are capable of catalysing the reduction of various isomers of Biliverdin. Biliverdin-IXα reductase (BVR-A) is most active with its physiological substrate Biliverdin-IXα, but can also reduce the three other Biliverdin isomers IXβ, IXδ and IXγ. Biliverdin-IXβ reductase (BVR-B) catalyses the reduction of only the IXβ, IXδ and IXγ isomers of Biliverdin. Therefore, the activity of BVR-A can be measured using Biliverdin-IXα as a specific substrate. We now show that the dimethyl esters of Biliverdin-IXβ and Biliverdin-IXδ 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-IXα for BVR-A and the dimethyl ester of either Biliverdin-IXβ or Biliverdin-IXδ 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-IXα are substrates for human BVR-A, and discuss the implications for the Biliverdin binding site. The oxidation of bilirubin-IXα ditaurate to Biliverdin-IXα ditaurate is also described. We show that Biliverdin-IXα ditaurate is a substrate for human BVR-A and discuss the possibility of using a competing substrate, which is reduced to a water soluble and excretable rubin, as a prototypic inhibitor of BVR-A.
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computational and experimental studies on the catalytic mechanism of Biliverdin ixβ reductase
Biochemical Journal, 2008Co-Authors: Liam J Smith, Seamus Browne, Adrian J Mulholland, Timothy J. MantleAbstract:BVR-B (Biliverdin-IXbeta reductase) also known as FR (flavin reductase) is a promiscuous enzyme catalysing the pyridine-nucleotide-dependent reduction of a variety of flavins, Biliverdins, PQQ (pyrroloquinoline quinone) and ferric ion. Mechanistically it is a good model for BVR-A (Biliverdin-IXalpha reductase), a potential pharmacological target for neonatal jaundice and also a potential target for adjunct therapy to maintain protective levels of Biliverdin-IXalpha during organ transplantation. In a commentary on the structure of BVR-B it was noted that one outstanding issue remained: whether the mechanism was a concerted hydride transfer followed by protonation of a pyrrolic anion or protonation of the pyrrole followed by hydride transfer. In the present study we have attempted to address this question using QM/MM (quantum mechanics/molecular mechanics) calculations. QM/MM potential energy surfaces show that the lowest energy pathway proceeds with a positively charged pyrrole intermediate via two transition states. These initial calculations were performed with His(153) as the source of the proton. However site-directed mutagenesis studies with both the H153A and the H153N mutant reveal that His(153) is not required for catalytic activity. We have repeated the calculation with a solvent hydroxonium donor and obtain a similar energy landscape indicating that protonation of the pyrrole is the most likely first step followed by hydride transfer and that the required proton may come from bulk solvent. The implications of the present study for the design of inhibitors of BVR-A are discussed.
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structure of human Biliverdin ixβ reductase an early fetal bilirubin ixβ producing enzyme
Nature Structural & Molecular Biology, 2001Co-Authors: Pedro Pereira, Timothy J. Mantle, Orla Cunningham, Sandra Macedoribeiro, Antonio Parraga, Rosa Perezluque, Kevin J Darcy, Miquel CollAbstract:Biliverdin IXβ reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXβ, the major heme catabolite during early fetal development. BVR-B displays a preference for Biliverdin isomers without propionates straddling the C10 position, in contrast to Biliverdin IXα reductase (BVR-A), the major form of BVR in adult human liver. In addition to its tetrapyrrole clearance role in the fetus, BVR-B has flavin and ferric reductase activities in the adult. We have solved the structure of human BVR-B in complex with NADP+ at 1.15 A resolution. Human BVR-B is a monomer displaying an α/β dinucleotide binding fold. The structures of ternary complexes with mesoBiliverdin IVα, Biliverdin IXα, FMN and lumichrome show that human BVR-B has a single substrate binding site, to which substrates and inhibitors bind primarily through hydrophobic interactions, explaining its broad specificity. The reducible atom of both Biliverdin and flavin substrates lies above the reactive C4 of the cofactor, an appropriate position for direct hydride transfer. BVR-B discriminates against the Biliverdin IXα isomer through steric hindrance at the bilatriene side chain binding pockets. The structure also explains the enzyme's preference for NADP(H) and its B-face stereospecificity.
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studies on the specificity of the tetrapyrrole substrate for human Biliverdin ixα reductase and Biliverdin ixβ reductase structure activity relationships define models for both active sites
Journal of Biological Chemistry, 2000Co-Authors: Orla Cunningham, Aisling Dunne, Portia Sabido, David A Lightner, Timothy J. MantleAbstract:Abstract A comparison of the initial rate kinetics for human Biliverdin-IXα reductase and Biliverdin-IXβ reductase with a series of synthetic Biliverdins with propionate side chains “moving” from a bridging position across the central methene bridge (α isomers) to a “γ-configuration” reveals characteristic behavior that allows us to propose distinct models for the two active sites. For human Biliverdin-IXα reductase, as previously discussed for the rat and ox enzymes, it appears that at least one “bridging propionate” is necessary for optimal binding and catalytic activity, whereas two are preferred. All other configurations studied were substrates for human Biliverdin-IXα reductase, albeit poor ones. In the case of mesoBiliverdin-XIIIα, extending the propionate side chains to hexanoate resulted in a significant loss of activity, whereas the butyrate derivative retained high activity. For human Biliverdin-IXα reductase, we suggest that a pair of positively charged side chains play a key role in optimally binding the IXα isomers. In the case of human Biliverdin-IXβ reductase, the enzyme cannot tolerate even one propionate in the bridging position, suggesting that two negatively charged residues on the enzyme surface may preclude productive binding in this case. The flavin reductase activity of Biliverdin-IXβ reductase is potently inhibited by mesoBiliverdin-XIIIα and protohemin, which is consistent with the hypothesis that the tetrapyrrole and flavin substrate bind at a common site.
David R Britt - One of the best experts on this subject based on the ideXlab platform.
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structure of the Biliverdin radical intermediate in phycocyanobilin ferredoxin oxidoreductase identified by high field epr and dft
Journal of the American Chemical Society, 2009Co-Authors: Stefan Stoll, Clark J Lagarias, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C Kohler, Andrew Ozarowski, Andrew J Fisher, David R BrittAbstract: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).
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Biliverdin reduction by cyanobacterial phycocyanobilin ferredoxin oxidoreductase pcya proceeds via linear tetrapyrrole radical intermediates
Journal of the American Chemical Society, 2004Co-Authors: Shihlong Tu, Alexander Gunn, Michael D Toney, David R Britt, Clark J LagariasAbstract:Cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the four electron reduction of Biliverdin IXα (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 ≈ 2 EPR signal measured at low temperature. Absorption spectral simulations of Biliverdin XIIIα reduction support a mechanism involving direct electron transfers from ferredoxin to protonated bilin:PcyA complexes.
Amanda C Kohler - One of the best experts on this subject based on the ideXlab platform.
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structure of the Biliverdin radical intermediate in phycocyanobilin ferredoxin oxidoreductase identified by high field epr and dft
Journal of the American Chemical Society, 2009Co-Authors: Stefan Stoll, Clark J Lagarias, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C Kohler, Andrew Ozarowski, Andrew J Fisher, David R BrittAbstract: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).
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structure of the Biliverdin radical intermediate in phycocyanobilin ferredoxin oxidoreductase identified by high field epr and dft
Journal of the American Chemical Society, 2009Co-Authors: Stefan Stoll, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C Kohler, Andrew Ozarowski, Andrew J Fisher, Clark J LagariasAbstract:The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two-step four-electron reduction of Biliverdin IXα 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 s...
Marcin Brynda - One of the best experts on this subject based on the ideXlab platform.
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structure of the Biliverdin radical intermediate in phycocyanobilin ferredoxin oxidoreductase identified by high field epr and dft
Journal of the American Chemical Society, 2009Co-Authors: Stefan Stoll, Clark J Lagarias, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C Kohler, Andrew Ozarowski, Andrew J Fisher, David R BrittAbstract: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).
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structure of the Biliverdin radical intermediate in phycocyanobilin ferredoxin oxidoreductase identified by high field epr and dft
Journal of the American Chemical Society, 2009Co-Authors: Stefan Stoll, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C Kohler, Andrew Ozarowski, Andrew J Fisher, Clark J LagariasAbstract:The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two-step four-electron reduction of Biliverdin IXα 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 s...