Stereoselectivity of each of the three steps of the heme oxygenase reaction: hemin to meso-hydroxyhemin,meso-hydroxyhemin to verdoheme,and verdoheme to biliverdin

Heme oxygenase catalyzes the regiospecific oxidation of hemin to biliverdin IXalpha with concomitant liberation of CO and iron by three sequential monooxygenase reactions. The alpha-regioselectivity of heme oxygenase has been thought to result from the regioselective oxygenation of the heme alpha-meso position at the first step, which leads to the reaction pathway via meso-hydroxyheme IXalpha and verdoheme IXalpha intermediates. However, recent reports concerning heme oxygenase forming biliverdin isomers other than biliverdin IXalpha raise a question whether heme oxygenase can degrade meso-hydroxyhemin and isomers other than the alpha-isomers. In this paper, we investigated the stereoselectivity of each of the two reaction steps from meso-hydroxyhemin to verdoheme and verdoheme to biliverdin by using a truncated form of rat heme oxygenase-1 and the chemically synthesized four isomers of meso-hydroxyhemin and verdoheme. Heme oxygenase-1 converted all four isomers of meso-hydroxyhemin to the corresponding isomers of verdoheme. In contrast, only verdoheme IXalpha was converted to the corresponding biliverdin IXalpha. We conclude that the third step, but not the second, is stereoselective for the alpha-isomer substrate. The present findings on regioselectivities of the second and the third steps have been discussed on the basis of the oxygen activation mechanisms of these steps.     

Human heme oxygenase oxidation of 5- and 15-phenylhemes

Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.     

Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.     

O(2)- and H(2)O(2)-dependent verdoheme degradation by heme oxygenase: reaction mechanisms and potential physiological roles of the dual pathway degradation

Heme oxygenase (HO) catalyzes the catabolism of heme to biliverdin, CO, and a free iron through three successive oxygenation steps. The third oxygenation, oxidative degradation of verdoheme to biliverdin, has been the least understood step despite its importance in regulating HO activity. We have examined in detail the degradation of a synthetic verdoheme IXalpha complexed with rat HO-1. Our findings include: 1) HO degrades verdoheme through a dual pathway using either O(2) or H(2)O(2); 2) the verdoheme reactivity with O(2) is the lowest among the three O(2) reactions in the HO catalysis, and the newly found H(2)O(2) pathway is approximately 40-fold faster than the O(2)-dependent verdoheme degradation; 3) both reactions are initiated by the binding of O(2) or H(2)O(2) to allow the first direct observation of degradation intermediates of verdoheme; and 4) Asp(140) in HO-1 is critical for the verdoheme degradation regardless of the oxygen source. On the basis of these findings, we propose that the HO enzyme activates O(2) and H(2)O(2) on the verdoheme iron with the aid of a nearby water molecule linked with Asp(140). These mechanisms are similar to the well established mechanism of the first oxygenation, meso-hydroxylation of heme, and thus, HO can utilize a common architecture to promote the first and third oxygenation steps of the heme catabolism. In addition, our results infer the possible involvement of the H(2)O(2)-dependent verdoheme degradation in vivo, and potential roles of the dual pathway reaction of HO against oxidative stress are proposed.     

Structure of human biliverdin IXbeta reductase, an early fetal bilirubin IXbeta producing enzyme

Biliverdin IXbeta reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXbeta, 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 IXalpha 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 alpha/beta dinucleotide binding fold. The structures of ternary complexes with mesobiliverdin IValpha, biliverdin IXalpha, 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 IXalpha 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.     

Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex

Biliverdin reductase (BVR) catalyzes the last step in heme degradation by reducing the gamma-methene bridge of the open tetrapyrrole, biliverdin IXalpha, to bilirubin with the concomitant oxidation of a beta-nicotinamide adenine dinucleotide (NADH) or beta-nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. Bilirubin is the major bile pigment in mammals and has antioxidant and anticompliment activity. We have determined X-ray crystal structures of apo rat BVR and its complex with NADH at 1.2 A and 1.5 A resolution, respectively. In agreement with an independent structure determination of the apo-enzyme, BVR consists of an N-terminal dinucleotide-binding domain (Rossmann-fold) and a C-terminal domain that contains a six-stranded beta-sheet that is flanked on one face by several alpha-helices. The C-terminal and N-terminal domains interact extensively, forming the active site cleft at their interface. The cofactor complex structure reported here reveals that the cofactor nicotinamide ring extends into the active site cleft, where it is adjacent to conserved amino acid residues and, consistent with the known stereochemistry of the reaction catalyzed by BVR, the si face of the ring is accessible for hydride transfer. The only titratable side-chain that appears to be suitably positioned to function as a general acid in catalysis is Tyr97. This residue, however, is not essential for catalysis, since the Tyr97Phe mutant protein retains 50% activity. This finding suggests that the dominant role in catalysis may be performed by hydride transfer from the cofactor, a process that may be promoted by proximity of the invariant residues Glu96, Glu123, and Glu126, to the nicotinamide ring.     

Insights into phycoerythrobilin biosynthesis point toward metabolic channeling

Phycoerythrobilin is a linear tetrapyrrole molecule found in cyanobacteria, red algae, and cryptomonads. Together with other bilins such as phycocyanobilin it serves as a light-harvesting pigment in the photosynthetic light-harvesting structures of cyanobacteria called phycobilisomes. The biosynthesis of both pigments starts with the cleavage of heme by heme oxygenases to yield biliverdin IXalpha, which is further reduced at specific positions by ferredoxin-dependent bilin reductases (FDBRs), a new family of radical enzymes. The biosynthesis of phycoerythrobilin requires two subsequent two-electron reductions, each step being catalyzed by one FDBR. This is in contrast to the biosynthesis of phycocyanobilin, where the FDBR phycocyanobilin: ferredoxin oxidoreductase (PcyA) catalyzes a four-electron reduction. The first reaction in phycoerythrobilin biosynthesis is the reduction of the 15,16-double bond of biliverdin IXalpha by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated pi -electron system thereby blue-shifting the absorbance properties of the linear tetrapyrrole. The second FDBR, phycoerythrobilin:ferredoxin oxidoreductase (PebB), then reduces the A-ring 2,3,3(1),3(2)-diene structure of 15,16-dihydrobiliverdin to yield phycoerythrobilin. Both FDBRs from the limnic filamentous cyanobacterium Fremyella diplosiphon and the marine cyanobacterium Synechococcus sp. WH8020 were recombinantly produced in Escherichia coli and purified, and their enzymatic activities were determined. By using various natural bilins, the substrate specificity of each FDBR was established, revealing conformational preconditions for their unique specificity. Preparation of the semi-reduced intermediate, 15,16-dihydrobiliverdin, enabled us to perform steady state binding experiments indicating distinct spectroscopic and fluorescent properties of enzyme.bilin complexes. A combination of substrate/product binding analyses and gel permeation chromatography revealed evidence for metabolic channeling.     

The heme oxygenase(s)-phytochrome system of Pseudomonas aeruginosa

For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXbeta and IXdelta. The gene for a second putative heme oxygenase in P. aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome. Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P. aeruginosa. HPLC, (1)H, and (13)C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IXalpha. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other alpha-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.     

Comparison of HMOX1 expression and enzyme activity in blue-shelled chickens and brown-shelled chickens

Blue egg coloring is attributed to biliverdin derived from the oxidative degradation of heme through catalysis by heme oxygenase (HO). The pigment is secreted into the eggshell by the shell gland. There is uncertainty as to whether the pigment is synthesized in the shell gland or in other tissues. To investigate the site of pigment biosynthesis, the expression of heme oxygenase (decycling) 1 (HMOX1), a gene encoding HO, and HO activity in liver and spleen were compared between blue-shelled chickens (n = 12) and brown-shelled chickens (n = 12). There were no significant differences in HMOX1 expression and HO activity in these tissues between the two groups. Since the liver and spleen, two important sites outside the shell gland where heme is degraded into biliverdin, CO and Fe²⁺, did not differ in HO expression and activity we conclude that the pigment is most likely synthesized in the shell gland.     

A conserved histidine-aspartate pair is required for exovinyl reduction of biliverdin by a cyanobacterial phycocyanobilin:ferredoxin oxidoreductase

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.     

Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis

Phytochromobilin (PPhiB) is an open chain tetrapyrrole molecule that functions as the chromophore of light-sensing phytochromes in plants. Derived from heme, PPhiB is synthesized through an open chain tetrapyrrole intermediate, biliverdin IXalpha (BV), in the biosynthesis pathway. BV is subsequently reduced by the PPhiB synthase HY2 in plants. HY2 is a ferredoxin-dependent bilin reductase that catalyzes the reduction of the A-ring 2,3,3(1),3(2)-diene system to produce an ethylidene group for assembly with apophytochromes. In this study, we sought to determine the catalytic mechanism of HY2. Data from UV-visible and EPR spectroscopy showed that the HY2-catalyzed BV reaction proceeds via a transient radical intermediate. Site-directed mutagenesis showed several ionizable residues that are involved in the catalytic steps. Detailed analysis of these site-directed mutants highlighted a pair of aspartate residues central to proton donation and substrate positioning. A mechanistic prediction for the HY2 reaction is proposed. These results support the hypothesis that ferredoxin-dependent bilin reductases reduce BV through a radical mechanism, but their double bond specificity is decided by strategic placement of different proton-donating residues surrounding the bilin substrate in the active sites.     

Fungal heme oxygenases: Functional expression and characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans

Heme oxygenases convert heme to free iron, CO, and biliverdin. Saccharomyces cerevisiae and Candida albicans express putative heme oxygenases that are required for the acquisition of iron from heme, a critical process for fungal survival and virulence. The putative heme oxygenases Hmx1 and CaHmx1 from S. cerevisiae and C. albicans, respectively, minus the sequences coding for C-terminal membrane-binding domains, have been expressed in Escherichia coli. The C-terminal His-tagged, truncated enzymes are obtained as soluble, active proteins. Purified ferric Hmx1 and CaHmx1 have Soret absorption maxima at 404 and 410 nm, respectively. The apparent heme binding Kd values for Hmx1 and CaHmx1 are 0.34 +/- 0.09 microM and 1.0 +/- 0.2 microM, respectively. The resonance Raman spectra of Hmx1 reveal a heme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants, including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxin reductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin production by Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation by Hmx1 and CaHmx1 regiospecifically produces biliverdin IXalpha. Spectroscopic analysis of aerobic reactions with H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal heme oxygenases to be heterologously overexpressed and characterized. Their heme degradation activity is consistent with a role in iron acquisition.     

Degradation of heme in gram-negative bacteria: the product of the hemO gene of Neisseriae is a heme oxygenase

A full-length heme oxygenase gene from the gram-negative pathogen Neisseria meningitidis was cloned and expressed in Escherichia coli. Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the E. coli cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen Corynebacterium diphtheriae and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXalpha and carbon monoxide as the final products. Homologs of the hemO gene were identified and characterized in six commensal Neisseria isolates, Neisseria lactamica, Neisseria subflava, Neisseria flava, Neisseria polysacchareae, Neisseria kochii, and Neisseria cinerea. All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.     

Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied.

We cloned a cDNA for a Drosophila melanogaster homologue of mammalian heme oxygenase (HO) and constructed a bacterial expression system of a truncated, soluble form of D. melanogaster HO (DmDeltaHO). The purified DmDeltaHO degraded hemin to biliverdin, CO and iron in the presence of reducing systems such as NADPH/cytochrome P450 reductase and sodium ascorbate, although the reaction rate was slower than that of mammalian HOs. Some properties of DmHO, however, are quite different from other known HOs. Thus DmDeltaHO bound hemin stoichiometrically to form a hemin-enzyme complex like other HOs, but this complex did not show an absorption spectrum of hexa-coordinated heme protein. The absorption spectrum of the ferric complex was not influenced by changing the pH of the solution. Interestingly, an EPR study revealed that the iron of heme was not involved in binding heme to the enzyme. Hydrogen peroxide failed to convert it into verdoheme. A spectrum of the ferrous-CO form of verdoheme was not detected during the reaction from hemin under oxygen and CO. Degradation of hemin catalyzed by DmDeltaHO yielded three isomers of biliverdin, of which biliverdin IXalpha and two other isomers (IXbeta and IXdelta) accounted for 75% and 25%, respectively. Taken together, we conclude that, although DmHO acts as a real HO in D. melanogaster, its active-site structure is quite different from those of other known HOs.     

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.     

Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.     

Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

Heme oxidation in a chimeric protein of the alpha-selective Neisseriae meningitidis heme oxygenase with the distal helix of the delta-selective Pseudomonas aeruginosa

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.     

Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic

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.