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.     

Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis

The HY1 gene of Arabidopsis encodes a plastid heme oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXalpha from heme with the concomitant production of carbon monoxide. Heme oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme oxygenases differ substantially.     

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.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization.

A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE. The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous-CO and ferrous-O2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain, and that the two domains are folded almost independently of each other. An NADPH-cytochrome-P-450 reductase system composed of NADPH and detergent-solubilized NADPH-cytochrome-P-450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.     

The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase

The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.     

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.     

Crystallization and preliminary X-ray diffraction analysis of a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O is a 24-kDa soluble bacterial heme degradation enzyme found in the pathogen Corynebacterium diphtheriae, the causative agent of diphtheria. Similar to the mammalian heme oxygenase, it binds hemin stoichiometrically and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Iron is an essential nutrient for bacteria and especially important for pathogenesis. Here we report the first crystallization and preliminary crystallographic study of the heme-Hmu O complex formed from hemin and a recombinant Hmu O, which was expressed in Escherichia coli from a synthetic gene based on the putative hmu O gene sequence. Crystals of the heme-Hmu O complex were obtained by the sitting drop vapor diffusion method using a precipitant solution containing 18% (w/v) PEG 8000 and 0.2 M calcium acetate in 0.1 M sodium cacodylate (pH 6.5). Using synchrotron radiation, the heme-Hmu O crystal diffracted to 2.8 A resolution. It belongs to the monoclinic space group C2, with unit cell parameters a = 123.18 A, b = 44.51 A, c = 92.10 A, and beta = 123.3 degrees. Assuming one molecule of the heme-Hmu O complex per asymmetric unit, the calculated value of Vm is 2.89 A3/Da.     

Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin‐dependent heme oxygenase from Synechocystis sp. PCC 6803

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXα, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IXα. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin-dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IXα by spectrophotometry and reverse-phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100 000 g. Expression of HO1 increased several-fold during incubation of the cells for 72 h in iron-deficient medium.     

Heme catabolism in cultured hepatocytes: evidence that heme oxygenase is the predominant pathway and that a proportion of synthesized heme is converted rapidly to biliverdin

Heme oxygenase has been considered to be involved in the predominant pathway of heme degradation in vivo. However, alternative pathways involving cytochrome P-450 reductase, and lipid peroxidation, have previously been demonstrated in vitro, and studies with cultured rat hepatocytes were interpreted to show a majority of endogenous hepatic heme breakdown by non-heme oxygenase pathways. To clarify the pathway of heme breakdown in hepatocytes and the role of heme oxygenase in this process, cultured hepatocytes were pre-labelled with 5-[5-14C]aminolevulinate [( 14C]ALA). Radioactivity in heme, carbon monoxide, and bile pigments was measured for 8-24 h after the removal of [14C]ALA. In cultured chick embryo hepatocytes, which lack biliverdin reductase, the rate of production of biliverdin IXa was closely similar to the rate of catabolism of exogenous heme and radioactivity in carbon monoxide and biliverdin IXa was similar to the loss of radioactivity from endogenous heme. These results support the conclusion that heme breakdown occurred predominantly, if not solely, by heme oxygenase. Also, no evidence of non-heme oxygenase pathways was found in the presence of tin protoporphyrin, an inhibitor of heme oxygenase or mephenytoin, an inducer of both cytochrome P-450 and heme oxygenase. Similarly, in untreated cultured rat hepatocytes, radioactivity in carbon monoxide corresponded with loss of radioactivity in endogenous heme. In other experiments with chick hepatocyte cultures, rates of heme synthesis and breakdown were measured, and data were fitted to various models of hepatic heme metabolism. The results observed were consistent only with models in which an appreciable fraction (control cells, 17%, mephenytoin treated cells, 41%) of the newly synthesized heme was degraded rapidly to biliverdin.     

Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa

The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.     

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.     

Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean)--coordination structure of the heme complex and catabolism of heme

Heme oxygenase converts heme into biliverdin, CO, and free iron. In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IX(alpha) as a direct synthetic resource. In this study, a higher plant heme oxygenase, GmHO-1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. The similarity in the amino acid sequence between GmHO-1 and heme oxygenases from other biological species is low, and GmHO-1 binds heme with 1 : 1 stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments. The heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid-base transition and is converted to biliverdin IX(alpha) in the presence of NADPH/ferredoxin reductase/ferredoxin, or ascorbate. During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme-heme oxygenase or CO-verdoheme-heme oxygenase complexes was observed and was extracted as a bis-imidazole complex; it was identified as verdoheme. A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation. Thus, the mechanism of heme degradation by GmHO-1 appears to be similar to that of known heme oxygenases, despite the low sequence homology. The heme conversion by GmHO-1 is as fast as that by SynHO-1 in the presence of NADPH/ferredoxin reductase/ferredoxin, thereby suggesting that the latter is the physiologic electron-donating system.     

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.     

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its precursor expressed in Escherichia coli are associated with groEL protein

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is synthesized in the cytoplasm as a precursor which is transported into the chloroplast. During or after transport the precursor is processed to its mature size by removal of an amino-terminal transit peptide. Eight small subunits and eight large subunits (synthesized in the chloroplast) assemble to form the holoenzyme. We have expressed the precursor of the small subunit in Escherichia coli as a fusion to the carboxyl terminus of staphylococcal protein A'. The fusion protein was recovered from the bacterial lysate by chromatography on IgG-agarose. A 58-kDa protein copurified with the fusion protein in approximately equal amounts. Much less of the 58-kDa protein copurified with a fusion in which the transit peptide was deleted, and it did not copurify with protein A'. The 58-kDa protein was identified as the E. coli groEL gene product with antibodies directed against a homologous mitochondrial heat shock protein. This finding is particularly interesting because a chloroplast protein involved in the assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase also is homologous to the groEL protein. These homologs could modulate protein-protein interactions during folding and assembly of subunits into native complexes.     

Reaction of the microsomal heme oxygenase with cobaltic protoporphyrin IX, and extremely poor substrate.

A reconstituted heme oxygenase system which was composed of a purified heme oxygenase from pig spleen microsomes and a partially purified NADPH-cytochrome c reductase from pig liver microsomes could not catalyze the conversion of cobaltic protoporphyrin IX (Co-heme) to biliverdin, although Co-heme could bind with the heme oxygenase protein to form a complex. The heme oxygenase system in the microsomes from pig spleen, rat spleen, and rat kidney also failed to oxidize Co-heme to biliverdin. Properties of the complex of Co-heme and heme oxygenase closely resembled those of cobalt myoglobin and cobalt hemoglobin; the Co-heme bound to the heme oxygenase protein did not react with cyanide and azide, the Co-heme moiety was reduced but only slowly with sodium dithionite, and the reduced form of the Co-heme did not appear to bind carbon monoxide. The co-heme bound to heme oxygenase was not reduced with the NADPH-cytochrome c reductase system in air. These findings further support the views that heme oxygenase may have a heme-binding crevice similar to those of myoglobin and hemoglobin and that reduction of heme is the prerequisite for the oxidative degradation of heme in the heme oxygenase reaction.     

The hmuQ and hmuD genes from Bradyrhizobium japonicum encode heme-degrading enzymes

Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ (bll7075) and hmuD (bll7423) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staphylococcus aureus. The hmuQ gene is clustered with known heme transport genes in the genome. Recombinant HmuQ bound heme with a K(d) value of 0.8 microM and showed spectral properties consistent with a heme oxygenase. In the presence of a reductant, HmuQ catalyzed the degradation of heme and the formation of biliverdin. The hmuQ and hmuD genes complemented a Corynebacterium ulcerans heme oxygenase mutant in trans for utilization of heme as the sole iron source for growth. Furthermore, homologs of hmuQ and hmuD were identified in many bacterial genera, and the recombinant homolog from Brucella melitensis bound heme and catalyzed its degradation. The findings show that hmuQ and hmuD encode heme oxygenases and indicate that the IsdG family of heme-degrading monooxygenases is not restricted to gram-positive pathogenic bacteria.     

Methene bridge carbon atom elimination in oxidative heme degradation catalyzed by heme oxygenase and NADPH-cytochrome P-450 reductase

Physiological heme degradation is mediated by the heme oxygenase system consisting of heme oxygenase and NADPH-cytochrome P-450 reductase. Biliverdin IX alpha is formed by elimination of one methene bridge carbon atom as CO. Purified NADPH-cytochrome P-450 reductase alone will also degrade heme but biliverdin is a minor product (15%). The enzymatic mechanisms of heme degradation in the presence and absence of heme oxygenase were compared by analyzing the recovery of 14CO from the degradation of [14C]heme. 14CO recovery from purified NADPH-cytochrome P-450 reductase-catalyzed degradation of [14C]methemalbumin was 15% of the predicted value for one molecule of CO liberated per mole of heme degraded. 14CO2 and [14C]formic acid were formed in amounts (18 and 98%, respectively), suggesting oxidative cleavage of more than one methene bridge per heme degraded, similar to heme degradation by hydrogen peroxide. The reaction was strongly inhibited by catalase, but superoxide dismutase had no effect. [14C]Heme degradation by the reconstituted heme oxygenase system yielded 33% 14CO. Near-stoichiometric recovery of 14CO was achieved after addition of catalase to eliminate side reactions. Near-quantitative recovery of 14CO was also achieved using spleen microsomal preparations. Heme degradation by purified NADPH-cytochrome P-450 reductase appeared to be mediated by hydrogen peroxide. The major products were not bile pigments, and only small amounts of CO were formed. The presence of heme oxygenase, and possibly an intact membrane structure, were essential for efficient heme degradation to bile pigments, possibly by protecting the heme from indiscriminate attack by active oxygen species.