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The retinal pigment epithelium (RPE) is essential for maintenance of photoreceptors and normally functions under conditions enriched with reactive oxygen species. RPE therefore expresses various defense enzymes against oxidative stress, including heme oxygenase-1 (HO-1). HO-1 catalyzes heme breakdown to release iron, carbon monoxide, and biliverdin, which is reduced to bilirubin, a potent radical scavenger. HO-1 expression is induced by various environmental factors, which has been established as a defense mechanism. To explore the hypothesis that the expression level of HO-1 is reduced in those RPE cells under certain conditions, we analyzed the effects of interferon-gamma and hypoxia, each of which represses the expression of HO-1 mRNA in other types of human cells. Expression levels of HO-1 mRNA were reduced by interferon-gamma in two human RPE cell lines, D407 and ARPE-19, which was consistently associated with the induction of mRNA for Bach1, a transcriptional repressor for the HO-1 gene. On the other hand, HO-1 and Bach1 mRNAs were induced by hypoxia in D407 cells but remained unchanged in ARPE-19 cells, suggesting that Bach1 is not a sole regulator for HO-1 expression. The hypoxia-mediated induction of HO-1 mRNA in D407 cells depends on gene transcription and protein synthesis, as judged by the effects of their inhibitors. The half-life of HO-1 mRNA did not change during hypoxia. Thus, hypoxia may increase transcription of the HO-1 gene through a certain protein factor in RPE cells. These results indicate that RPE cells maintain retinal homeostasis by repressing or inducing the expression of HO-1, depending on the microenvironment.

Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors. Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection. This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB. CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore. Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity. The tight binding of BV by CphB prevents PCB from competing for the binding cavity. Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV. The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding. Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group. The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein.

Recently we have shown that ferric α-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196–18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric α-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous α-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203–213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. α-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of α-hydroxyhaem with haem oxygenase may be small and a substantial amount of free α-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase.

The phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 forms holoprotein adducts with close spectral similarity to plant phytochromes when autoassembled in vitro with bilin chromophores. Cph1 is a 85-kDa protein that acts as a light-regulated histidine kinase seemingly involved in 'two-component' signalling. This paper describes the improvement of Cph1 purification, estimation of the extinction coefficient of holo-Cph1, spectral analyses of the assembly procedure and studies on quaternary structure. During assembly with the natural chromophore phycocyanobilin (PCB), a red-shifted intermediate is observed. A similar result was obtained when phycoerythrobilin was used as chromophore. As shown by SDS/PAGE and Zn2+ fluorescence, the covalent attachment of PCB is blocked by 1 mM iodoacetamide, a cysteine-derivatizing agent. When PCB was incubated with blocked apo-Cph1, again a shoulder at longer wavelengths appeared. It is therefore proposed that the long-wavelength-absorbing form represents the protonated, noncovalently bound bilin. Biliverdin, which is neither protonated nor covalently attached, undergoes spectral changes in its blue-absorbing band upon incubation with apo-Cph1. On the basis of these data we therefore propose a three-step model for phytochrome autoassembly. Size-exclusion chromatography revealed different mobilities for the apoprotein, red-absorbing Cph1-PCB and far-red-absorbing Cph1-PCB. The major peaks of both holoprotein adducts had apparent molecular masses approximately 200 kDa, a result in agreement with the notion that autophosphorylation in sensory histidine kinases requires dimerization. When Cph1-PCB was further purified by preparative native electrophoresis, the mobility on size-exclusion chromatography was approximately 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.

Subarachnoid haemorrhage is often followed by haemolysis and concomitant oxidative stress, and is frequently complicated by pathological vasoconstriction or cerebral vasospasm. It is known that upregulation of haem oxygenase (HO-1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here we report biomimetic studies aimed at modelling pathological conditions leading to oxidative degradation of BR. Oxidative degradation products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biological activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biological activity was observed with vasoactive cerebrospinal fluid from subarachnoid haemorrhage patients. Three degradation products of BR were isolated: two were assigned as isomeric monopyrrole (C9H11N2O2) derivatives, 4-methyl-5-oxo-3-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and 3-methyl-5-oxo-4-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and the third was 4-methyl-3-vinylmaleimide (MVM), a previously isolated photodegradation product of biliverdin. Possible mechanisms of oxidative degradation of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degradation products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathological vasoconstriction associated with haemorrhage.

Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.

Biliverdin reductase is unique among all enzymes described to date in having two pH optima, 6.75 and 8.7, at which NADH or NADPH, respectively, are required for activity. The enzyme converts biliverdin to bilirubin in mammals. The mature enzyme, which is 293 amino acids long, has 3 cysteine residues, and is sulfhydryl dependent. To understand the role of the cysteine residues in enzyme activity, we examined the effects of the neutral substitution with alanine of each of three residues, individually and in combination, by site-directed mutagenesis. These residues in the predicted amino acid sequence of rat biliverdin reductase correspond to amino acids 73, 280 and 291. The modification of the amino-proximal cysteine (Cys73), which is flanked by a tyrosine residue, completely inactivated the enzyme with NADH at pH 6.75 and NADPH at pH 8.7. The loss of reductase activity was not due to changes in three-dimensional characteristics of the protein as suggested by its mobility in a non-denaturing gel. Although modification of either of the two cysteines located near the C-terminus (Cys280 and Cys291) significantly reduced activity with both cofactors, these mutations did not inactivate the enzyme. Comparison of Km values for the Cys280Ala and Cys291Ala mutants with the wild type protein, at pH 8.7, suggests that Cys280 principally functions in substrate binding while Cys291 is predominantly involved in cofactor binding. This assignment probably also applies at pH 6.75. Comparison of kcat of the mutants with wild type shows that mutation of Cys280 decreases Vmax of the enzyme. Mutation of both C-terminal cysteines caused inactivation of the enzyme, comparable to that produced by mutation of Cys73. Analysis by circular dichroism at far-ultraviolet wavelengths suggests that the alterations in activity are not the result of changes in the secondary structure of these mutants. These results are consistent with Cys73 having a central role in substrate/cofactor binding while biliverdin reductase can function, albeit at a reduced rate, with only one of the near C-terminus cysteines. The results are further consistent with the suggestion that although the two C-terminal cysteines have preferential affinities, they can serve similar functions in the interaction with substrate/cofactor.

A plasmid, pKK-RHO, was constructed by incorporating the coding sequence of a cDNA for rat heme oxygenase into the expression vector pKK233-2. Escherichia coli strain XL1-blue transformed with pKK-RHO produced a catalytically active, full-length heme oxygenase. The 32-kDa native enzyme expressed, was localized in the bacterial membranes, possibly due to the spontaneous membrane-binding properties of a hydrophobic segment in its C-terminal region. During cultivation, a few degraded forms of heme oxygenase that had lost their membrane-associative properties appeared. Probably, some bacterial proteases cut the native heme oxygenase at sites near its C-terminus and so release hydrophilic peptides of heme oxygenase from the membranes. A 30-kDa polypeptide, one of the degraded forms of heme oxygenase, retained ability to accept electrons from NADPH-cytochrome P450 reductase and also activity for catalyzing breakdown of heme to biliverdin. The cultured cells were pale green. From them we extracted green pigment(s), of which the absorption spectrum closely resembled that of biliverdin, suggesting that a large amount of the endogenous heme of E. coli was actually degraded to biliverdin by the expressed heme oxygenase.

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.

A major inducible form of heme oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pretreated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33,000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase, NADPH-cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 +/- 0.5 microM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 +/- 0.8 microM; and the Km for NADPH was 6.1 +/- 0.4 microM (all values mean +/- SD, n = 3). Oxygen concentration as low as 15 microM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4; at 37 degrees C, the apparent Vmax was 580 +/- 44 nmol biliverdin.(mg protein)-1.min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heme to non-biliverdin products and led to nearly stoichiometric conversion of heme to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin greater than tin protoporphyrin greater than zinc protoporphyrin greater than manganese protoporphyrin greater than cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 microM) (and several other porphyrins) and metallic ions (100 microM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 microM and totally at 15 microM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

We report the molecular defect of albumin Herborn, a new genetic variant of human serum albumin which has been found in Germany. Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr 3 (residues 124-298). This fragment was isolated on a preparative scale and subjected to tryptic and V8 protease digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution Lys240-->Glu. The -2 charge change of albumin Herborn, which is probably due to a A-->G transition in the first position of the corresponding codon in the structural gene, has no significant effect on its electrophoretic mobility under non-denaturating conditions. Therefore we have assumed that residue 240, which has been implicated in the bilirubin primary binding site (Jacobsen, C. (1978) Biochem. J. 171, 453-459), is buried. The binding of bilirubin and biliverdin by albumin Herborn was quantified using the fluorescence quenching method. The apparent equilibrium association constants (Ka +/- SD) and the number of high-affinity binding sites (n) of the defatted variant for bilirubin and biliverdin were Ka = 1.03 +/- 0.18 x 10(8) M-1, n = 1.07; and Ka = 7.48 +/- 1.10 x 10(6) M-1, n = 1.01, respectively. The Ka values are about 93.3% and 99.1% of the values found for the normal protein under the same conditions. These results strongly suggest that Lys240 of human serum albumin is not the basic residue involved in ion pairing with one of the carboxylate groups of bilirubin at its high-affinity site.

The recently described biliverdin-apomyoglobin complex has been investigated by two-dimensional NMR methods and molecular modeling with respect to the geometry of the chromophore and its position within the myoglobin pocket. Nuclear Overhauser effect correlations between the ligand and apoprotein amino acid residues prove that the bile pigment assumes a cyclic helical conformation and a position similar to heme in native myoglobin.

In the revised recommendations two new trivial names (isobacteriochlorin and sirohydrochlorin) are defined. Isobacteriochlorin is 2,3,7,8-tetrahydroporphyrin and is isomeric with bacteriochlorin. An example of an isobacteriochlorin is sirohydrochlorin, an intermediate in the biosynthesis of corrinoids. The names of linear tetrapyrroles are amended. The name bilin is now restricted to the fully oxidized parent, and less oxidized forms are named bilane, bilene, and biladiene. Names are now provided for the analogous dipyrrole system based on the parent dipyrrin. Tables are provided to show the structures of the more commonly encountered compounds using the Fischer system for denoting isomers (the fifteen isomers of mesoporphyrin defined by Fischer, and some isomers of biliverdin). Experience has shown that these names continue to be useful because of their brevity.

The red pigment uroerythrin, a chromophore known to be adsorbed by the amorphous urate sediments (sedimentum lateritium), has been isolated from human urine and further purified as its trimethyl derivative. Urine was applied to a column of Amberlite XAD-2 resin on which uroerythrin and other pigments were adsorbed. The pigments were eluted with methanol and uroerythrin was further purified by extraction with ether at pH 4.0, repeated chromatography on lipophilic Sephadex LH-20 and thin-layer chromatography on silica gel. For optimal purification uroerythrin was converted into the trimethyl derivative and chromatographed on silical gel thin-layer plates. The structure of the pigment has been studied by chromate degradation followed by identification of the imide products by thin-layer chromatography. From these results and from infrared, mass spectral and nuclear magnetic resonance data a tripyrrole structure for uroerythrin is concluded. The proposed structure for the chromophore is related to that of the bile pigment biliverdin consisting, however, only of the rings A, B and C.

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.

The following agents were found to oxidize bilirubin in vitro: hemoglobin and horse-radish peroxidase (both with hydrogen peroxide), cytochrome c, xanthine oxidase, and an insoluble oxidase, present in brain and other tissues. Kinetic constants were determined. The process with hemoglobin was inhibited competitively by two product molecules. The insoluble oxidase from brain was present in mitochondria. The supernatant fraction contained an inhibitor. The oxidase was inactive in the absence of salt and was unspecifically activated by a number of salts, the activity depending upon ionic strength, irrespective of which ions were present. Reaction products included biliverdin and a yellow, diazo-negative, polar pigment with the same oxidation level as bilirubin.