Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPR-supported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling.     

Reduction of oxaporphyrin ring of CO-bound α-verdoheme complexed with heme oxygenase-1 by NADPH-cytochrome P450 reductase

Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, carbon monoxide (CO) and ferrous iron via three successive monooxygenase reactions, using electrons provided by NADPH-cytochrome P450 reductase (CPR) and oxygen molecules. For cleavage of the oxaporphyrin ring of ferrous α-verdoheme, an intermediate in the HO reaction, involvement of a verdoheme π-neutral radical has been proposed. To explore this hypothetical mechanism, we performed electrochemical reduction of ferrous α-verdoheme-rat HO-1 complex under anaerobic conditions. Upon binding of CO, an O₂ surrogate, the midpoint potential for one-electron reduction of the oxaporphyrin ring of ferrous α-verdoheme was increased from −0.465 to −0.392 V vs the normal hydrogen electrode. Because the latter potential is close to that of the semiquinone/reduced redox couple of FAD in CPR, the one-electron reduction of the oxaporphyrin ring of CO-bound verdoheme complexed with HO-1 is considered to be a thermodynamically likely process. Indeed the one-electron reduced species, [Fe^II(verdoheme•)], was observed spectroscopically in the presence of CO in both NADPH/wild-type and FMN-depleted CPR systems under anaerobic conditions. Under physiological conditions, therefore, it is possible that O₂ initially binds to the ferrous iron of α-verdoheme in its complex with HO-1 and an electron is subsequently transferred from CPR, probably via FAD, to the oxaporphyrin ring.     

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.     

Human biliverdin reductase is a leucine zipper-like DNA-binding protein and functions in transcriptional activation of heme oxygenase-1 by oxidative stress.

Human biliverdin reductase (hBVR) is a serine/threonine kinase that catalyzes reduction of the heme oxygenase (HO) activity product, biliverdin, to bilirubin. A domain of biliverdin reductase (BVR) has primary structural features that resemble leucine zipper proteins. A heptad repeat of five leucines (L(1)--L(5)), a basic domain, and a conserved alanine characterize the domain. In hBVR, a lysine replaces L(3). The secondary structure model of hBVR predicts an alpha-helix-turn-beta-sheet for this domain. hBVR translated by the rabbit reticulocyte lysate system appears on a nondenaturing gel as a single band with molecular mass of approximately 69 kDa. The protein on a denaturing gel separates into two anti-hBVR immunoreactive proteins of approximately 39.9 + 34.6 kDa. The dimeric form, but not purified hBVR, binds to a 100-mer DNA fragment corresponding to the mouse HO-1 (hsp32) promoter region encompassing two activator protein (AP-1) sites. The specificity of DNA binding is suggested by the following: (a) hBVR does not bind to the same DNA fragment with one or zero AP-1 sites; (b) a 56-bp random DNA with one AP-1 site does not form a complex with hBVR; (c) in vitro translated HO-1 does not interact with the 100-mer DNA fragment with two AP-1 sites; (d) mutation of Lys(143), Leu(150), or Leu(157) blocks both the formation of the approximately 69-kDa specimens and hBVR DNA complex formation; and (e) purified preparations of hBVR or hHO-1 do not bind to DNA with two AP-1 sites. The potential significance of the AP-1 binding is suggested by the finding that the response of HO-1, in COS cells stably transfected with antisense hBVR, with 66% reduced BVR activity, to superoxide anion (O(2)()) formed by menadione is attenuated, whereas induction by heme is not affected. We propose a role for BVR in the signaling cascade for AP-1 complex activation necessary for HO-1 oxidative stress response.     

Biliverdin Reductase Is Heat Resistant and Coexpressed with Constitutive and Heat Shock Forms of Heme Oxygenase in Brain

Abstract: Two heme oxygenase (HO) isozymes—HO-1, which is a heat shock protein (HSP32), and HO-2—catalyze the isomer-specific production of biliverdin IXα and carbon monoxide. The latter has the potential of functioning as a neurotransmitter, whereas the reduced form of biliverdin, bilirubin, has potent antioxidant activity. Formation of bilirubin is catalyzed by biliverdin reductase (BVR). The reductase is a unique enzyme in being dual pyridine nucleotide and dual pH dependent. Here, we show that the reductase is resistant to thermal stress at both the protein and message level. We further demonstrate that the reductase is coexpressed in cells that display HO-1 and/or HO-2 under normal conditions, as well as in regions and cell types that have the potential to express heat shock-inducible HO-1 protein. Exposure of male rats to 42°C for 20 min did not decrease brain BVR activity, but caused a slight increase in NADPH-and NADH-dependent activities at 1 and 6 h following hyperthermia. High levels of the ～ 1.5-kb BVR mRNA were detected in control brain; it too displayed thermal tolerance. Similarly, the pattern of multiplicity of net charge variants of the enzyme purified from brain of heat-shocked rats did not differ from the control pattern. Immunochemical localization of BVR protein in normal brain correlated well with the presence of HO-1 and/or HO-2 throughout the forebrain, diencephalon, cerebellum, and brainstem regions. There were select neuronal and nonneuronal cells in the substantia nigra and cerebellum that did express the reductase under normal conditions, wherein no HO isozymes could be detected. The same population, however, responded to heat shock by an intense increase in the level of HO-1. We postulate that the constitutive presence of the reductase in this cell population and the overall thermal stability of the enzyme represent a safeguard mechanism in the brain for the prompt conversion of biliverdin to bilirubin under conditions when oxidation of the heme moiety of denatured hemoproteins by HO-1 is accelerated.     

The reactions of heme- and verdoheme-heme oxygenase-1 complexes with FMN-depleted NADPH-cytochrome P450 reductase. Electrons required for verdoheme oxidation can be transferred through a pathway not involving FMN

Electrons utilized in the heme oxygenase (HO) reaction are provided by NADPH-cytochrome P450 reductase (CPR). To investigate the electron transfer pathway from CPR to HO, we examined the reactions of heme and verdoheme, the second intermediate in the heme degradation, complexed with rat HO-1 (rHO-1) using a rat FMN-depleted CPR; the FMN-depleted CPR was prepared by dialyzing the CPR mutant, Y140A/Y178A, against 2 m KBr. Degradation of heme in complex with rHO-1 did not occur with FMN-depleted CPR, notwithstanding that the FMN-depleted CPR was able to associate with the heme-rHO-1 complex with a binding affinity comparable with that of the wild-type CPR. Thus, the first electron to reduce the ferric iron of heme complexed with rHO-1 must be transferred from FMN. In contrast, verdoheme was converted to the ferric biliverdin-iron chelate with FMN-depleted CPR, and this conversion was inhibited by ferricyanide, indicating that electrons are certainly required for conversion of verdoheme to a ferric biliverdin-iron chelate and that they can be supplied from the FMN-depleted CPR through a pathway not involving FMN, probably via FAD. This conclusion was supported by the observation that verdoheme dimethyl esters were accumulated in the reaction of the ferriprotoporphyrin IX dimethyl ester-rHO-1 complex with the wild-type CPR. Ferric biliverdin-iron chelate, generated with the FMN-depleted CPR, was converted to biliverdin by the addition of the wild-type CPR or desferrioxamine. Thus, the final electron for reducing ferric biliverdin-iron chelate to release ferrous iron and biliverdin is apparently provided by the FMN of CPR.     

Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation

Biliverdin reductase (BVR) reduces heme oxygenase (HO) activity product, biliverdin, to bilirubin. BVR is unique in having dual pH/dual cofactor requirements. Using Escherichia coli-expressed human BVR and COS cells, we show that BVR is autophosphorylated and that phosphorylation is required for its activity. An "in blot" autophosphorylation assay showed that BVR is a renaturable phosphoprotein. Controls for the experiments were HO-1 and HO-2; both are phosphoproteins but are not autophosphorylated. Autophosphorylation was pH-dependent, with activity at pH 8.7 being most prominent. In addition, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate fluorescence titration of BVR gave a lower K(d) at pH 8.7 than at pH 7.4 (15.5 versus 28.0 micrometer). Mn(2+) was required for binding of the ATP analogue and for autophosphorylation; the autokinase activity was lost when treated at 60 degrees C for 10 min. The loss of transferred phosphates by alkaline treatment suggested that BVR is a serine/threonine kinase. Potato acid phosphatase treatment reversibly inactivated the enzyme. The enzyme was also inactivated by treatment with the serine/threonine phosphatase, protein phosphatase 2A; okadaic acid attenuated the inhibition. Titration of protein phosphatase 2A-released phosphates indicated a 1:6 molar ratio of BVR to phosphate. The BVR immunoprecipitated from COS cell lysates was a phosphoprotein, and its activity and phosphorylation levels increased in response to H(2)O(2). The results define a previously unknown mechanism for regulation of BVR activity and are discussed in the context of their relevance to heme metabolism.     

Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-¹³C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys²⁸² and Cys²⁶⁵ are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys²⁶⁵ and Cys²⁸² varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys²⁶⁵ appears to be in a relatively hydrophilic, oxidizable environment compared with Cys¹²⁷ and Cys²⁸². Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.     

Immunohistochemical localization of the antioxidant enzymes biliverdin reductase and heme oxygenase-2 in human and pig gastric fundus

The intrinsic antioxidant capacities of the bile pigments biliverdin and bilirubin are increasingly recognized since both heme degradation products can exert beneficial cytoprotective effects due to their scavenging of oxygen free radicals and interaction with antioxidant vitamins. Several studies have been published on the localization of the carbon monoxide producing enzyme heme oxygenase-2 (HO-2), which concomitantly generates biliverdin; histochemical data on the distribution of biliverdin reductase (BVR), converting biliverdin to bilirubin, are still very scarce in large mammals including humans. The present study revealed by means of immunohistochemistry the presence of BVR and HO-2 in mucosal epithelial cells and in the endothelium of intramural vessels of both human and porcine gastric fundus. In addition, co-labeling with the specific neural marker protein-gene product 9.5 (PGP 9.5) demonstrated that both BVR and HO-2 were present in all intrinsic nerve cell bodies of both submucous and myenteric plexuses, while double labeling with c-Kit antibody confirmed their presence in intramuscular interstitial cells of Cajal (ICC). Our results substantiate the hypothesis that BVR, through the production of the potent antioxidant bilirubin, might be an essential component of normal physiologic gastrointestinal defense in man and pig.     

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased (P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

Heme Regulatory Motifs in Heme Oxygenase-2 Form a Thiol/Disulfide Redox Switch That Responds to the Cellular Redox State

Heme oxygenase (HO) catalyzes the rate-limiting step in heme catabolism to generate CO, biliverdin, and free iron. Two isoforms of HO have been identified in mammals: inducible HO-1 and constitutively expressed HO-2. HO-1 and HO-2 share similar physical and kinetic properties but have different physiological roles and tissue distributions. Unlike HO-1, which lacks cysteine residues, HO-2 contains three Cys-Pro signatures, known as heme regulatory motifs (HRMs), which are known to control processes related to iron and oxidative metabolism in organisms from bacteria to humans. In HO-2, the C-terminal HRMs constitute a thiol/disulfide redox switch that regulates affinity of the enzyme for heme (Yi, L., and Ragsdale, S. W. (2007) J. Biol. Chem. 282, 20156–21067). Here, we demonstrate that the thiol/disulfide switch in human HO-2 is physiologically relevant. Its redox potential was measured to be −200 mV, which is near the ambient intracellular redox potential. We expressed HO-2 in bacterial and human cells and measured the redox state of the C-terminal HRMs in growing cells by thiol-trapping experiments using the isotope-coded affinity tag technique. Under normal growth conditions, the HRMs are 60–70% reduced, whereas oxidative stress conditions convert most (86–89%) of the HRMs to the disulfide state. Treatment with reductants converts the HRMs largely (81–87%) to the reduced dithiol state. Thus, the thiol/disulfide switch in HO-2 responds to cellular oxidative stress and reductive conditions, representing a paradigm for how HRMs can integrate heme homeostasis with CO signaling and redox regulation of cellular metabolism.Heme oxygenase (HO³ ; EC 1.14.99.3) catalyzes the O₂- and NADPH-dependent conversion of heme to biliverdin, carbon monoxide (CO), and iron in a reaction that is coupled to cytochrome P450 reductase. Then, biliverdin reductase catalyzes the NADPH-dependent reduction of biliverdin to the antioxidant bilirubin. Several recent reviews on HO (1–5) and biliverdin reductase (6) are available. HO is present in organisms from bacteria to eukaryotes and, as the only known enzyme that can degrade heme, plays a critical role in heme and iron homeostasis.There are two major HO isoforms in mammals: inducible HO-1, which is ancient and widely distributed among organisms from bacteria to man, and constitutively expressed HO-2, which emerged 250 million years ago with the amniotes (7). HO-1 is found in most tissues and is highly expressed in spleen and liver (8). Conversely, HO-2 has a narrow tissue distribution, exhibiting high expression levels in the brain, testes, and carotid body (8, 9). Both HO-1 and HO-2 catalyze the NADPH- and cytochrome P450 reductase-dependent degradation of heme to CO, iron, and biliverdin, which is quickly reduced to bilirubin in the presence of biliverdin reductase (10). Controlling cellular heme concentrations is crucial because heme is required as a prosthetic group by regulatory and redox proteins, yet concentrations higher than 1 μm free heme are toxic (11). Thus, as the only mammalian proteins known to degrade heme, HOs play a key role in cellular heme homeostasis; furthermore, in vitro and in vivo studies of cellular and tissue injuries, such as oxidative stress and hemin-induced cytotoxicity, indicate that HO is cytoprotective (9).HO-1 and HO-2 share high sequence and three-dimensional structural homology in their core domains (12, 13); however, their sequences diverge near their C termini, in which HO-2 contains two conserved heme regulatory motifs (HRMs), involving Cys²⁶⁵ in HRM1 and Cys²⁸² in HRM2⁴ (12, 14) (Fig. 1). It was shown recently that the HRMs in HO-2 do not bind heme per se but instead form a reversible thiol/disulfide redox switch that indirectly regulates the affinity of HO-2 for heme (14). However, for this redox switch to have any physiological consequence, the midpoint redox potential of the thiol/disulfide couple must be near the ambient intracellular redox potential, estimated to range from −170 to −250 mV (15).Open in a separate windowFIGURE 1.Major structural regions in HO-1 and HO-2. His²⁵ in HO-1 or His⁴⁵ in HO-2 is the heme-binding ligand.The HRM has been proposed to constitute a heme-binding site (16, 17) that regulates key metabolic processes from bacteria to humans. The HRM consists of a conserved Cys-Pro core sequence that is usually flanked at the N terminus by basic amino acids and at the C terminus by a hydrophobic residue. HRM/heme interactions have been proposed to regulate the activity and/or stability of proteins that play central roles in respiration and oxidative damage (18, 19), coordination of protein synthesis and heme availability in reticulocytes (20, 21), and controlling iron and heme homeostasis (22–26). An important component of the last process is HO-2.Here, we demonstrate that the C-terminal HRMs, which form a thiol/disulfide redox switch between Cys²⁶⁵ and Cys²⁸², exhibit a redox potential that falls well within the ambient cellular redox potential. By expressing HO-2 in bacterial and human cells and trapping the thiols using the isotope-coded affinity tag (ICAT) technique, it was shown that the redox state of the C-terminal HRMs in growing cells responds to the cellular redox state. The disulfide state is favored under oxidative conditions, and the dithiol state is predominant under reducing conditions. Thus, the HRMs act as a molecular rheostat that responds to the ambient intracellular redox potential and, based on earlier studies (14), controls activity of HO-2 by regulating heme binding to the enzyme.     

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.     

Comparison of the heme-free and -bound crystal structures of human heme oxygenase-1

Heme oxygenase (HO) catalyzes the degradation of heme to biliverdin. The crystal structure of human HO-1 in complex with heme reveals a novel helical structure with conserved glycines in the distal helix, providing flexibility to accommodate substrate binding and product release (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). To structurally understand the HO catalytic pathway in more detail, we have determined the crystal structure of human apo-HO-1 at 2.1 A and a higher resolution structure of human HO-1 in complex with heme at 1.5 A. Although the 1.5-A heme.HO-1 model confirms our initial analysis based on the 2.08-A model, the higher resolution structure has revealed important new details such as a solvent H-bonded network in the active site that may be important for catalysis. Because of the absence of the heme, the distal and proximal helices that bracket the heme plane in the holo structure move farther apart in the apo structure, thus increasing the size of the active-site pocket. Nevertheless, the relative positioning and conformation of critical catalytic residues remain unchanged in the apo structure compared with the holo structure, but an important solvent H-bonded network is missing in the apoenzyme. It thus appears that the binding of heme and a tightening of the structure around the heme stabilize the solvent H-bonded network required for proper catalysis.     

Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso-hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasite’s genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.     

Distinct protective mechanisms of HO-1 and HO-2 against hydroperoxide-induced cytotoxicity

Heme oxygenases (HO-1 and HO-2) catalyze the NADPH-cytochrome P(450) reductase (CPR)-dependent degradation of heme into iron, carbon monoxide, and biliverdin, which is reduced into bilirubin. Under basal conditions, HO-1 is often undetected and can be induced by numerous stress conditions. Although HO-2 is constitutively expressed, its activity appears to be regulated by post-translational modifications. HO activity has been associated with cellular protection, by which it degrades heme, a prooxidant, into bioactive metabolites. Under given circumstances, overexpression of HO-1 can render cells more sensitive to free radicals. Here, we investigated the properties of human HO isoforms that protect against oxidative stress. Considering that CPR can be a limiting factor for optimal HO activity, we tested stable HO-1 and HO-2 cell lines that derived from the CPR cells. Results indicate that the HO-1 and HO-2 cells are more resistant than controls to hemin and to the organic tert-butyl hydroperoxide, t-BuOOH. However, HO-1 cells are less resistant than HO-2 cells to hydrogen peroxide (H(2)O(2)). The levels of oxidatively modified proteins of HO-1 and HO-2 cells in response to t-BuOOH toxicity are identical, but the level of oxidatively modified proteins of HO-2 cells is less than that of HO-1 cells in response to H(2)O(2) toxicity. Performing subcellular fractionations revealed that HO-2 and CPR are found together in the microsomal fractions, whereas HO-1 is partially present in the microsome and also found in other fractions, such as the cytosol. These same findings were observed in non-transfected primary neurons where HO-1 proteins were chemically induced with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)). The differences in subcellular localization of HO-1 and HO-2 could explain some of the discrepancies in their cellular activity and enzymatic protective mechanisms.     

Backbone assignments of the apo and Zn(II) protoporphyrin IX-bound states of the soluble form of rat heme oxygenase-1

In nature, heme is a prosthetic group that is universally used as a cofactor for heme proteins. It is necessary for the execution of fundamental biological processes including electron transfer, oxidation and metabolism. However, free heme is toxic to cells, because of its capability to enhance oxidative stress, hence its cellular concentration is strictly regulated through multiple mechanisms. Heme oxygenase (HO) serves as an irreplaceable member in the heme degradation system. It is a ubiquitous protein, existing in many species including mammals, higher plants, and interestingly, certain pathogenic bacteria. In the HO reaction, HO catalyzes oxidative cleavage of heme to generate biliverdin and release carbon monoxide and ferrous iron. Because of the beneficial effects of these heme catabolism products, HO plays a key role in iron homeostasis and in defense mechanism against oxidative stress. HO is composed of an N-terminal structured region and a C-terminal membrane-bound region. Furthermore, the soluble form of HO, which is obtainable by excision of the membrane-bound region, retains its catalytic activity. Here, we present the backbone resonance assignments of the soluble form (residues 1–232) of HO-1 in the free and Zn(II) protoporphyrin IX (ZnPP)-bound states, and analyzed the structural differences between the states. ZnPP is a potent enzyme inhibitor, and the ZnPP-bound structure of HO-1 mimics the heme-bound structure. These assignments provide the structural basis for a detailed investigation of the HO-1 function.     

Limited Role for the Bilirubin-Biliverdin Redox Amplification Cycle in the Cellular Antioxidant Protection by Biliverdin Reductase

In mammalian cells, heme is degraded by heme oxygenase to biliverdin, which is then reduced to bilirubin by biliverdin reductase (BVR). Both bile pigments have reducing properties, and bilirubin is now generally considered to be a potent antioxidant, yet it remains unclear how it protects cells against oxidative damage. A presently popular explanation for the antioxidant function of bilirubin is a redox cycle in which bilirubin is oxidized to biliverdin and then recycled by BVR. Here, we reexamined this putative BVR-mediated redox cycle. We observed that lipid peroxidation-mediated oxidation of bilirubin in chloroform, a model of cell membrane-bound bilirubin, did not yield biliverdin, a prerequisite for the putative redox cycle. Similarly, H₂O₂ did not oxidize albumin-bound bilirubin to biliverdin, and in vitro oxidation of albumin or ligandin-bound bilirubin by peroxyl radicals gave modest yields of biliverdin. In addition, decreasing cellular BVR protein and activity in HeLa cells using RNA interference did not alter H₂O₂-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H₂O₂-mediated damage, irrespective of whether bilirubin or biliverdin were added to the cells as substrate for the putative redox cycle. Similarly, transformation of human BVR into hmx1 (heme oxygenase) mutant yeast did not provide protection against H₂O₂ toxicity above that seen in hmx1 mutant yeast expressing human heme oxygenase-1. Together, these results argue against the BVR-mediated redox cycle playing a general or important role as cellular antioxidant defense mechanism.Biliverdin reductase (BVR)³ forms part of the major pathway for the disposition of cellular heme in mammalian cells. This pathway is initiated by heme oxygenase, which converts heme to carbon monoxide, iron, and biliverdin, which in turn is reduced to bilirubin by BVR at the expense of NADPH. Because of its intramolecular hydrogen bonding, the bilirubin produced is sparingly soluble in water at physiological pH and ionic strength (1). Hence, bilirubin is usually tightly bound to albumin in order to be transported within the blood circulation (2), from which it is removed mainly through uptake by hepatocytes. Once bilirubin is transferred across the cell membrane of hepatocytes, it binds glutathione S-transferases before being transformed to water-soluble derivatives by conjugation of one or both of its propionyl groups before its excretion into bile and then the intestine (3).Under physiological conditions, plasma bilirubin concentrations in humans range from ∼5 to 20 μm, practically all of which is unconjugated pigment bound to albumin (1). Abnormally high plasma concentrations are associated with the risk of developing neurologic dysfunction due to preferential deposition of bilirubin in brain and its toxic effects on cell functions. In fact for many years, biliverdin and bilirubin were generally regarded as waste products of heme metabolism in higher animals, although earlier work suggested that these bile pigments might play a role as natural antioxidants, since small quantities of the pigment stabilize vitamin A and β-carotene during intestinal uptake, and animals with low plasma bilirubin showed early signs of vitamin E deficiency (4, 5).In a series of in vitro studies, Stocker et al. (6–8) demonstrated that unconjugated bilirubin, at micromolar concentrations, efficiently scavenged peroxyl radicals in homogenous solution or multilamellar liposomes. At physiologically relevant oxygen tension, bilirubin surpassed α-tocopherol as an antioxidant in liposomes (8), and it is thought to protect plasma proteins and lipids from many but not all oxidants (9). However, it is less clear whether this antioxidant activity extends to in vivo situations or protection of cells from oxidative stress. Although produced in essentially all cells, the normal range of cellular bilirubin concentrations is unknown. However, it is probably in the low nanomolar range, well below that of established cellular antioxidants, such as glutathione and ascorbate, arguing against bilirubin being an important cellular antioxidant. Nonetheless, in vitro studies with rat neuronal cultures showed that the presence of 10 nm bilirubin in the culture medium protected cells against 10,000-fold higher concentrations of hydrogen peroxide (10). Later, Barañano et al. (11) confirmed such observations in HeLa cells and demonstrated that BVR depletion increased reactive oxygen species (ROS) and cell death. This led to the following proposal of the BVR-amplified redox cycle. While acting as an antioxidant, bilirubin is oxidized to biliverdin that is then reduced back to bilirubin by the ubiquitous and abundant BVR.An important underlying assumption of this amplification cycle is that ROS-mediated bilirubin oxidation in cells is specific and yields substantial if not stoichiometric amounts of biliverdin. Inconsistent with this assumption, however, earlier studies showed that high yields of biliverdin formation are limited to certain oxidants (i.e. peroxyl radicals) and albumin-bound bilirubin. In cells, bilirubin is probably present in membranes, bound to proteins other than albumin, or present in conjugated form. Therefore, we reexamined the putative redox amplification cycle. Our results show that reaction of these forms of bilirubin with 1e- or 2e-oxidants at best generates modest amounts of biliverdin. Furthermore, overexpression of BVR does not protect mammalian or yeast cells from hydrogen peroxide-mediated damage, thereby casting doubt on the importance of the putative BVR redox cycle for cellular antioxidant protection.