Crystal structure of human heme oxygenase-1 in a complex with biliverdin

Heme oxygenase oxidatively cleaves heme to biliverdin, leading to the release of iron and CO through a process in which the heme participates both as a cofactor and as a substrate. Here we report the crystal structure of the product, iron-free biliverdin, in a complex with human HO-1 at 2.19 A. Structural comparisons of the human biliverdin-HO-1 structure with its heme complex and the recently published rat HO-1 structure in a complex with the biliverdin-iron chelate [Sugishima, M., Sakamoto, H., Higashimoto, Y., Noguchi, M., and Fukuyama, K. (2003) J. Biol. Chem. 278, 32352-32358] show two major differences. First, in the absence of an Fe-His bond and solvent structure in the active site, the distal and proximal helices relax and adopt an "open" conformation which most likely encourages biliverdin release. Second, iron-free biliverdin occupies a different position and orientation relative to heme and the biliverdin-iron complex. Biliverdin adopts a more linear conformation and moves from the heme site to an internal cavity. These structural results provide insight into the rate-limiting step in HO-1 catalysis, which is product, biliverdin, release.     

Crystal structure of heme oxygenase-1 from cyanobacterium Synechocystis sp. PCC 6803 in complex with heme.

Heme oxygenase (HO) catalyzes the oxidative degradation of heme utilizing molecular oxygen and reducing equivalents. In photosynthetic organisms, HO functions in the biosynthesis of such open-chain tetrapyrroles as phyto-chromobilin and phycobilins, which are involved in the signal transduction for light responses and light harvesting for photosynthesis, respectively. We have determined the first crystal structure of a HO-1 from a photosynthetic organism, Synechocystis sp. PCC 6803 (Syn HO-1), in complex with heme at 2.5 A resolution. Heme-Syn HO-1 shares a common folding with other heme-HOs. Although the heme pocket of heme-Syn HO-1 is, for the most part, similar to that of mammalian HO-1, they differ in such features as the flexibility of the distal helix and hydrophobicity. In addition, 2-propanol derived from the crystallization solution occupied the hydrophobic cavity, which is proposed to be a CO trapping site in rat HO-1 that suppresses product inhibition. Although Syn HO-1 and mammalian HO-1 are similar in overall structure and amino acid sequence (57% similarity vs. human HO-1), their molecular surfaces differ in charge distribution. The surfaces of the heme binding sides are both positively charged, but this patch of Syn HO-1 is narrow compared to that of mammalian HO-1. This feature is suited to the selective binding of ferredoxin, the physiological redox partner of Syn HO-1; the molecular size of ferredoxin is approximately 10 kDa whereas the size of NADPH-cytochrome P450 reductase, a reducing partner of mammalian HO-1, is approximately 77 kDa. A docking model of heme-Syn HO-1 and ferredoxin suggests indirect electron transfer from an iron-sulfur cluster in ferredoxin to the heme iron of heme-Syn HO-1.     

Crystal structure of human heme oxygenase-1.

Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the alpha-meso heme carbon is due primarily to steric influence of the distal helix.     

Crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate. Conformational change of the distal helix during the heme cleavage reaction

The crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate (biliverdin(Fe)-HO-1), the immediate precursor of the final product, biliverdin, has been determined at a 2.4-A resolution. The electron density in the heme pocket clearly showed that the tetrapyrrole ring of heme is cleaved at the alpha-meso edge. Like the heme bound to HO-1, biliverdin-iron chelate is located between the distal and proximal helices, but its accommodation state seems to be less stable in light of the disordering of the solvent-exposed propionate and vinyl groups. The middle of the distal helix is shifted away from the center of the active site in biliverdin(Fe)-HO-1, increasing the size of the heme pocket. The hydrogen-bonding interaction between Glu-29 and Gln-38, considered to restrain the orientation of the proximal helix in the heme-HO-1 complex, was lost in biliverdin(Fe)-HO-1, leading to relaxation of the helix. Biliverdin has a distorted helical conformation; the lactam oxygen atom of its pyrrole ring-A interacted with Asp-140 through a hydrogen-bonding solvent network. Because of the absence of a distal water ligand, the iron atom is five-coordinated with His-25 and four pyrrole nitrogen atoms. The coordination geometry deviates considerably from a square pyramid, suggesting that the iron may be readily dissociated. We speculate that the opened conformation of the heme pocket facilitates sequential product release, first iron then biliverdin, and that because of biliverdin's increased flexibility, iron release triggers its slow dissociation.     

Crystal structure of dimeric heme oxygenase-2 from Synechocystis sp. PCC 6803 in complex with heme

Phycobiliproteins, light-harvesting proteins in cyanobacteria, red algae, and cryptophytes, contain phycobilin pigments. Phycobilins are synthesized from biliverdin, which is produced by the oxidative cleavage of the heme porphyrin ring catalyzed by heme oxygenase (HO). Two paralogs of ho (ho1 and ho2) have been identified in the genome of the cyanobacterium, Synechocystis sp. PCC 6803. The recombinant proteins of both paralogs (Syn HO-1 and Syn HO-2) possess in vitro heme degradation activity. We have determined the crystal structures of Syn HO-2 in complex with heme (heme-Syn HO-2) and its reduced and NO bound forms. The heme-Syn HO-2 crystal was a nonmerohedral twin, and detwinned diffraction data were used to refine the structure. Although heme-Syn HO-2 shares common folding with other HOs, the C-terminal segment is ordered and turns back to the heme-binding side. Gel-filtration chromatography analysis and molecular packing in the crystal indicate that heme-Syn HO-2 forms a homodimer, in which the C-terminal ordered segments interact with each other. Because Syn HO-2 is a monomer in the apo state, the dimeric interaction may aid in the selection of the reducing partner but likely does not interfere with heme binding. The heme iron is coordinated by a water molecule in the ferric form, but the distal water is absent in the ferrous form. In all of the Syn HO-2 structures, several water molecules form a hydrogen-bond network at the distal hemepocket, which is involved in HO activity. Upon NO binding, the side-chain conformation of Tyr 156 changes. Tyr 156 is located at the hydrophobic cluster, which interrupts the possible H(+) pathway from the molecular surface to the hemepocket. Thus, Tyr 156 may function as a H(+) shuttle by changing conformation.     

Crystal structure of rat apo-heme oxygenase-1 (HO-1): mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms

Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin by utilizing O(2) and NADPH. HO (apoHO) was crystallized as twinned P3(2) with three molecules per asymmetric unit, and its crystal structure was determined at 2.55 A resolution. Structural comparison of apoHO and its complex with heme (HO-heme) showed three distinct differences. First, the A helix of the eight alpha-helices (A-H) in HO-heme, which includes the proximal ligand of heme (His25), is invisible in apoHO. In addition, the B helix, a portion of which builds the heme pocket, is shifted toward the heme pocket in apoHO. Second, Gln38 is shifted toward the position where the alpha-meso carbon of heme is located in HO-heme. Nepsilon of Gln38 is hydrogen-bonded to the carbonyl group of Glu29 located at the C-terminal side of the A helix in HO-heme, indicative that this hydrogen bond restrains the angle between the A and B helices in HO-heme. Third, the amide group of Gly143 in the F helix is directed outward from the heme pocket in apoHO, whereas it is directed toward the distal ligand of heme in HO-heme. This means that the F helix around Gly143 must change its conformation to accommodate heme binding. The apoHO structure has the characteristic that the helix on one side of the heme pocket fluctuates, whereas the rest of the structure is similar to that of HO-heme, as observed in such hemoproteins as myoglobin and cytochromes b(5) and b(562). These structural features of apoHO suggest that the orientation of the proximal helix and the position of His25 are fixed upon heme binding.     

Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1

We report the crystal structure of heme oxygenase from the pathogenic bacterium Neisseria meningitidis at 1.5 A and compare and contrast it with known structures of heme oxygenase-1 from mammalian sources. Both the bacterial and mammalian enzymes share the same overall fold, with a histidine contributing a ligand to the proximal side of the heme iron and a kinked alpha-helix defining the distal pocket. The distal helix differs noticeably in both sequence and conformation, and the distal pocket of the Neisseria enzyme is substantially smaller than in the mammalian enzyme. Key glycine residues provide the flexibility for the helical kink, allow close contact of the helix backbone with the heme, and may interact directly with heme ligands.     

Crystal structures of ferrous and ferrous-NO forms of verdoheme in a complex with human heme oxygenase-1: catalytic implications for heme cleavage

Heme oxygenase oxidatively degrades heme to biliverdin resulting in the release of iron and CO through a process in which the heme participates both as a cofactor and substrate. One of the least understood steps in the heme degradation pathway is the conversion of verdoheme to biliverdin. In order to obtain a better understanding of this step we report the crystal structures of ferrous-verdoheme and, as a mimic for the oxy-verdoheme complex, ferrous-NO verdoheme in a complex with human HO-1 at 2.20 and 2.10 A, respectively. In both structures the verdoheme occupies the same binding location as heme in heme-HO-1, but rather than being ruffled verdoheme in both sets of structures is flat. Both structures are similar to their heme counterparts except for the distal helix and heme pocket solvent structure. In the ferrous-verdoheme structure the distal helix moves closer to the verdoheme, thus tightening the active site. NO binds to verdoheme in a similar bent conformation to that found in heme-HO-1. The bend angle in the verodoheme-NO structure places the terminal NO oxygen 1 A closer to the alpha-meso oxygen of verdoheme compared to the alpha-meso carbon on the heme-NO structure. A network of water molecules, which provide the required protons to activate the iron-oxy complex of heme-HO-1, is absent in both ferrous-verdoheme and the verdoheme-NO structure.     

Crystal structure of guaiacol and phenol bound to a heme peroxidase

Guaiacol is a universal substrate for all peroxidases, and its use in a simple colorimetric assay has wide applications. However, its exact binding location has never been defined. Here we report the crystal structures of guaiacol bound to cytochrome c peroxidase (CcP). A related structure with phenol bound is also presented. The CcP-guaiacol and CcP-phenol crystal structures show that both guaiacol and phenol bind at sites distinct from the cytochrome c binding site and from the δ-heme edge, which is known to be the binding site for other substrates. Although neither guaiacol nor phenol is seen bound at the δ-heme edge in the crystal structures, inhibition data and mutagenesis strongly suggest that the catalytic binding site for aromatic compounds is the δ-heme edge in CcP. The functional implications of these observations are discussed in terms of our existing understanding of substrate binding in peroxidases [Gumiero A et al. (2010) Arch Biochem Biophys 500, 13-20].     

Electrochemical reduction of ferrous alpha-verdoheme in complex with heme oxygenase-1

The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to alpha-hydroxyheme, alpha-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme pi-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be -0.47+/-0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the pi-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O(2) to the ferrous iron of verdoheme is the first step in the degradation of verdoheme.     

Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors

The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole “anchor” which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole “anchor” may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via “induced fit” to accommodate bulky substituents at the 4-position of the dioxolane ring.     

Hydroxylamine and hydrazine bind directly to the heme iron of the heme-heme oxygenase-1 complex

We investigated whether or not hydroxylamine (HA) and hydrazine (HZ) interact with heme bound to heme oxygenase-1. Anaerobic addition of either HA or HZ to the ferric heme-enzyme complex produced a low-spin heme species. Titration studies at different pHs revealed that the neutral form of each of HA and HZ selectively binds to the heme with dissociation constants of 9.8 and 1.8 mM, respectively. Electron spin resonance analysis suggested that the nitrogen atom of each amine is coordinated to the ferric heme iron. With a concentrated solution of the heme-enzyme complex, however, another species of HA binding appeared, in which the oxygen atom of HA is coordinated to the iron. This species showed an unusual low-spin signal which is similar to that of the ferric hydroperoxide species in the heme oxygenase reaction.     

Up-regulation of heme oxygenase-1 in rat spleen after aniline exposure

The splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis, and the development of a variety of sarcomas in rats. However, the underlying mechanisms by which aniline elicits splenotoxic response are not well understood. Previously we have shown that aniline exposure causes oxidative damage to the spleen. To further explore the oxidative mechanism of aniline toxicity, we evaluated the potential contribution of heme oxygenase-1 (HO-1), which catalyzes heme degradation and releases free iron. Male SD rats were given 1 mmol/kg/day aniline in water by gavage for 1, 4, or 7 days, and respective controls received water only. Aniline exposure led to significant increases in HO-1 mRNA expression in the spleen (2-and 2.4-fold at days 4 and 7, respectively) with corresponding increases in protein expression, as confirmed by ELISA and Western blot analysis. Furthermore, immunohistochemical assessment of spleen showed stronger immunostaining for HO-1 in the spleens of rats treated for 7 days, confined mainly to the red pulp areas. No changes were observed in mRNA and protein levels of HO-1 after 1 day exposure. The increase in HO-1 expression was associated with increases in total iron (2.4-and 2.7-fold), free iron (1.9-and 3.5-fold), and ferritin levels (1.9-and 2.1-fold) at 4 and 7 days of aniline exposure. Our data suggest that HO-1 up-regulation in aniline-induced splenic toxicity could be a contributing pro-oxidant mechanism, mediated through iron release, and leading to oxidative damage.     

血红素氧合酶-1在离体大鼠心肌缺血预适应中的作用

目的:分别观察给予HO-1诱导剂和抑制剂对心肌相对缺血再灌注损伤和缺血预适应的影响,探讨HO-1在缺血预适应中的作用.方法:实验动物随机分为对照组(CN)、缺血/再灌损伤组(I/R)、缺血预适应 缺血/再灌损伤组(PC)、HO-1诱导剂 缺血/再灌损伤组(HM)、HO-1抑制剂 缺血预适应组(ZP).心肌缺血/再灌损伤采用相对缺血/再灌损伤模型,缺血预适应则为相对缺血5min恢复5min,反复2次.测定心功能、MDA及HO-1活性变化.结果:HM组HO-1活性升高,心功能恢复率均显著高于IR组(P＜0.01),MDA含量显著低于IR组(P＜0.05).ZP组活性降低,心功能恢复率显著低于PC组(P＜0.05),MDA含量显著高于PC组(P＜0 05).结论:HO-1是缺血预适应释放的内源性活性物质之一.     

Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines

Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2.     

Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia

Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions.     

内毒素引起的乳鼠心肌细胞血红素加氧酶—1基因的表达

为了探讨在内毒素作用下的乳鼠心肌细胞（neonatal rat cardiomyocytes,NRCMs）血红素加氧酶－1（heme oxygenase-1,HO-1）基因的表达及其在细胞损伤中的作用,分别用10、30及50μg/ml的脂多糖（lipopolysaccharide,LPS）,10μg/ml LPS 10μmol/ml锌原卟啉Ⅸ（Zn-protoporphyrin-Ⅸ,ZnPPⅨ）和单纯10μmol/ml ZnPPⅨ与培养的NRCMs共同孵育6h,以及10μg/ml LPS与NRCMs共同孵育9h和18h。分别观察细胞HO－1 mRNA表达、MDA含量、LDH释放量与台盼蓝摄取率的变化。结果显示,同样与细胞孵育6h,LPS10μg/ml时HO－1 mRNA表达比对照组增加81.2％,30μg/ml时表达量增加126.3％,50μg/ml时表达量增加92.8％;LPS为10μg/ml时,孵育9h后HO－1 mRNA的表达量比对照组增加93.6％,孵育18h后一增加105.8％。LPS30、50μg/ml,10μg/ml LPS＋10μmol/ml ZnPPⅨ与细胞孵育6h及LPS 10μg/ml孵育18h后,细胞MDA含量、LDH释放量与台盼蓝摄取率明显增加（P＜0.01）;单纯10μg/ml LPS与单纯10μmol/ml ZnPPⅨ孵育6h后,上述指标均无明显升高。结果表明,LPS可诱导NRCMs HO－1 mRNA的表达,且在较低LPS剂量范围内具有时间依赖性和浓度依赖性;NRCMs HO-1 mRNA的表达可减低LPS引起的细胞损伤,这可能是细胞产生的一种自身保护性反应。     

Effects of heme oxygenase-1 expression on sterol homeostasis in rat astroglia

Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. Central oxidation of cholesterol to oxysterols has been implicated in neuroembryogenesis, synaptic plasticity, and membrane repair. In the current study, we demonstrated that transient transfection of rat astroglia with human (h)ho-1 cDNA for 3 days significantly decreased intracellular cholesterol concentrations and increased levels of four oxysterol species (measured by GC/MS) compared to untreated control cultures and HO-1-transfected cells exposed to the HO inhibitor, tin mesoporphyrin (SnMP). Relative to control preparations, oxidative stress was augmented in mitochondria (isolated by subcellular fractionation) and culture media derived from HO-1-transfected astrocytes, as evidenced by enhanced oxidation of the synthetic reporter molecules, linoleoyl tyrosine (LT), linoleoyl tyrosine cholesterol ester (LTC), or linoleoyl tyrosine deoxyguanosyl ester (LTG; measured by GC/MS and LC/MS/MS). We also observed enhanced oxidation of exogenous LTC in human neuroblastoma (M17) cells exposed for 18 h to conditioned media collected from HO-1-transfected astrocytes relative to control media. In AD and other pathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g., beta-amyloid) into altered patterns of glial sterol metabolism which, in turn, may affect neuronal membrane turnover, survival, and adaptability.