Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O*-2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p < 0.05). High glucose decreased HO-1 promoter activity (p < 0.05). Hyperglycemia increased O*-2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p < 0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O*-2 production, and a decrease in endothelial cell sloughing.     

Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats

Heme oxygenase-1 (HO-1), an inducible stress protein, has been implicated in cytoprotection against oxidative stress in vitro and in vivo. Estrogens also have antioxidant effects. This study investigated the time course of HO-1 and inducible nitric oxide synthase (iNOS) expression in the aortas of ovariectomized rats, and the regulatory relationship between the NO/NOS and the carbon monoxide/HO systems. HO-1 and iNOS protein expression was induced by ovariectomy (Ovx) and was extremely high 2-6 weeks after Ovx compared with the sham-operated group. Expression of the constitutive enzymes HO-2 and endothelial NOS did not differ significantly between sham-operated and Ovx rats. 17beta-Estradiol (E(2)) replacement reversed these changes in rats after Ovx. Long-term treatment with the antioxidant tempol significantly inhibited HO-1 and iNOS expression. The iNOS inhibitor aminoguanidine significantly suppressed the induction of HO-1. Oxidized glutathione in the hearts of Ovx rats increased gradually, with significant elevation at 3-6 weeks after Ovx compared with the sham-operated group, whereas plasma levels of NO metabolites were significantly reduced 4-6 weeks after Ovx. Treatment with the HO inhibitor zinc protoporphyrin IX blocked HO-1 induction, but significantly increased the plasma levels of NO metabolites. In conclusion, HO-1 is induced by oxidative stress resulting from E(2) depletion. The NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause.     

Regulation of heme oxygenase expression by alcohol, hypoxia and oxidative stress

AIM:To study the effect of both acute and chronic alcohol exposure on heme oxygenases(HOs) in the brain,liver and duodenum.METHODS:Wild-type C57BL/6 mice,heterozygous Sod2 knockout mice,which exhibit attenuated manganese superoxide dismutase activity,and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia.For acute alcohol exposure,ethanol was administered in the drinking water for 1 wk.Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies.HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting.RESULTS:Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice.It did not alter liver HO-1 expression,nor HO-2 expression in the brain,liver or duodenum.In contrast,acute alcohol exposure decreased both liver HO-1 and HO-2 expression,and HO-2 expression in the duodenum of wild-type mice.The decrease in liver HO-1 expression was abolished in ARNT+/-mice.Sod2+/-mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression.However,alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2+/-mice.Collectively,these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner.Chronic alcohol exposure alters brain and duodenal,but not liver HO expression.However,acute alcohol exposure inhibits liver HO-1 and HO-2,and also duodenal HO-2 expression.CONCLUSION:The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.     

Biliverdin protects against polymicrobial sepsis by modulating inflammatory mediators

Highly inducible heme oxygenase (HO)-1 is protective against acute and chronic inflammation. HO-1 generates carbon monoxide (CO), ferrous iron, and biliverdin. The aim of this study was to investigate the protective effects of biliverdin against sepsis-induced inflammation and intestinal dysmotility. Cecal ligation and puncture (CLP) was performed on Sprague-Dawley rats under isoflurane anesthesia with and without intraperitoneal biliverdin injections, which were done before, at the time of CLP, and after CLP. In vivo gastrointestinal transit was carried out with fluorescein-labeled dextran. Jejunal circular muscle contractility was quantified in vitro using organ bath-generated bethanechol dose-response curves. Neutrophilic infiltration into the muscularis externa was quantified. The jejunal muscularis was studied for cytokine mRNA expressions [interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, inducible nitric oxide synthase, cyclooxygenase-2, biliverdin, IL-10, and HO-1] using real-time RT-PCR. Biliverdin treatment prevented the sepsis-induced suppression of gastrointestinal muscle contractility in vivo and in vitro and significantly decreased neutrophilic infiltration into the jejunal muscularis. Inflammatory mRNA expressions for small bowel IL-6 and MCP-1 were significantly reduced after biliverdin treatment in CLP-induced septic animals compared with untreated septic animals. The anti-inflammatory mediator expression of small bowel IL-10 was significantly augmented after CLP at 3 h compared with untreated septic animals. These findings demonstrate that biliverdin attenuates sepsis-induced morbidity to the intestine by selectively modulating the inflammatory cascade and its subsequent sequelae on intestinal muscularis function.     

Ferritin in Parkinsonian and control brains

Heme oxygenase-1 (HO-1) is a stress protein expressed in various pathological conditions associated with oxidative stress. Brain HO-1 expression and activity in response to LPS treatment showed regional variability with the highest levels in the substantia nigra (SN) and hippocampus. HO-1 induction by LPS was redox-sensitive and associated with increased levels of NO synthase and arginase, two proteins involved in the regulation of cellular redox state. Brain HO-2 and HO-3 expression, studied by quantitative RT-PCR, did not show significant changes. Our data suggest an interaction between NO and the HO system in the brain after LPS treatment. As SN and hippocampus are involved in Parkinson's and Alzheimer's diseases, understanding interaction of these proteins in the brain will help to elucidate the mechanisms involved in neurodegeneration.     

Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2alpha (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemic rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemic animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemic rats brings about a reduction in O2- production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes.     

MiR-150-5p protects against septic acute kidney injury via repressing the MEKK3/JNK pathway

BackgroundSeptic acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. MicroRNA is reportedly involved in sepsis-induced organ dysfunction, while the role of miR-150 in septic AKI remains ambiguous.MethodsQuantitative real-time PCR (qRT-PCR) was carried out to examine miR-150-5p expression in both septic AKI patients and volunteers without septic AKI. Lipopolysaccharide (LPS) was used to treat renal tubular epithelial cell line HK-2 and C57/BL6 mice to establish in vitro and in vivo sepsis-induced AKI models. Cell apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Renal pathological changes were examined via Hematoxylin-Eosin (H&E) staining, and renal function was measured via blood urea nitrogen (BUN) and creatinine (Cre) measurements. The MEKK3/JNK profile and oxidative stress markers (including COX2 and iNOS) were examined by immunoblot analysis, and the expression levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and oxidative stress markers (MDA, SOD, and CAT) were evaluated by ELISA.ResultsMiR-150-5p was down-regulated in the serum of patients with septic AKI (compared to healthy volunteers). Moreover, miR-150-5p levels were lower in LPS-treated HK-2 cell lines and in the septic AKI mouse model. Additionally, Stat-3 activation mediated the decrease of miR-150-5p. Functionally, miR-150-5p agomir attenuated LPS-induced apoptosis in HK-2 cells, in addition to renal inflammatory responses and oxidative stress. In contrast, inhibition of miR-150-5p aggravated LPS-induced apoptosis, inflammatory reactions and oxidative stress. Furthermore, miR-150-5p agomir decreased BUN and Scr levels in the septic AKI mice model repressed TNF-α, IL-6 and IL-1β, and up-regulated SOD and CAT down-regulated MDA in the kidney tissues. Moreover, miR-150-5p was identified as a target gene for Stat3, and the overexpression of Stat3 partially promoted the effect of down-regulating miR-150-5p on LPS-induced HK2 cell injury. Mechanistically, the MEKK3/JNK pathway was identified as a functional target of miR-150-5p, and the knockdown of MEKK3 showed protective effects against LPS mediated HK-2 cell apoptosis.ConclusionStat3-mediated miR-150-5p exerted protective effects in sepsis-induced acute kidney injury by regulating the MEKK3/JNK pathway.     

内、外源性硫化氢在脂多糖所致大鼠急性肺损伤中的作用

目的：探讨内、外源性硫化氢（H2S）在脂多糖（LPS）所致大鼠急性肺损伤（ALI）中的作用并初探其机制。方法：将120只SD大鼠随机分为对照组、IPS组（经气管内滴注LPS复制ALI模型）、NaHS＋LPS组和炔丙基甘氨酸（PPG）＋LPS组。给药后4h或8h处死动物,测定肺系数;光镜观察肺组织形态学改变;化学法检测血浆H2S、NO和CO含量、肺组织丙二醛（MDA）含量、胱硫醚-γ-裂解酶（CSE）、诱导型一氧化氮合酶（iNOS）和血红素加氧酶（HO）活性以及支气管肺泡灌洗液（BALF）中中性粒细胞（PMN）数目和蛋白含量的变化;用免疫组织化学法检测肺组织iNOS、HO-1蛋白表达。再将血浆H2S含量与上述指标进行相关性分析。结果：气管内滴注LPS可引起肺组织明显的形态学改变;肺系数和肺组织MDA含量增加;BALF中PMN数目和蛋白含量增加;血浆H2S含量和肺组织CSE活性下降;肺组织iNOS活性、HO活性和iNOS蛋白表达、HO-1蛋白表达增强,血浆NO含量、CO含量增加。预先给予NaHS可显著减轻LPS所致上述指标的改变;而预先给予PIG可加重LPS所致肺损伤,使BALF中PMN数目和蛋白含量、血浆NO含量、肺组织iNOS活性和iNOS蛋白表达进一步增加,但对血浆CO含量、肺组织HO活性和HO-1蛋白表达无明显影响。HS含量与CSE活性、血浆CO含量、肺组织HO-1活性呈正相关（r值=0．945—0．987,P均〈0．01）;与其他指标呈负相关（r值=-0．994～-0．943,P均〈0．01）。结论：H2S／CSE体系的下调在LPS所致大鼠Ⅲ的发病学中有一定作用,内、外源性H2S具有抗LPS所致Au的作用,该作用可能与其抗氧化效应、减轻PMN所致肺过度的炎症反应以及下调NO／iNOS体系、上调CO／HO—1体系有一定关系。     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

HO-1 induction attenuates renal damage and oxidative stress induced by K2Cr2O7

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme; its inducible isozyme HO-1 protects against some types of acute tissue injury. The expression and functional role of HO-1 in rats with renal injury induced by potassium dichromate (K(2)Cr(2)O(7)) was investigated in this work. Rats were studied 24 h after a single injection of K(2)Cr(2)O(7). To address the possible protective effect of HO-1 in this experimental model, this enzyme was induced by an injection of stannous chloride (SnCl(2)) 12 h before K(2)Cr(2)O(7) administration. The functional role of HO-1 in K(2)Cr(2)O(7) + SnCl(2)-treated animals was tested by inhibiting HO activity with an injection of zinc (II) protoporphyrin IX (ZnPP) 18 h before K(2)Cr(2)O(7). In K(2)Cr(2)O(7)-treated rats: (i) renal HO-1 content, measured by Western blot, increased 2.6-fold; and, (ii) renal nitrotyrosine and protein carbonyl content, markers of oxidative stress, increased 3.5- and 1.36-fold, respectively. Renal damage and oxidative stress were ameliorated and HO-1 content was increased in the K(2)Cr(2)O(7) + SnCl(2) group. The attenuation of renal injury and oxidative stress was lost by the inhibition of HO activity in K(2)Cr(2)O(7) + SnCl(2) + ZnPP-treated animals. Our data suggest that HO-1 overexpression induced by SnCl(2) is responsible for the attenuation of renal damage and oxidative stress induced by K(2)Cr(2)O(7).     

Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer’s disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H₂O₂-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-κB (NF-κB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-κB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.     

性激素对血红素氧化酶在大鼠前列腺腹侧叶表达的影响

血红素氧化酶(heme oxygenase,HO)是产生内源性一氧化碳(carbon monoxide,CO)的限速酶,最近发现内源性CO在调节平滑肌张力方面起重要作用。而人的良性前列腺增生(benign prostates hyperplasia,BPH)所致的膀胱出口梗阻与前列腺平滑肌张力有密切关系,但还不清楚内源性HO/CO系统是否介导了前列腺平滑肌的活动。为了观察性激素对大鼠前列腺腹侧叶中血红素氧化酶-1(heme oxygenase-1,HO-1)和血红素氧化酶-2(heme oxygenase-2,HO-2)基因表达的影响,我们采用睾丸切除术建立雄性SD大鼠去势模型,用RT-PCR方法观察HO-1和HO-2的转录水平,应用免疫组织化学结合图像分析技术,观察去势、外源性雄激素和雌激素对前列腺腹侧叶中HO—1和HO-2蛋白水平的影响。结果表明,HO-1和HO-2在正常大鼠前列腺腹侧叶中都有表达,腺上皮细胞和纤维平滑肌间质呈现HO-1的免疫活性,HO-2的免疫染色仅在腺上皮细胞内检测到;去势组HO-1的mRNA和蛋白表达水平显著低于正常对照组(P<0.01):外源性给予雄激素组和雌激素组的HO-1表达水平明显增高(P<0.01),且雌激素主要增加前列腺纤维平滑肌间质的HO-1表达:HO-2在各组间的表达无明显差异(P>0.05)。这些结果提示,性激素对HO-1有诱导作用,但对HO-2无明显的影响,因此推测一氧化碳-血红素氧化酶(CO—HO)     

Activated microglia are less vulnerable to hemin toxicity due to nitric oxide-dependent inhibition of JNK and p38 MAPK activation

In intracerebral hemorrhage, microglia become rapidly activated and remove the deposited blood and cellular debris. To survive in a harmful hemorrhagic or posthemorrhagic condition, activated microglia must be equipped with appropriate self-defensive mechanism(s) to resist the toxicity of hemin, a component released from damaged RBCs. In the current study, we found that activation of microglia by pretreatment with LPS markedly reduced their vulnerability to hemin toxicity in vitro. Similarly, intracorpus callosum microinjection of LPS prior to hemin treatment reduced the brain tissue damage caused by hemin and increased microglial density in the penumbra in rats. LPS induced the expressions of inducible NO synthase (iNOS) and heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation in microglia. The preventive effect by LPS was significantly diminished by an iNOS inhibitor, L-N(6)-(1-iminoethyl)lysine, whereas it was mimicked by a NO donor, diethylamine-NONOate, both suggesting the crucial role of NO in the modulation of hemin-induced toxicity in activated microglia. We further found that NO reduced hemin toxicity via inhibition of hemin-induced activation of JNK and p38 MAPK pathways in microglia. Whereas HO-1 expression in LPS-stimulated microglia was markedly blocked by L-N(6)-(1-iminoethyl)lysine, the HO-1 inhibitor, tin protoporphyrin, increased iNOS expression and decreased the susceptibility of LPS-activated microglia to hemin toxicity. The data indicate that the mutual interaction between NO and HO-1 plays a critical role in modulating the adaptive response of activated microglia to hemin toxicity. Better understanding of the survival mechanism of activated microglia may provide a therapeutic strategy to attenuate the devastating intracerebral hemorrhagic injury.     

Heme oxygenase-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells

Hyperglycaemia is associated with oxidative stress. The inducible isoform of heme oxygenase (HO-1) is an effective system to counteract oxidative stress, yet it is unclear how hyperglycaemia affects HO-1. In this study, we explored: 1) the HO-1 protein content and HO activity in human umbilical vein endothelial cells (HUVECs) exposed to different glucose concentrations, and 2) the mechanisms which account for the high glucose-induced effects on HO-1. We evaluated HO-1 protein expression, HO activity, apoptosis and reactive oxygen species (ROS) in HUVECs treated for 48 h with 5.5, 10 and 20 mM glucose. A dose-dependent production of reactive oxygen species was observed. At 10 mM glucose, an increase of HO-1 protein expression and HO activity was observed, whereas at 20 mM, there was no change in protein content and activity relative to at 5.5 mM glucose. HO-1 protein expression in HUVECs exposed to 20 mM of glucose was increased in the presence of 20 U/ml superoxide dismutase (SOD). HO-1 gene silencing augments ROS production both at 5.5 and 10 mM glucose, leading to an increased apoptosis. We conclude that, in endothelial cells, the regulation of HO-1 by glucose is dependent upon levels of glucose itself. Lack of homeostatic HO-1 upregulation fails to protect from oxidative damage and results in a higher rate of apoptotic cell death.     

Heme-oxygenase-mediated iron accumulation in the liver

Heme oxygenase (HO) isozymes, HO-1 and HO-2, catalyze the conversion of heme to iron, carbon monoxide, and biliverdin. The present study was aimed at elucidating the role of the HO system in iron accumulation and oxidative stress in the liver. We have also studied the regulation of an iron exporter, ferroportin-1 (FPN-1), as an adaptive response mechanism to increased iron levels. Sprague-Dawley rats were treated with HO inducer hemin or HO inhibitor tin-protoporphyrin IX (SnPPIX) for 1 month. A portion of liver tissues was subjected to RT-PCR for HO-1, HO-2, and FPN-1 gene expression as well as an HO activity assay. Paraffin-embedded tissues were stained for iron with Prussian blue. Hepatic iron concentration was measured by High Resolution-Inductively Coupled Plasma-Mass Spectrometry. 8-hydroxy-2'-deoxyguanosine (8-OHdG) stain, a sensitive and specific marker of oxidative DNA damage, was performed to assess oxidative stress. Hemin treatment led to augmented HO expression and activity in association with increased iron accumulation and oxidative stress. FPN-1 expression was also found to be upregulated. SnPPIX treatment reduced HO activity, intracellular iron levels, and oxidative stress as compared to controls. Our data provides evidence of increased HO activity as an important pro-oxidant mechanism leading to iron accumulation in the liver.     

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.