Mammalian Heme Oxygenase Research Articles

Molecular Characterization of Spodoptera frugiperda Heme Oxygenase and Its Involvement in Susceptibility to Chlorantraniliprole

The mammalian heme oxygenase (HO) plays an important role in cytoprotection against oxidative-stress-induced cell damage; however, functional characterization of insect HO is still limited. In this study, cDNA encoding a HO, named SfHO, was cloned from Spodoptera frugiperda. Analysis of the transcription level and enzymatic activity showed that exposure of the LC30 concentration of chlorantraniliprole to the third instar larvae significantly upregulated both the mRNA level and enzymatic activity of SfHO at 24 h after treatment. Further injection of the HO activator, hemin, into the third instar larvae led to the upregulation of SfHO as well as decreased susceptibility of S. frugiperda to chlorantraniliprole. Consistently, overexpression of SfHO increased the Sf9 cell viability under chlorantraniliprole treatment. Strikingly, both RNAi and the dual-luciferase reporter assay in Sf9 cells revealed that, unlike mammalian HO that is regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), SfHO was not subject to the regulation by cap 'n' collar isoform C (CncC), the Nrf2 homologue in insects. These data provide insights into the function and regulatory mechanism of insect HOs and had applied implications for the control of S. frugiperda.

Zebrafish Heme Oxygenase 1a Is Necessary for Normal Development and Macrophage Migration.

Heme oxygenase function is highly conserved between vertebrates where it plays important roles in normal embryonic development and controls oxidative stress. Expression of the zebrafish heme oxygenase 1 genes is known to be responsive to oxidative stress suggesting a conserved physiological function. In this study, we generate a knockout allele of zebrafish hmox1a and characterize the effects of hmox1a and hmox1b loss on embryonic development. We find that loss of hmox1a or hmox1b causes developmental defects in only a minority of embryos, in contrast to Hmox1 gene deletions in mice that cause loss of most embryos. Using a tail wound inflammation assay we find a conserved role for hmox1a, but not hmox1b, in normal macrophage migration to the wound site. Together our results indicate that zebrafish hmox1a has clearly a partitioned role from hmox1b that is more consistent with conserved functions of mammalian Heme oxygenase 1.

Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.

Activation of artemisinin and heme degradation in Leishmania tarentolae promastigotes: A possible link

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.

Role of His63 in HutZ from Vibrio cholerae in the heme degradation reaction and heme binding.

HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes oxygen-dependent degradation of heme via a similar catalytic mechanism to mammalian heme oxygenase. However, HutZ oxidizes the β- or δ-meso position of heme at a ∼1 : 1 ratio distinct from heme oxygenase, which initiates the degradation of heme solely at the α-meso position. His63 is a residue that potentially forms hydrogen bond with the heme 7-propionate group. To establish the role of His63 in regioselectivity of heme degradation by HutZ and heme binding, we constructed mutants of His63. Interestingly, the H63L mutant retained a comparable level of β- or δ-regioselectivity as wild-type HutZ. Ascorbic acid-assisted heme degradation by HutZ is pH-dependent, showing activity at pH 6.0 but not above pH 8.0. Compared to the wild-type protein, the H63L mutant was inactive, even at pH 6.0, and affinity for heme was significantly decreased in contrast with a comparable heme binding affinity at pH 8.0, as observed for the mutant of Asp132 to Val, which is located within hydrogen bonding distance of the heme axial ligand His170, but in a different protomer. In addition, the distance between heme and Trp109 increased from 16-18 Å for wild-type HutZ to 24-28 Å for the H63L mutant, indicating that protomer orientation is altered by the mutation, since Trp109 is in another subunit of the heme axial ligand. Our results collectively suggest that His63 positioned near heme does not contribute to regioselectivity of heme degradation but plays a key role in maintaining the orientation of subunits for HutZ to function of heme degradation.

Hydrogen sulfide bypasses the rate-limiting oxygen activation of heme oxygenase

Discovery of unidentified protein functions is of biological importance because it often provides new paradigms for many research areas. Mammalian heme oxygenase (HO) enzyme catalyzes the O2-dependent degradation of heme into carbon monoxide (CO), iron, and biliverdin through numerous reaction intermediates. Here, we report that H2S, a gaseous signaling molecule, is part of a novel reaction pathway that drastically alters HO's products, reaction mechanism, and catalytic properties. Our prediction of this interplay is based on the unique reactivity of H2S with one of the HO intermediates. We found that in the presence of H2S, HO produces new linear tetrapyrroles, which we identified as isomers of sulfur-containing biliverdin (SBV), and that only H2S, but not GSH, cysteine, and polysulfides, induces SBV formation. As BV is converted to bilirubin (BR), SBV is enzymatically reduced to sulfur-containing bilirubin (SBR), which shares similar properties such as antioxidative effects with normal BR. SBR was detected in culture media of mouse macrophages, confirming the existence of this H2S-induced reaction in mammalian cells. H2S reacted specifically with a ferric verdoheme intermediate of HO, and verdoheme cleavage proceeded through an O2-independent hydrolysis-like mechanism. This change in activation mode diminished O2 dependence of the overall HO activity, circumventing the rate-limiting O2 activation of HO. We propose that H2S could largely affect O2 sensing by mammalian HO, which is supposed to relay hypoxic signals by decreasing CO output to regulate cellular functions. Moreover, the novel H2S-induced reaction identified here helps sustain HO's heme-degrading and antioxidant-generating capacity under highly hypoxic conditions.

Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae.

HutZ is a heme-degrading enzyme in Vibrio cholerae. It converts heme to biliverdin via verdoheme, suggesting that it follows the same reaction mechanism as that of mammalian heme oxygenase. However, none of the key intermediates have been identified. In this study, we applied steady-state and time-resolved UV-vis absorption and resonance Raman spectroscopy to study the reaction of the heme-HutZ complex with H2O2 or ascorbic acid. We characterized three intermediates: oxyferrous heme, meso-hydroxyheme, and verdoheme complexes. Our data support the view that HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence and structural homology.

Escherichia coli heme oxygenase modulates host innate immune responses.

Induction of mammalian heme oxygenase (HO)-1 and exposure of animals to carbon monoxide (CO) ameliorates experimental colitis. When enteric bacteria, including Escherichia coli, are exposed to low iron conditions, they express an HO-like enzyme, chuS, and metabolize heme into iron, biliverdin and CO. Given the abundance of enteric bacteria residing in the intestinal lumen, our postulate was that commensal intestinal bacteria may be a significant source of CO and those that express chuS and other Ho-like molecules suppress inflammatory immune responses through release of CO. According to real-time PCR, exposure of mice to CO results in changes in enteric bacterial composition and increases E. coli 16S and chuS DNA. Moreover, the severity of experimental colitis correlates positively with E. coli chuS expression in IL-10 deficient mice. To explore functional roles, E. coli were genetically modified to overexpress chuS or the chuS gene was deleted. Co-culture of chuS-overexpressing E. coli with bone marrow-derived macrophages resulted in less IL-12p40 and greater IL-10 secretion than in wild-type or chuS-deficient E. coli. Mice infected with chuS-overexpressing E. coli have more hepatic CO and less serum IL-12 p40 than mice infected with chuS-deficient E. coli. Thus, CO alters the composition of the commensal intestinal microbiota and expands populations of E. coli that harbor the chuS gene. These bacteria are capable of attenuating innate immune responses through expression of chuS. Bacterial HO-like molecules and bacteria-derived CO may represent novel targets for therapeutic intervention in inflammatory conditions.

Role of Propionates in Substrate Binding to Heme Oxygenase from Neisseria meningitidis: A Nuclear Magnetic Resonance Study

Heme oxygenase (HO) cleaves hemin into biliverdin, iron, and CO. For mammalian HOs, both native hemin propionates are required for substrate binding and activity. The HO from the pathogenic bacterium Neisseria meningitidis (NmHO) possesses a crystallographically undetected C-terminal fragment that by solution (1)H nuclear magnetic resonance (NMR) is found to fold and interact with the active site. One of the substrate propionates has been proposed to form a salt bridge to the C-terminus rather than to the conventional buried cationic side chain in other HOs. Moreover, the C-terminal dipeptide Arg208His209 cleaves spontaneously over ~24 h at a rate dependent on substituent size. Two-dimensional (1)H NMR of NmHO azide complexes with hemins with selectively deleted or rearranged propionates shows that all bind to NmHO with a structurally conserved active site as reflected in optical spectra and NMR nuclear Overhauser effect spectroscopy cross-peak and hyperfine shift patterns. In contrast to mammalian HOs, NmHO requires only a single propionate interacting with the buried terminus of Lys16 to exhibit full activity and tolerates the existence of a propionate at the exposed 8-position. The structure of the C-terminus is qualitatively retained upon deletion of the 7-propionate, but a dramatic change in the 7-propionate carboxylate (13)C chemical shift upon C-terminal cleavage confirms its role in the interaction with the C-terminus. The stronger hydrophobic contacts between pyrroles A and B with NmHO contribute more substantially to the substrate binding free energy than in mammalian HOs, "liberating" one propionate to stabilize the C-terminus. The functional implications of the C-terminus in product release are discussed.

Influence of Substrate Modification and C-Terminal Truncation on the Active Site Structure of Substrate-Bound Heme Oxygenase from Neisseriae meningitidis. A 1H NMR Study

Heme oxygenase (HO), from the pathogenic bacterium N. meningitidis(NmHO), which secures host iron, shares many properties with mammalian HOs but also exhibits some key differences. The crystal structure appears more compact, and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D (1)H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross-peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin, and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~10(2) increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed.

Decreased heme oxygenase‐1 expression distinguishes human melanomas from melanocytic nevi

Dear Sir, Heme oxygenase (HO) is a microsomal enzyme that catalyzes the first, rate-limiting step in the degradation of heme, and plays an important role in its recycling (Maines, 1997). It cleaves the a-meso carbon bridge of heme, yielding equimolar quantities of carbon monoxide, Fe, and biliverdin (Tenhunen et al., 1968). The enzymatic activity of HO results in decreased oxidative stress, an attenuated inflammatory response, and a lower rate of apoptosis. Three distinct mammalian HO isoforms (HO-1, HO-2, and HO-3) have been identified. They are the products of three different genes (Maines, 1988). Heme oxygenase-1, the inducible 32-kDa isoform, also known as heat shock protein 32, is highly expressed in liver and spleen, but can also be detected in many other tissues. It can be induced strongly in response to cellular stress and diverse oxidative stimuli, including its heme substrate, heat shock, ultraviolet (UV) irradiation, reactive oxygen species (ROS), nitric oxide (NO), inflammatory cytokines, prostaglandins, ethanol, heavy metals, and hypoxia (Applegate et al., 1991; Choi and Alam, 1996). Most of the known HO-1 inducers stimulate the production of ROS or lead to the depletion of glutathione levels, suggesting the involvement of HO-1 activity in cellular protection against oxidative stress (Ryter and Choi, 2002). Heme oxygenase-1 is known to play an important role in resistance to apoptosis and in the rapid growth of several solid tumors. It is often up-regulated in tumor tissues, and its expression can be further increased in response to therapy (Jozkowicz et al., 2007). Induction of HO-1 has been recognized as one of the most promising targets for chemoprevention and chemoprotection research (Prawan et al., 2005). Few studies of HO-1 expression in melanoma have been reported. Okamoto et al. (2006) suggested that HO-1 is an important candidate gene in the pathogenesis and growth of melanomas. Was et al. (2006) found that overexpression of HO-1 increased the viability, proliferation, and angiogenic potential of melanoma cells, augmented metastasis, and decreased survival of melanoma-bearing mice. In this study, we examined the expression of HO-1 in melanocytic nevi and melanomas by immunohistochemical staining, and in cultured normal human melanocytes and melanoma cell lines. We enrolled 38 patients, 20 with melanocytic nevi and 18 with melanomas. Patient characteristics are shown in Table S1. The mean age of the patients was 42 ± 23 yr (range 6–80). The diagnoses of the 20 melanocytic nevi were as follows: compound nevi (12; 60%), junctional nevi (4; 20%), intradermal nevus (1; 5%), and Spitz nevi (3; 15%). The histopathological types of the 18 melanomas were superficial spreading type (7; 39%), acral lentiginous type (7; 39%), lentigo maligna type (3; 17%), and metastatic melanoma (1; 6%). Immunohistochemical staining was performed using formalin-fixed, paraffin-embedded tissue specimens from 20 patients with melanocytic nevi and 18 with melanomas, using the LSAB2 System-HRP (DakoCytomation, Carpenteria, CA, USA). The intensity of HO-1 immunoreactivity was graded using the following scale: 0 = negative (no staining of melanocytic cells, but staining of the epidermis); 1 = weakly positive (staining intensity was markedly weaker than that of the epidermis); 2 = moderately positive (staining intensity was mildly weaker than that of the epidermis); and 3 = strongly positive (staining intensity was at least equal to that of the epidermis). The Mann–Whitney test (performed using SPSS version 12.0; SPSS Inc., Chicago, IL, USA) was used to analyze differences in HO-1 staining intensity between melanocytic nevi and melanomas. Additionally, HO-1 expression in melanocytic nevi and melanomas from sun-exposed and sun-protected areas was compared. Expression of HO-1 was detected in 19 of 20 melanocytic nevi (95%) and in 11 of 18 melanomas (61%). Of melanomas, one superficial spreading type, four acral lentiginous type, one lentigo maligna type, and one metastatic type did not express HO-1. Note that the intensity of HO-1 expression was markedly different between melanocytic nevi and melanomas (Figure 1). The mean intensity in melanocytic nevi was 1.82 ± 0.86. There was no difference in staining intensity among the types of melanocytic nevi. The intensity of HO-1 expression in the nearby epidermis in melanocytic nevi was 2.30 ± 0.64 (Figure S1). Only 13 of 20

Candida albicans heme oxygenase and its product CO contribute to pathogenesis of candidemia and alter systemic chemokine and cytokine expression

Mammalian heme oxygenases play important roles in immune regulation by producing immunosuppressive CO. The pathogenic yeast Candida albicans encodes a heme oxygenase, Hmx1, that is specifically induced by the host protein hemoglobin, suggesting a role in the pathogenesis of disseminated bloodstream infections. We show that exposing mice to therapeutic levels of CO increases C. albicans virulence, whereas an HMX1 null strain has decreased virulence in murine disseminated candidiasis. Levels of several regulatory cytokines and chemokines are decreased in mice infected with the null strain, and initial lesions in the kidney are more rapidly cleared after polymorphonuclear leukocyte infiltration. Reconstitution of one or both alleles restores virulence to the level of wild type. Growth in vitro and initial organ burdens in infected mice are not decreased and host iron overload does not restore virulence for the null strain, suggesting that early growth in the host is not limited by Hmx1-mediated iron scavenging. In contrast, inhaled CO partially reverses the virulence defect of the null strain and restores several host cytokine responses to wild-type levels. Collectively, these results show that C. albicans Hmx1 expression and CO production limit the host immune response and contribute to the pathogenesis of candidemia.

Coupling of the Distal Hydrogen Bond Network to the Exogenous Ligand in Substrate-Bound, Resting State Human Heme Oxygenase

Mammalian heme oxygenase (HO) possesses catalytically implicated distal ordered water molecules within an extended H-bond network, with one of the ordered water molecules (#1) providing a bridge between the iron-coordinated ligand and the catalytically critical Asp140, that, in turn, serves as an acceptor for the Tyr58 OH H-bond. The degree of H-bonding by the ligated water molecule and the coupling of this water molecule to the H-bond network are of current interest and are herein investigated by (1)H NMR. Two-dimensional NMR allowed sufficient assignments to provide both the H-bond strength and hyperfine shifts, the latter of which were used to quantify the magnetic anisotropy in both the ferric high-spin aquo and low-spin hydroxo complexes. The anisotropy in the aquo complex indicates that the H-bond donation to water #1 is marginally stronger than in a bacterial HO, while the anisotropy for the hydroxo complex reveals a conventional (d(xz), d(yz))(1) ground state indicative of only moderate to weak H-bond acceptance by the ligated hydroxide. Mapping out the changes of the H-bond strengths in the network during the ligated water --> hydroxide conversion by correcting for the effects of magnetic anisotropy reveals a very substantial change in H-bond strength for Tyr58 OH and lesser effects on nearby H-bonds. The effect of pH on the H-bonding network in human HO is much larger and transmitted much further from the iron than in a pathogenic bacterial HO. The implications for the HO mechanism of the H-bond of Tyr58 to Asp140 are discussed.

Comparison of Apo- and Heme-bound Crystal Structures of a Truncated Human Heme Oxygenase-2

Heme oxygenase (HO) catalyzes the first step in the heme degradation pathway. The crystal structures of apo- and heme-bound truncated human HO-2 reveal a primarily alpha-helical architecture similar to that of human HO-1 and other known HOs. Proper orientation of heme in HO-2 is required for the regioselective oxidation of the alpha-mesocarbon. This is accomplished by interactions within the heme binding pocket, which is made up of two helices. The iron coordinating residue, His(45), resides on the proximal helix. The distal helix contains highly conserved glycine residues that allow the helix to flex and interact with the bound heme. Tyr(154), Lys(199), and Arg(203) orient the heme through direct interactions with the heme propionates. The rearrangements of side chains in heme-bound HO-2 compared with apoHO-2 further elucidate HO-2 heme interactions.

Heme Oxygenase-1 as a Potential Therapeutic Target for Hepatoprotection

Heme oxygenase (HO), the rate limiting enzyme in the breakdown of heme into carbon monoxide (CO), iron and bilirubin, has recently received overwhelming research attention. To date three mammalian HO isozymes have been identified, and the only inducible form is HO-1 while HO-2 and HO-3 are constitutively expressed. Advances in unveiling signal transduction network indicate that a battery of redox-sensitive transcription factors, such as activator protein-1 (AP-1), nuclear factor-kappa B (NF-kappaB) and nuclear factor E2-related factor-2 (Nrf2), and their upstream kinases including mitogen-activated protein kinases play an important regulatory role in HO-1 gene induction. The products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin have been shown to exert protective effects in several organs against oxidative and other noxious stimuli. In this context, it is interesting to note that induction of HO-1 expression contributes to protection against liver damage induced by several chemical compounds such as acetaminophen, carbon tetrachloride and heavy metals, suggesting HO-1 induction as an important cellular endeavor for hepatoprotection. The focus of this review is on the significance of targeted induction of HO-1 as a potential therapeutic strategy to protect against chemically-induced liver injury as well as hepatocarcinogenesis.

Heme oxygenase-1: a new therapeutic target for inflammatory bowel disease.

Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, followed by production of biliverdin, free iron and carbon monoxide (CO). Three mammalian HO isozymes have been identified, one of which, HO-1, is a stress-responsive protein induced by various oxidative agents. HO-2 and HO-3 genes are constitutively expressed. Recent studies demonstrate that the expression of HO-1 in response to different inflammatory mediators may contribute to the resolution of inflammation and have protective effects in several organs against oxidative injury. Although the mechanism underlying the anti-inflammatory actions of HO-1 remains poorly defined, both CO and biliverdin/bilirubin have been implicated in this response. In the intestinal tract, HO-1 is shown to be transcriptionally induced in response to oxidative stress, preconditioning and acute inflammation. Recent studies suggest that the induction of HO-1 expression plays a critical protective role in intestinal damage models induced by trinitrobenzene sulphonic acid or dextran sulphate sodium, indicating that activation of HO-1 may act as an endogenous defensive mechanism to reduce inflammation and tissue injury in the intestinal tract. These in vitro and in vivo data suggest that HO-1 may be a novel therapeutic target in patients with inflammatory bowel disease.

Solution 1H NMR Investigation of the Active Site Molecular and Electronic Structures of Substrate-bound, Cyanide-inhibited HmuO, a Bacterial Heme Oxygenase fromCorynebacterium diphtheriae

The molecular structure and dynamic properties of the active site environment of HmuO, a heme oxygenase (HO) from the pathogenic bacterium Corynebacterium diphtheriae, have been investigated by (1)H NMR spectroscopy using the human HO (hHO) complex as a homology model. It is demonstrated that not only the spatial contacts among residues and between residues and heme, but the magnetic axes that can be related to the direction and magnitude of the steric tilt of the FeCN unit are strongly conserved in the two HO complexes. The results indicate that very similar contributions of steric blockage of several meso positions and steric tilt of the attacking ligand are operative. A distal H-bond network that involves numerous very strong H-bonds and immobilized water molecules is identified in HmuO that is analogous to that previously identified in hHO (Li, Y., Syvitski, R. T., Auclair, K., Wilks, A., Ortiz de Montellano, P. R., and La Mar, G. N. (2002) J. Biol. Chem. 277, 33018-33031). The NMR results are completely consistent with the very recent crystal structure of the HmuO.substrate complex. The H-bond network/ordered water molecules are proposed to orient the distal water molecule near the catalytically key Asp(136) (Asp(140) in hHO) that stabilizes the hydroperoxy intermediate. The dynamic stability of this H-bond network in HmuO is significantly greater than in hHO and may account for the slower catalytic rate in bacterial HO compared with mammalian HO.

Solution NMR Characterization of an Unusual Distal H-bond Network in the Active Site of the Cyanide-inhibited, Human Heme Oxygenase Complex of the Symmetric Substrate, 2,4-Dimethyldeuterohemin

The presence of variable static hemin orientational disorder about the alpha-gamma-meso axis in the substrate complexes of mammalian heme oxygenase, together with the incomplete averaging of a second, dynamic disorder, for each hemin orientation, has led to NMR spectra with severe spectral overlap and loss of key two-dimensional correlations that seriously interfere with structural characterization in solution. We demonstrate that the symmetric substrate, 2,4-dimethyldeuterohemin, yields a single solution species for which the dynamic disorder is sufficiently rapid to allow effective and informative (1)H NMR structural characterization. A much more extensive, effective, and definitive NMR characterization of the cyanide-inhibited, symmetric heme complex of human heme oxygenase shows that the active site structure, with some minor differences, is essentially the same as that for the native protohemin in solution and crystal. A unique distal network that involves particularly strong hydrogen bonds, as well as inter-aromatic contacts, is described that is proposed to stabilize the position of the catalytically critical distal helix Asp-140 carboxylate (Liu, Y., Koenigs Lightning, L., Huang, H., Moënne-Loccoz, P., Schuller, D. J., Poulos, T. L., Loehr, T. M., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 34501-34507). The potential role of this network in placing a water molecule to stabilize the hydroperoxy species and as a template for the condensation of the distal helix upon substrate binding are discussed.

Heme oxygenase-1: a novel anti-inflammatory mediator

Heme oxygenase (HO) is the rate limiting enzyme in heme catabolism, which leads to the generation of biliverdin, free iron and carbon monoxide (CO). Three mammalian HO isoforms have been identified, one of which, HO-1, is a stress-responsive protein induced by various oxidative agents. Over the past few years, HO-1 has been implicated in the cytoprotective defence response against oxidative injury. There is increasing interest in the potential effects of CO because, like nitric oxide, it can activate the cGMP pathway and elicit neurotransmission and vasodilation. It also inhibits the lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines and increases LPS-induced expression of interleukin (IL)-10 in macrophages, suggesting that it is involved in the anti-inflammatory actions of HO-1. An exciting interplay between IL-10 and HO-1 in the regulation of inflammatory responses has now been revealed [1].

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

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