Thermoregulatory response to hypoxia after inhibition of the central heme oxygenase–carbon monoxide pathway

(1) Centrally acting carbon monoxide (CO) seems to play thermoregulatory actions, but no report exists about its role in hypoxia-induced anapyrexia. (2) CO arises from the catabolism of heme by heme oxygenase (HO), an enzyme that is overexpressed during hypoxia. Thus, we tested the hypothesis that the central HO–CO pathway modulates hypoxia-induced anapyrexia by means of intracerebroventricular injection of the HO inhibitor ZnDPBG. (3) Core temperature (T_C) of awake rats was determined by biotelemetry. ZnDPBG did not alter basal T_c, but it exacerbated hypoxia-induced anapyrexia, indicating that the central HO–CO pathway is a modulator of hypoxia-induced anapyrexia, probably preventing excessive decreases in T_c.     

Modification of the heme·heme oxygenase system with iron and cobalt tetrasulfonated phthalocyanines

Modification of heme·heme oxygenase by iron(III) and cobalt(II) tetrasulfonated phthalocyanines has been performed. New compounds have been isolated and their properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, electron paramagnetic resonance (EPR) and carboxymethylation at histidyl groups. Spectrophotometric titration data indicate the ratio of the reagents in this process to be 1:1. The visible absorption spectra show the main peak at 650 nm for the iron compound and 682 nm for the cobalt one. Electrophoresis and molecular weight estimation show both complexes to be monomers. Cobalt(II) tetrasulfonated phthalocyanine, under aerobic conditions with heme oxygenase protein, undergoes autooxidation to the cobalt(III) complex, as has been proved by EPR and spectroscopic data. Iron and cobalt phthalocyanine modified heme·heme oxygenase with excess dithionite is reduced at the phthalocyanine ligand. In the presence of oxygen, the reduction product transforms into oxygenated Fe(III)Lheme oxygenase or Co(III)heme oxygenase, respectively. Reduction of the iron(III) model complex with ascorbic acid under anaerobic conditions leads to degradation of the phthalocyanine moiety, while Co(III)heme oxygenase with ascorbic acid is reduced to Co(II)Lheme oxygenase. As has been shown by carboxymethylation of the heme oxygenase protein at the histidine residues, the predominant binding site of both phthalocyanine complexes is the heme-binding histidyl residue. There is evidence that there is a second binding site with lower affinity towards Co(II)L on the heme oxygenase protein. Iron and cobalt tetrasulfonated phthalocyanines are not able to displace heme from the heme·heme oxygenase complex. In this reaction the iron complex undergoes degradation and the cobalt one gives a hybrid compound with heme·heme oxygenaseHeme oxygenase protein complexes with iron and cobalt tetrasulfonated phthalocyanines do not exhibit activity in their oxidative degradation.     

Structural characterization of a carbon monoxide adduct of a heme–DNA complex

Abstract  

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3′-terminal G-quartet of the DNA though a π–π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π–π stacking interaction between the pseudo-C ₂-symmetric heme and the C ₄-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C ₂ axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe–CO bond in the complex is similar to that of the Fe–CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.     

Generation of cyclic guanosine monophosphate by heme oxygenases in the human testis--a regulatory role for carbon monoxide in Sertoli cells?

Previous studies have demonstrated that cGMP is produced by nitric oxide-mediated activation of soluble guanylyl cyclase (sGC) in seminiferous tubules of the human testis. It is not known, however, whether carbon monoxide (CO), another activator of sGC, is also involved in testicular function. To address this issue, testicular probes from 65- to 75-yr-old men have been examined. The CO-generating enzyme, heme oxygenase-1 (HO-1), could be localized by immunohistochemical and immunoblot analyses to Sertoli cells. In these cells, HO-1 is detectable in adluminal cell compartments, whereas sGC immunoreactivity is distributed exclusively in basal compartments. Treatments of isolated tubules with either sodium arsenite, known to induce HO-1, or hematin, an HO substrate, resulted in 4.4- and 1.8-fold, respectively, increases in cGMP levels. ODQ, a specific sGC inhibitor, inhibited completely the sodium arsenite-stimulated cGMP production. Moreover, the HO inhibitor zinc protoporphyrin-IX and the CO scavenger hemoglobin both significantly reduced (77% or 46% of control, respectively) tubular cGMP generation. These findings, demonstrating for the first time a link between HO-1 activity in Sertoli cells and sGC-dependent cGMP production in seminiferous tubules, suggest a functional role of CO in the human testis.     

Influence of cobalt substitution on the activity of iron-type nitrile hydratase: are cobalt type nitrile hydratases regulated by carbon monoxide?

Comamonas testosteroni Ni1 nitrile hydratase is a Fe-type nitrile hydratase whose native and recombinant forms are identical. Here, the iron of Ni1 nitrile hydratase was replaced by cobalt using a chaperone based Escherichia coli expression system. Cobalt (CoNi1) and iron (FeNi1) enzymes share identical Vmax (30 nmol min(-1) mg(-1)) and Km (200 microM) toward their substrate and identical Ki values for the known competitive inhibitors of FeNi1. However, nitrophenols used as inhibitors do display a different inhibition pattern on both enzymes. Furthermore, CoNi1 and FeNi1 are also different in their sensitivity to nitric oxide and carbon monoxide, CO being selective of the cobalt enzyme. These differences are rationalized in relation to the nature of the catalytic metal center in the enzyme.     

Effect of carbon source on the β-glucosidase system of the thermophilic fungus Humicola grisea

H. grisea produced an extracellular -glucosidase (EC 3.2.1.21) at high activity in media supplemented with carboxymethyl cellulose (CMC) or cellobiose. Cellobiose-induced -glucosidase was insensitive to glucose repression whereas that of CMC-supplemented cultures was partially repressed. Molecular sieving revealed three main active components (M_r 50, 128 and 240 kDa). Glucose competitively inhibited -glucosidase activities with K_i values of 0.9mM and 3.3mM (extracellular) and 10.2mM and 22.6mM (cytosolic), induced in the presence of CMC or cellobiose respectively.The authors are with the Departamento de Biologia, Faculdade de Filosofia. Ciências e Letras de Ribeirão Preto, Universidade de São Paulo-14040-901 Ribeirão Preto, São Paulo, Brasil;     

Reciprocal regulation of Ca²+-activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide

? The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. ? In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. ? Heme (1 μM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 μM intracellular free Ca2+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. ? These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.     

Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO?

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

Structural and functional reconstruction in situ of the [CuSMoO2] active site of carbon monoxide dehydrogenase from the carbon monoxide oxidizing eubacterium Oligotropha carboxidovorans

Carbon monoxide dehydrogenase from the bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO₂ at a unique [CuSMoO₂] cluster. In the bacteria the cluster is assembled post-translational. The integration of S, and particularly of Cu, is rate limiting in vivo, which leads to CO dehydrogenase preparations containing the mature and fully functional enzyme along with forms of the enzyme deficient in one or both of these elements. The active sites of mature and immature forms of CO dehydrogenase were converted into a [MoO₃] centre by treatment with potassium cyanide. We have established a method, which rescues 50% of the CO dehydrogenase activity by in vitro reconstitution of the active site through the supply of sulphide first and subsequently of Cu(I) under reducing conditions. Immature forms of CO dehydrogenase isolated from the bacterium, which were deficient in S and/or Cu at the active site, were similarly activated. X-ray crystallography and electron paramagnetic resonance spectroscopy indicated that the [CuSMoO₂] cluster was properly reconstructed. However, reconstituted CO dehydrogenase contains mature along with immature forms. The chemical reactions of the reconstitution of CO dehydrogenase are summarized in a model, which assumes resulphuration of the Mo-ion at both equatorial positions at a 1:1 molar ratio. One equatorial Mo–S group reacts with Cu(I) in a productive fashion yielding a mature, functional [CuSMoO₂] cluster. The other Mo–S group reacts with Cu(I), then Cu₂S is released and an oxo group is introduced from water, yielding an inactive [MoO₃] centre.     

Synthesis and evaluation of imidazole–dioxolane compounds as selective heme oxygenase inhibitors: Effect of substituents at the 4-position of the dioxolane ring

Several imidazole–dioxolane compounds were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). These compounds, which include a series of substituted thiophenol and substituted phenol derivatives of (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[(phenylsulfanyl)methyl]-1,3-dioxolane hydrochloride (3), in addition to smaller functionalized derivatives, continue our structure–activity studies by exploration of the aminothiophenol region (‘northeastern region’) in our original target structure azalanstat (1). In vitro, most of the compounds in this series were found to be highly potent inhibitors of the stress-induced isozyme HO-1 and the constitutive isozyme HO-2, showing only moderate selectivity for HO-1. Nevertheless, a few of the compounds displayed higher selectivity toward HO-1. None of the compounds having a larger appendage in the northeastern region were inhibitors of CYP2E1, whereas a compound having a relatively small fluorine substituent in this region did inhibit CYP2E1; all of the compounds tested exhibited high inhibitory potency against CYP3A1/3A2.     

Effect of carbon monoxide,hydrogen and sulfate on thermophilic (55°C) hydrogenogenic carbon monoxide conversion in two anaerobic bioreactor sludges

The conversion routes of carbon monoxide (CO) at 55°C by full-scale grown anaerobic sludges treating paper mill and distillery wastewater were elucidated. Inhibition experiments with 2-bromoethanesulfonate (BES) and vancomycin showed that CO conversion was performed by a hydrogenogenic population and that its products, i.e. hydrogen and CO₂, were subsequently used by methanogens, homo-acetogens or sulfate reducers depending on the sludge source and inhibitors supplied. Direct methanogenic CO conversion occurred only at low CO concentrations [partial pressure of CO (P _CO) <0.5 bar (1 bar=10⁵ Pa)] with the paper mill sludge. The presence of hydrogen decreased the CO conversion rates, but did not prevent the depletion of CO to undetectable levels (<400 ppm). Both sludges showed interesting potential for hydrogen production from CO, especially since after 30 min exposure to 95°C, the production of CH₄ at 55°C was negligible. The paper mill sludge was capable of sulfate reduction with hydrogen, tolerating and using high CO concentrations (P _CO>1.6 bar), indicating that CO-rich synthesis gas can be used efficiently as an electron donor for biological sulfate reduction.     

Dominant-negative Inhibitors of the Clostridium perfringens ?-Toxin

The Clostridium perfringens ϵ-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ϵ-toxin. Site-specific mutations were introduced into the gene encoding ϵ-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wild-type ϵ-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ϵ-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric pore-forming toxins and provide the basis for developing new therapeutics for treating intoxication by ϵ-toxin.The Clostridium perfringens ϵ-toxin is one of the most potent bacterial toxins (1, 2). The ϵ-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3–6). The disease most frequently affects livestock animals, though the toxin may also affect humans (7–9). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ϵ-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10–13). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ϵ-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin.One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins are mixed together. Such dominant-negative inhibitors have been described for a diverse set of toxins, including Helicobacter pylori VacA (15–19), Bacillus anthracis anthrax toxin protective antigen (20–25), Bacillus thuringiensis Cry1Ab (26), and Escherichia coli ClyA cytotoxin (27). Like VacA, protective antigen, Cry1Ab, and ClyA, the ϵ-toxin assembles into oligomeric complexes containing multiple toxin monomers (28–30). In the case of VacA and protective antigen, the most extensively studied examples of toxins inhibited by dominant-negative mutants, the number of mutations that inactivate the toxins is substantially greater than the number of mutations that lead to a dominant-negative phenotype (16, 17, 24, 31, 32). Although many of the mutations leading to dominant-negative toxins are located within regions of the toxins that are believed to form the membrane insertion domain, some mutations that inactivate the toxins (but are not dominant-negative) also map within the predicted membrane insertion domains (24, 32). Thus, a deeper understanding of the nature of the dominant-negative phenotype is needed.In this study, we sought to generate dominant-negative mutants of the ϵ-toxin. We hypothesized that mutations within the membrane insertion domain of ϵ-toxin, particularly mutations that are expected to restrict movement of this domain, would lead to dominant-negative inhibitors. We expressed wild-type and site-specific mutants of the ϵ-toxin as recombinant proteins in E. coli. The recombinant proteins were purified, and cytotoxicity was assessed using an established cell culture assay. Using this approach, we identified mutant proteins that inhibited the activity of wild-type ϵ-toxin in vitro and determined the mechanism of inhibition.     

Does superoxide channel between the copper centers in peptidylglycine monooxygenase? A new mechanism based on carbon monoxide reactivity

Peptidylglycine monooxygenase (PHM) carries out the hydroxylation of the alpha-C atom of glycine-extended propeptides, the first step in the amidation of peptide hormones by the bifunctional enzyme peptidyl-alpha-amidating monooxygenase (PAM). Since PHM is a copper-containing monooxygenase, a study of the interaction between the reduced enzyme and carbon monoxide has been carried out as a probe of the interaction of the Cu(I) sites with O(2). The results show that, in the absence of peptide substrate, reduced PHM binds CO with a stoichiometry of 0.5 CO/Cu(I), indicating that only one of the two copper centers, Cu(B), forms a Cu(I)-carbonyl. FTIR spectroscopy shows a single band in the 2200-1950 cm(-)(1) energy region with nu(CO) = 2093 cm(-)(1) assigned to the intraligand C-O stretch via isotopic labeling with (13)CO. A His242Ala mutant of PHM, which deletes the Cu(B) site by replacing one of its histidine ligands, completely eliminates CO binding. EXAFS spectroscopy is consistent with binding of a single CO ligand with a Cu-C distance of 1.82 +/- 0.03 A. The Cu-S(met) distance increases from 2.23 +/- 0. 02 A in the reduced unliganded enzyme to 2.33 +/- 0.01 A in the carbonylated enzyme, suggesting that the methionine-containing Cu(B) center is the site of CO binding. The binding of the peptide substrate N-Ac-tyr-val-gly perturbs the CO ligand environment, eliciting an IR band at 2062 cm(-)(1) in addition to the 2093 cm(-)(1) band. (13)CO isotopic substitution assigns both frequencies as C-O stretching bands. The CO:Cu binding stoichiometry and peptide/CO FTIR titrations indicate that the 2062 cm(-)(1) band is due to binding of CO at a second site, most likely at the Cu(A) center. This suggests that peptide binding may activate the Cu(A) center toward O(2) binding and reduction to superoxide. As a result of these findings, a new mechanism is proposed involving channeling of superoxide across the 11 A distance between the two copper centers.     

Inhibitors of the p53/hdm2 protein–protein interaction—path to the clinic

A growing number of the elements identified in intracellular signaling events that affect cell growth and transformation are proteins that physically interact with each other via domains or specifically recognized amino acid sequences. Some of these intracellular protein–protein interactions are attractive targets for anticancer targeted therapy, but progress in this field has been compromised by the paucity of compounds with suitable biological profiles and pharmacological properties. This Letter covers salient achievements in the identification and development of inhibitors of the p53–hdm2 protein–protein interaction, and highlights different screening techniques and structure-based design approaches that may be brought to bear on the discovery and development of inhibitors of other therapeutically relevant intracellular protein–protein interactions.     

Evidence for pH-dependent multiple conformers in iron(II) heme–human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.