Inhibitors of the heme oxygenase - carbon monoxide system: on the doorstep of the clinic?

The past decade has seen substantial developments in our understanding of the physiology, pathology, and pharmacology of heme oxygenases (HO), to the point that investigators in the field are beginning to contemplate therapies based on administration of HO agonists or HO inhibitors. A significant amount of our current knowledge is based on the judicious application of metalloporphyrin inhibitors of HO, despite their limitations of selectivity. Recently, imidazole-based compounds have been identified as potent and more selective HO inhibitors. This 'next generation' of HO inhibitors offers a number of desirable characteristics, including isozyme selectivity, negligible effects on HO protein expression, and physicochemical properties favourable for in vivo distribution. Some of the applications of HO inhibitors that have been suggested are treatment of hyperbilirubinemia, neurodegenerative disorders, certain types of cancer, and bacterial and fungal infections. In this review, we address various approaches to altering HO activity with a focus on the potential applications of second-generation inhibitors of HO.     

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.     

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.     

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.     

Tunnel mutagenesis and Ni-dependent reduction and methylation of the α subunit of acetyl coenzyme A synthase/carbon monoxide dehydrogenase

Two isolated alpha subunit mutants (A110C and A222L) of the alpha(2)beta(2) acetyl coenzyme A synthase (ACS)/carbon monoxide dehydrogenase (CODH) from Moorella thermoacetica were designed to block the CO-migrating tunnel in the alpha subunit, allowing comparison with equivalent mutants in ACS/CODH. After Ni activation, both mutants exhibited electron paramagnetic resonance spectra indicating that the A-cluster was properly assembled. ACS activities were similar to those of the wild-type recombinant Ni-activated alpha subunit, suggesting that CO diffuses directly to the A-cluster from solvent rather than through the tunnel as is observed for the "majority" activity of ACS/CODH. Thus, CO appears to migrate to the A-cluster through two pathways, one involving and one not involving the tunnel. The kinetics and extent of reduction of the Fe(4)S(4) cubane in the apo-alpha subunit and the Ni-activated alpha subunit upon exposure to titanium(III) citrate were examined using the stopped-flow method. The extent of reduction was independent of Ni, whereas the kinetics of reduction was Ni-dependent. Apo-alpha subunit reduction was monophasic while Ni-activated alpha subunit reduction was biphasic, with the more rapid phase coincident with that of apo-alpha subunit reduction. Thus, binding of Ni to the A-cluster slows the reduction kinetics of the [Fe(4)S(4)](2+) cubane. An upper limit of two electrons per alpha subunit are transferred from titanium(III) citrate to the Ni subcomponent of the A-cluster during reductive activation. These electrons are accepted quickly relative to the reduction of the [Fe(4)S(4)](2+) cubane. This reduction is probably a prerequisite for methyl group transfer. CO appears to bind to reduced nonfunctional subunits, thereby inhibiting reduction (or promoting reoxidation) of the cubane subcomponent of the A-cluster.     

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

The study of Ni–Fe–Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O₂. In this work we studied these inhibition processes for the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni–Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni–Fe–Se hydrogenase is more protected against the attack by O₂. The inhibition of the Ni–Fe–Se hydrogenase activity by O₂ could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O₂ on the bridging site between Ni and Fe.     

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.     

Should intake of carbon monoxide be used as a guide to intake of other smoke constituents?

The relation between blood carboxyhaemoglobin (COHb) and plasma nicotine concentrations was studied in a group of 12 smokers smoking cigarettes of three levels of standard delivery. While the intake of carbon monoxide from a single cigarette was unrelated to the intake of nicotine, presmoking "trough" concentrations of the two substances (reflecting longer-term exposure) were highly correlated. Various other measures of nicotine exposure were at best only moderately correlated with blood nicotine concentrations. Thus trough COHb concentrations might be used to provide a reliable indication of the exposure to nicotine of individual smokers smoking the same type of cigarette, and of the relative exposure to nicotine of populations smoking cigarettes of different standard deliveries.     

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.     

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.     

The structure of carbonyl horseradish peroxidase: spectroscopic and kinetic characterization of the carbon monoxide complexes of His-42 → Leu and Arg-38 → Leu mutants

Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the involvement of two key distal heme cavity residues, histidine 42 and arginine 38, in the formation and structure of the carbon monoxide complex of HRPC (carbonyl HRPC). The rates of CO binding to the wild-type glycosylated and non-glycosylated recombinant (HRPC*) ferrous enzymes were essentially identical and exhibited the same pH dependence with pK _as at 7.4 and 4.0. Data obtained with the His-42?→?Leu [(H42L)HRPC*)] and Arg-38?→?Leu [(R38L)HRPC*] mutants allowed the pK _a at 7.4 in ferrous HRPC to be assigned to His-42. The infra-red and electronic absorption spectra of HRPC-CO, HRPC*-CO, (R38L)HRPC*-CO and (H42L)HRPC*-CO have been investigated over the pH range 3.0–10.0. HRPC*-CO exhibited two ν?(CO) bands at 1934?cm^–1 and 1905?cm^–1 whose relative intensity changed with pH, showing an acidic and a basic pK _a as previously reported for HRPC [IE Holzbaur; AM English, AA Ismail (1996) J Am Chem Soc 118?:?3354–3359]. (H42L)HRPC*-CO and (R38L)HRPC*-CO exhibited single infra-red bands at 1924.2?cm^–1 (pH?7.0) and 1941.5?cm^–1 (pH?5.0) respectively. Acidic and alkaline pK _as were determined from shifts in the infra-red frequencies and by UV-visible spectrophotometry at the Söret maxima. (H42L)HRPC*-CO exhibited a pK _a at ～pH?4.0 but no alkaline pK _a. (R38L)HRPC*-CO exhibited a single pK _a at pH?6.5. Shifts of 2–3?cm^–1 in ν?(CO) with (H42L)HRPC*-CO in D₂O show that a distal residue is H-bonding to the CO in this variant at both pD?7.5 and 3.9. However, with (R38L)HRPC*-CO, only a small shift of the ν?(CO) band was observed at pD?5.5. The results are consistent with the involvement of Arg-38 in H-bonding to the CO ligand in HRPC and with His-42 modulating the distribution of carbonyl HRPC conformers below pH?8.7. These data are discussed in terms of the importance of distal pocket polarity in HRPC. It is concluded that His-42 can have a pK _a between 4.0 and 8.7 depending on its environment and the nature of the distal ligand at position 38. This enables His-42 to carry out multiple functions during the catalytic cycle of HRPC.     

Endothelin-1 release from the lamb ductus arteriosus: are carbon monoxide and nitric oxide regulatory agents?

We have proposed that endothelin-1 (ET-1), formed through the activation of a cytochrome P450 (CYP450)-based monooxygenase reaction, is important for generation of contractile tone in the ductus arteriosus and, consequently, for closure of the vessel at birth. The present investigation was undertaken to ascertain, using an isolated ductus preparation from near-term fetal lambs, whether carbon monoxide (CO) and nitric oxide (NO) qualify as regulators of the CYP450/ET-1 system. Preparations released ET-1 at rest and its amount showed no significant reduction following removal of the endothelium. Basal release was not changed by the NO synthesis inhibitor, N(G)-nitro-L-arginine methylester (L-NAME, 100 microM), nor by agents altering cyclic GMP content (i.e. increase; ONO-1505, 1 microM) and action (i.e. decrease; LY-83583, 10 microM). These findings extend previous work showing no effect of the CO synthesis inhibitor zinc protoporphyrin IX (ZnPP, 10 microM) under the same conditions (10). Conversely, both CO (65 microM) and the NO donor, sodium nitroprusside (SNP, 10 microM), curtailed ET-1 release. ET-1 release was increased by oxygen and reduced by pyrogens (endotoxin and IL-1, both at 100 ng mL(-1)). The endotoxin effect tended to be reversed by L-NAME and ZnPP, used singly or in combination. We conclude that ET-1 is formed naturally in the ductus and that its formation may change in response to physiological (oxygen) and pathophysiological (pyrogens) stimuli. Endogenous CO and NO, however, appear to have little or no role as ET-1 regulators.     

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;     

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.     

Nitric oxide — a retrograde messenger for carbon monoxide signaling in ischemic heart

To examine the intracellular signaling mechanism of NO in ischemic myocardium, isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine in the presence or absence of 650 M of protoporphyrin, a heme oxygenase inhibitor for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor placed into the right atrium. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. Induction for the expression of heme oxygenase was studied by Northern hybridization. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [³H] myoinositol and [¹⁴C] arachidonic acid. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. Protoporphyrin modulated the effects of L-arginine. cGMP, which was remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The NO-mediated augmentation of cGMP was reduced by protoporphyrin suggesting that part of the effects may be mediated by CO generated through the heme oxygenase pathway. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [³H] inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [¹⁴C] arachidonic acid resulted in modest increases in [¹⁴C] diacylglycerol and [¹⁴C] phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfision the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. The results suggests for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. This signaling appears to be on- and off- nature, and linked with SOD content of the tissue. The signaling is transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. Our results also suggest that NO activates heme oxygenase which further stimulates the production of cGMP presumably by CO signaling. Thus, NO not only potentiates cGMP mediated intracellular signaling, it also functions as a retrograde messenger for CO signaling in heart.     

Can the restoration of natural vegetation be accelerated on the Chinese Loess Plateau? A study of the response of the leaf carbon isotope ratio of dominant species to changing soil carbon and nitrogen levels

For the heavily degraded ecosystem on the Chinese Loess Plateau, it would be of great significance if vegetation restoration could be accelerated anthropogenically. However, one major concern is that if the late successional species were planted or sown in degraded habitats, would they still be competitive in terms of some critical plant traits associated with specific habitats? Water use efficiency (WUE) is a major plant trait shaping the pattern of species turnover in vegetation secondary succession on the Loess Plateau. We hypothesized that if late successional stage plants could still hold a competitive advantage in terms of WUE, the prospects for an acceleration of succession by sowing these species in newly abandoned fields would be good. We tested this hypothesis by comparing the leaf C isotope ratio (δ¹³C) value (a surrogate of WUE) of dominant species from different successional stages at given soil C and N levels. Results indicated that leaf δ¹³C of the two dominant species that co-dominated in the second and third stages were significantly more positive than that of the dominant species from the first stage regardless of changing soil C and N. Yet the dominant species from the climax stage is a C₄ grass assumed to have the highest WUE. In addition, increasing soil nutrition had no effects on leaf δ¹³C of two dominant species in the late successional stage, indicating that dominant species from the late successional stages could still have a competitive advantage in terms of WUE in soil C- and N-poor habitats. Therefore, from the perspective of plant WUE, there are great opportunities for ecosystem restoration by sowing both dominant species and other species that co-occur in late successional stages in newly abandoned fields, for the purpose of enhancing species diversity and optimising species composition.     

Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoë

When intact Kalanchoë plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoë.