Anti-inflammatory effects of HO-1 activity in vascular endothelial cells, commentary on "Carbon monoxide donors or heme oxygenase (HO-1) overexpression blocks interleukin-18-mediated NF-kappaB-PTEN-dependent human cardiac endothelial cell death"

In this issue of Free Radical Biology & Medicine, Zabalgoitia et al. show that IL-18-dependent cell death of human microvascular endothelial cells (EC) is due to activation of p38alpha and NF-kappaB and suppression of p38beta activity. Most interestingly, IL-18 and heme oxygenase-1 (HO-1) activities appear to oppose each other in these cells. IL-18 suppresses HO-1, an effect that is mediated by instability of the HO-1 mRNA. Though the contribution of HO-1 metabolites remains somewhat a mystery, treatment with carbon monoxide releasing molecules (CORMs) also induces these same effects, implicating carbon monoxide (CO) as a major player. HO-1 and CO act to suppress IL-18-mediated activation of p38alpha and to restore p38beta activity, which is suppressed by IL-18. Furthermore, HO-1 and CO suppress NF-kappaB activation by IL-18. This suppression of NF-kappaB reduces levels of PTEN which relieves IL-18-mediated suppression of Akt activity. Thus, HO-1 and CO oppose multiple proinflammatory and pro-cell death effects of IL-18 in human microvascular endothelial cells. The results of this study imply that induction of HO-1 or application of CORMs should be protective to the microvascular endothelium. Clinical trials to test the effects of CORMs in pulmonary inflammation are ongoing. The study by Zabalgoitia et al. provides mechanistic information pertaining to the homeostatic balance of IL-18 and HO-1 activities and may be useful for designing new clinical studies and for interpretation of data from ongoing studies.     

Dicarbonyl-bis(cysteamine)iron(II): a light induced carbon monoxide releasing molecule based on iron (CORM-S1)

Carbon monoxide releasing molecules (CORMs) deliver controlled amounts of CO to biological targets and organs. The reaction of cysteamine with triirondodecacarbonyl yields dicarbonyl bis(aminoethylthiolato)iron(II) that represents an iron-based CORM with biogenic ligands. X-ray diffraction studies at a single crystal show a cis-arrangement of the carbonyl ligands in trans-position to the amino groups with average Fe-C and C-O distances of 176.8 and 114.8 pm. The CO release is mediated by irradiation with visible light (λ > 400 nm). Physiological tests using ion channels sensitive to CO revealed the light- and time-dependent decomposition of CORM-S1 without obvious adverse effects on the cellular level. CORM-S1 is thus suitable for selective CO release and possesses a high potential for therapeutic application.     

Application of carbon monoxide diffusing capacity in the mouse lung

In the past decade the mouse has become the primary animal model of a variety of lung diseases. To assess various mechanisms underlying such pathologies, it is essential to make functional measurements that can reflect the developing pathology. In this regard, the diffusing capacity for carbon monoxide is a variable that directly reflects structural changes in the lung. Although measurement of single-breath diffusing capacity of the lung for carbon monoxide (DL(CO)) has also been previously reported in mice by a number of investigators, a number of technical issues have precluded routine and widespread use of this metric in mouse models. In the present report, we describe a means to quickly and simply measure a dimensionless variable closely related to the DL(CO) in mice, termed a diffusion factor for carbon monoxide (DF(CO)). The DF(CO) procedure involves a 9-s lung inflation with tracer gases in an anesthetized mouse, followed by a 1-min gas analysis time. We have tested the approach with two common models of lung pathology, elastase-induced emphysema and bleomycin-induced fibrosis. Results show a significant 15% reduction in DF(CO) in emphysema, and a 41% reduction in the fibrosis model. Repeat measurements within a mouse were found to be highly reproducible. This pulmonary function test can thus be used to detect structural changes with these pathological models. The method can also be used to measure changes in pulmonary blood volume, since the uptake of CO is highly dependent on this variable in addition to the gas exchange surface area.     

Carbon Monoxide (CO) Is a Novel Inhibitor of Connexin Hemichannels

Hemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or “free HCs” at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions.     

Carbon monoxide-dependent energy metabolism in anaerobic bacteria and archaea

Despite its toxicity for the majority of living matter on our planet, numerous microorganisms, both aerobic and anaerobic, can use carbon monoxide (CO) as a source of carbon and/or energy for growth. The capacity to employ carboxidotrophic energy metabolism anaerobically is found in phylogenetically diverse members of the Bacteria and the Archaea. The oxidation of CO is coupled to numerous respiratory processes, such as desulfurication, hydrogenogenesis, acetogenesis, and methanogenesis. Although as diverse as the organisms capable of it, any CO-dependent energy metabolism known depends on the presence of carbon monoxide dehydrogenase. This review summarizes recent insights into the CO-dependent physiology of anaerobic microorganisms with a focus on methanogenic archaea. Carboxidotrophic growth of Methanosarcina acetivorans, thought to strictly rely on the process of methanogenesis, also involves formation of methylated thiols, formate, and even acetogenesis, and, thus, exemplifies how the beneficial redox properties of CO can be exploited in unexpected ways by anaerobic microorganisms.     

ATPace™: injectable adenosine 5′-triphosphate

Carbon monoxide (CO) is produced endogenously by heme oxygenase (HO) enzymes. HO-1 is highly expressed in many inflammatory disease states, where it is broadly protective. The protective effects of HO-1 expression can be largely mimicked by the exogenous application of CO and CO-releasing molecules (CORMs). Despite a dearth of pharmacological tools for their study, molecular methodologies have identified P2X4 receptors as a potential anti-nociceptive drug target. P2X4 receptors are up-regulated in animal models of inflammatory pain, and their knock-down reduces pain behaviours. In these same animal models, HO-1 expression is anti-nociceptive, and we therefore investigated whether P2X4 was a target for CO and tricarbonyldichlororuthenium (II) dimer (CORM-2). Using conventional whole-cell and perforated-patch recordings of heterologously expressed human P2X4 receptors, we demonstrate that CORM-2, but not CO gas, is an inhibitor of these channels. We also investigated the role of soluble guanylate cyclase and mitochondria-derived reactive oxygen species using pharmacological inhibitors but found that they were largely unable to affect the ability of CORM-2 to inhibit P2X4 currents. A control breakdown product of CORM-2 was also without effect on P2X4. These results suggest that P2X4 receptors are not a molecular target of endogenous CO production and are, therefore, unlikely to be mediating the anti-nociceptive effects of HO-1 expression in inflammatory pain models. However, these results show that CORM-2 is an effective antagonist at human P2X4 receptors and represents a useful pharmacological tool for the study of these receptors given the current dearth of antagonists.     

Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901

We report here the sequencing and analysis of the genome of the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. This species is a model for studies of hydrogenogens, which are diverse bacteria and archaea that grow anaerobically utilizing carbon monoxide (CO) as their sole carbon source and water as an electron acceptor, producing carbon dioxide and hydrogen as waste products. Organisms that make use of CO do so through carbon monoxide dehydrogenase complexes. Remarkably, analysis of the genome of C. hydrogenoformans reveals the presence of at least five highly differentiated anaerobic carbon monoxide dehydrogenase complexes, which may in part explain how this species is able to grow so much more rapidly on CO than many other species. Analysis of the genome also has provided many general insights into the metabolism of this organism which should make it easier to use it as a source of biologically produced hydrogen gas. One surprising finding is the presence of many genes previously found only in sporulating species in the Firmicutes Phylum. Although this species is also a Firmicutes, it was not known to sporulate previously. Here we show that it does sporulate and because it is missing many of the genes involved in sporulation in other species, this organism may serve as a “minimal” model for sporulation studies. In addition, using phylogenetic profile analysis, we have identified many uncharacterized gene families found in all known sporulating Firmicutes, but not in any non-sporulating bacteria, including a sigma factor not known to be involved in sporulation previously.     

The Bactericidal Activity of Carbon Monoxide–Releasing Molecules against Helicobacter pylori

Helicobacter pylori is a pathogen that establishes long life infections responsible for chronic gastric ulcer diseases and a proved risk factor for gastric carcinoma. The therapeutic properties of carbon-monoxide releasing molecules (CORMs) led us to investigate their effect on H. pylori. We show that H. pylori 26695 is susceptible to two widely used CORMs, namely CORM-2 and CORM-3. Also, several H. pylori clinical isolates were killed by CORM-2, including those resistant to metronidazole. Moreover, sub-lethal doses of CORM-2 combined with metronidazole, amoxicillin and clarithromycin was found to potentiate the effect of the antibiotics. We further demonstrate that the mechanisms underpinning the antimicrobial effect of CORMs involve the inhibition of H. pylori respiration and urease activity. In vivo studies done in key cells of the innate immune system, such as macrophages, showed that CORM-2, either alone or when combined with metronidazole, strongly reduces the ability of H. pylori to infect animal cells. Hence, CORMs have the potential to kill antibiotic resistant strains of H. pylori.     

Microbial CO Conversions with Applications in Synthesis Gas Purification and Bio-Desulfurization

ABSTRACT

Recent advances in the field of microbial physiology demonstrate that carbon monoxide is a readily used substrate by a wide variety of anaerobic micro-organisms, and may be employed in novel biotechnological processes for production of bulk and fine chemicals or in biological treatment of waste streams. Synthesis gas produced from fossil fuels or biomass is rich in hydrogen and carbon monoxide. Conversion of carbon monoxide to hydrogen allows use of synthesis gas in existing hydrogen utilizing processes and is interesting in view of a transition from hydrogen production from fossil fuels to sustainable (CO₂-neutral) biomass. The conversion of CO with H₂O to CO₂ and H₂ is catalyzed by a rapidly increasing group of micro-organisms. Hydrogen is a preferred electron donor in biotechnological desulfurization of wastewaters and flue gases. Additionally, CO is a good alternative electron donor considering the recent isolation of a CO oxidizing, sulfate reducing bacterium. Here we review CO utilization by various anaerobic micro-organisms and their possible role in biotechnological processes, with a focus on hydrogen production and bio-desulfurization.     

Microbial CO conversions with applications in synthesis gas purification and bio-desulfurization

Recent advances in the field of microbial physiology demonstrate that carbon monoxide is a readily used substrate by a wide variety of anaerobic micro-organisms, and may be employed in novel biotechnological processes for production of bulk and fine chemicals or in biological treatment of waste streams. Synthesis gas produced from fossil fuels or biomass is rich in hydrogen and carbon monoxide. Conversion of carbon monoxide to hydrogen allows use of synthesis gas in existing hydrogen utilizing processes and is interesting in view of a transition from hydrogen production from fossil fuels to sustainable (CO2-neutral) biomass. The conversion of CO with H2O to CO2 and H2 is catalyzed by a rapidly increasing group of micro-organisms. Hydrogen is a preferred electron donor in biotechnological desulfurization ofwastewaters and flue gases. Additionally, CO is a good alternative electron donor considering the recent isolation of a CO oxidizing, sulfate reducing bacterium. Here we review CO utilization by various anaerobic micro-organisms and their possible role in biotechnological processes, with a focus on hydrogen production and bio-desulfurization.     

Beneficial effects of carbon monoxide-releasing molecules on post-ischemic myocardial recovery

There is increasing evidence corroborating a protective role of carbon monoxide releasing molecules (CORMs) in injured tissues. Carbon monoxide (CO) carriers have been recently developed as a pharmacological tool to simulate the effect of heme oxygenase-1-derived CO. The effects of CORM-3, a water-soluble CO releaser, on the incidence of reperfusion-induced ventricular fibrillation (VF) and tachycardia (VT) were studied in isolated rat hearts. Hearts were treated with different doses of CORM-3 before the induction of 30 min global ischemia followed by 120 min reperfusion. We found that at concentrations of 25 μM and 50 μM of CORM-3 promoted a significant reduction in the incidence of VF and VT. Thus, the incidence of VF was reduced by 67% (p < 0.05) and 92% (p < 0.05) with 25 μM and 50 μM of CORM-3, respectively. The protective effect of CORM-3 on the incidence of VT followed the same pattern. The antiarrhythmic protection was associated with a marked attenuation in infarct size, significant decreases in cellular Na⁺ and Ca²⁺ gains and K⁺ loss. Consequently, the recovery of post-ischemic function was significantly improved. In conclusion, CORM-3 exerts beneficial effects against ischemia/reperfusion-induced injury through its abilities to release CO which mediates a cardioprotective action by regulating tissue Na⁺, K⁺, and Ca²⁺ levels.     

Decreased snake venom metalloproteinase effects via inhibition of enzyme and modification of fibrinogen

Since the introduction of antivenom administration 120 years ago to treat venomous snake bit, it has been the gold standard for saving life and limb. However, this therapeutic approach is not always effective and not without potential life-threatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases by modification of fibrinogen with iron and carbon monoxide and by inhibiting these Zn²⁺ dependent metalloproteinases directly with carbon monoxide exposure. Assessment of the fibrinogenolytic effects of venoms collected from Puff adder, Gaboon viper and Indian cobra snakes on plasmatic coagulation kinetics was performed with thrombelastography. Pretreatment of plasma with iron and carbon monoxide exposure markedly attenuated the effects of all three venoms, and direct pretreatment of each venom with carbon monoxide also significantly decreased the ability to compromise coagulation. These results demonstrated that the introduction of a transition metal (e.g., modulation of the α-chain of fibrinogen with iron), modulation of transition metal in heme (e.g., carbon monoxide modulation of fibrinogen-bound heme iron), and direct inhibition of transition metal containing venom enzymes (e.g., CO binding to Zn²⁺ or displacing Zn²⁺ from the catalytic site) significantly decreased fibrinogenolytic activity. This biometal modulation strategy to attenuate the anticoagulant effects of snake venom metalloproteinases could potentially diminish hemostatic injury in envenomed patients until antivenom can be administered.     

Complexes of horseradish peroxidase with formate, acetate, and carbon monoxide

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the "hydrated" form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP-acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463-468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron-ligand distances of 2.3 and 1.8 A, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 A from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.     

Infrared studies of carbon monoxide binding to carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme that catalyzes the reversible reduction of carbon dioxide into carbon monoxide and the coupled synthesis of acetyl-CoA from the carbon monoxide produced. Exposure of CODH/ACS from Moorella thermoacetica to carbon monoxide gives rise to several infrared bands in the 2100-1900 cm(-1) spectral region that are attributed to the formation of metal-coordinated carbon monoxide species. Infrared bands attributable to M-CO are not detected in the as-isolated enzyme, suggesting that the enzyme does not contain intrinsic metal-coordinated CO ligands. A band detected at 1996 cm(-1) in the CO-flushed enzyme is assigned as arising from CO binding to a metal center in cluster A of the ACS subunit. The frequency of this band is most consistent with it arising from a terminally coordinated Ni(I) carbonyl. Multiple infrared bands at 2078, 2044, 1970, 1959, and 1901 cm(-1) are attributed to CO binding at cluster C of the CODH subunit. All infrared bands attributed to metal carbonyls decay in a time-dependent fashion as CO(2) appears in the solution. These observations are consistent with the enzyme-catalyzed oxidation of carbon monoxide until it is completely depleted from solution during the course of the experiments.     

CO-metal interaction: Vital signaling from a lethal gas

The past few years have witnessed intense research into the biological significance of carbon monoxide (CO) as an essential signaling mediator in cells and tissues. To transduce the signal properly, CO must react selectively with functional and structural proteins containing moieties that show preferred reactivity towards this gaseous molecule. This selectivity is exemplified by the interaction of CO with iron- and heme-dependent proteins, although systems containing other transition metals can potentially become a preferential target for CO. Notably, transition metal carbonyls, which carry and liberate CO, are also emerging as a pharmacological tool to mimic the bioactivity of endogenously generated CO. Thus, exploring how CO binding to metal complexes is translated into a cytoprotective function is a challenging task and might open up opportunities for therapeutic applications based on CO delivery.     

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.     

Microbial growth on carbon monoxide

The utilization of carbon monoxide as energy and/or carbon source by different physiological groups of bacteria is described and compared. Utilitarian CO oxidation which is coupled to the generation of energy for growth is achieved by aerobic and anaerobic eu- and archaebacteria. They belong to the physiological groups of aerobic carboxidotrophic, facultatively anaerobic phototrophic, and anaerobic acetogenic, methanogenic or sulfate-reducing bacteria. The key enzyme in CO oxidation is CO dehydrogenase which is a molybdo iron-sulfur flavoprotein in aerobic CO-oxidizing bacteria and a nickel-containing iron-sulfur protein in anaerobic ones. In carboxidotrophic and phototrophic bacteria, the CO-born CO₂ is fixed by ribulose bisphosphate carboxylase in the reductive pentose phosphate cycle. In acetogenic, methanogenic, and probably in sulfate-reducing bacteria, CODH/acetyl-CoA synthase directly incorporates CO into acetyl-CoA.In plasmid-harbouring carboxidotrophic bacteria, CO dehydrogenase as well as enzymes involved in CO₂ fixation or hydrogen utilization are plasmid-encoded. Structural genes encoding CO dehydrogenase were cloned from carboxidotrophic, acetogenic and methanogenic bacteria. Although they are clustered in each case, they are genetically distinct.Soil is a most important biological sink for CO in nature. While the physiological microbial groups capable of CO oxidation are well known, the type and nature of the microorganisms actually representing this sink are still enigmatic. We also tried to summarize the little information available on the nutritional and physicochemical requirements determining the sink strength. Because CO is highly toxic to respiring organisms even in low concentrations, the function of microbial activities in the global CO cycle is critical.     

Athletic performance and urban air pollution.

Air pollution may affect athletic performance. In Los Angeles, contaminants include carbon monoxide, ozone, peroxyacetylnitrate (PAN) and nitrogen oxides, whereas in older European cities, such as Sarajevo, "reducing smog" of sulfur dioxide is the main hazard. The carbon monoxide and ozone levels expected in Los Angeles this summer could affect the athletes'' performance in endurance events at the Olympic Games. Carbon monoxide may also impair psychomotor abilities, and PAN causes visual disturbances. The only likely physiologic consequence from reducing smog is an increase in the workload of the respiratory system and thus a decrease in endurance performance. While carbon monoxide has been blamed for myocardial infarctions, nitrogen oxides for pulmonary edema and sulfur dioxide for deaths due to respiratory failure, the only illnesses that are likely to be more frequent than usual among young athletes exposed to high levels of these pollutants are upper respiratory tract infections. Therapeutic tactics include the avoidance of pollution, the administration of oxygen, vitamin C and vitamin E, and general reassurance.