Interaction of Carbon Monoxide with Transition Metals

Current Drug Targets, 2010, 11, 00-00

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Interaction of Carbon Monoxide with Transition Metals: Evolutionary Insights into Drug Target Discovery Roberta Foresti and Roberto Motterlini* Department of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, 16163 Genoa, Italy Abstract: The perception that carbon monoxide (CO) is poisonous and life-threatening for mammalian organisms stems from its intrinsic propensity to bind iron in hemoglobin, a reaction that ultimately leads to impaired oxygen delivery to tissues. From evolutionary and chemical perspectives, however, CO is one of the most essential molecules in the formation of biological components and its interaction with transition metals is at the origin of primordial cell signaling. Not surprisingly, mammals have gradually evolved systems to finely control the synthesis and the sensing of this gaseous molecule. Cells are indeed continuously exposed to small quantities of CO produced endogenously during the degradation of heme by constitutive and inducible heme oxygenase enzymes. We have gradually learnt that heme oxygenase-derived carbon monoxide (CO) serves as a ubiquitous signaling mediator which could be exploited for therapeutic purposes. The development of transition metal carbonyls as prototypic carbon monoxide-releasing molecules (CO-RMs) represents a novel stratagem for a safer delivery of CO-based pharmaceuticals in the treatment of various pathological disorders. This review looks back at evolution to analyze and argue that a dynamic interaction of CO with specific intracellular metal centers is the common denominator for the diversified beneficial effects mediated by this gaseous molecule.

Keywords: Carbon monoxide, transition metals, carbon monoxide-releasing molecules, hemoglobin, mitochondria. INTRODUCTION The constitutive (HO-2) and inducible (HO-1) heme oxygenase enzymes utilize the substrate heme to generate carbon monoxide (CO), biliverdin and iron [1, 2]. These proteins are highly conserved, they are ubiquitously present from algae to bacteria and their expression in mammals has been detected, albeit with different distribution, in all types of cells and tissues. Contrary to the initial assumption that these enzymes play essentially a catabolic role in the degradation of heme and other protoporphyrins, it is now clear that HO-1 and HO-2 directly regulate several physiological processes [3, 4] not only within the cardiovascular, nervous and immune systems, but also in other disparate tissues such as skin, bone and muscle [5-8]. The last ten years have witnessed an increasing interest in the field of heme oxygenase research and, as more and more information is gathered to give us a better understanding of how these enzymic pathways work, two important concepts have emerged: 1) the inducible isoform protein, HO-1, is a fundamental ‘sensor’ of cellular stress and actively contributes to reduce or prevent the damage caused by the stress itself and 2) the products of heme oxygenase enzymic activity dynamically participate in the control of cellular adaptation to stress and are inherently involved in the mechanisms of cytoprotection elicited by HO-1 and heme oxygenase-2 (HO-2). Among these products, CO is undoubtedly captivating both from chemical and biological perspectives. It is a small gaseous molecule with strong reducing capacities, it binds iron and other transition metals with high affinity and its physical properties make it potentially very dangerous [9-11]. It is the hazardous properties of CO that scientists know best and fear *Address correspondence to this author at the Department of Drug Discovery and Development, Italian Institute of Technology, Via Morego, 30, 16163 Genova, Italy; Tel: +39-01071781547; Fax: +39-010720321; E-mail: [email protected] 1389-4501/10 $55.00+.00

most, as for many it is still rather surprising that this gas can play after all any beneficial role; to this end, even more challenging is the idea to exploit CO for therapeutic purposes. This article is intended to broadly review the chemical properties and reactivity of CO with transition metals, integrate them into some of the most common reactions known for CO in physiology and rationalize why and how CO-metal interaction is gradually becoming an important element in drug target discovery. CO-IRON INTERACTION: AN OBLIGATORY STEP IN THE EVOLUTION OF BIOMOLECULES AND PRIMORDIAL INTRACELLULAR SIGNALLING It is well accepted that organic synthesis in the Earth’s primitive atmosphere was an opening step for the sequence of events that lead to the origin of life. The most convincing evidence supporting the synthesis of organic biomolecules from inorganic precursors has been put forward by Stanley Miller with his seminal experiment conducted in 1953. Using a mixture of methane (CH4), hydrogen (H2), water (H2O) and ammonia (NH4), Miller showed that these components of the early atmosphere could be converted into aminoacids when simply reacted with an electrical spark [12]. Remarkably, in recent years Miller and co-workers demonstrated that adding carbon monoxide (CO) gas to their original mixture of inorganic components, and thereby simulating a CO-dominant atmosphere, gave origin to a variety of bioorganic compounds, including aminoacids and nucleic acid bases (primarily uracil and cytosine), with yields comparable to those obtained from a strongly reducing environment [13]. It is also intriguing that photolysis of water vapour with CO, but not carbon dioxide (CO2), results in the production of alcohols, aldehydes and organic acids confirming that the presence of CO gas was essential for the © 2010 Bentham Science Publishers Ltd.

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formation of pool of organic material on the primitive Earth [14]. Another theory on the origin of life provides evidence that CO served as an important element in the formation of peptides from hydrothermal vents. In experiments modeling volcanic or hydrothermal settings, aminoacids were converted into their peptides by use of a cluster of iron (FeS) and nickel sulfide (NiS) in conjunction with CO gas under anaerobic and aqueous conditions at high temperatures and pressures [15, 16]. These results demonstrate that aminoacids can be activated under geochemically relevant conditions and that evolution of a primordial metabolism occurs in the presence of CO gas and transition metal minerals, primarily Fe and Ni. It is important to emphasize that, from a chemical perspective, CO is a highly reducing agent and the theory supporting a primitive reducing environment rich in this gas makes sense also in view of the fact that the early microorganisms appeared on Earth 3.5 billion years ago when the atmosphere still lacked of molecular oxygen (O2). In fact, O2 started to be produced in the atmosphere thanks to the advent of photosynthesis carried out initially by cyanobacteria (around 3.2 billion years ago) and later on by higher plants (480 million years ago). The gradual accumulation of O2 in the atmosphere is the crucial event that enabled the proliferation of aerobic organisms. Thus, the presence of CO (a reducing agent) and Fe alongside the absence of O2 (an oxidizing molecule) were essential conditions for the chemical evolution of early biomolecules and the development of primordial organisms. It is evident that as more complex forms of life slowly adapted to the new environment, even more sophisticated and integrated biochemical mechanisms were gradually required for cell signalling and function. At this point one can hypothesize that these signalling mechanisms might have relied on and involved the same “rudimentary” components that were so essential for the chemical evolution of aminoacids and DNA/RNA bases. If so, it is then not surprising that the interaction between biologically relevant transition metals (essentially Fe) and CO gas represents one of the principal modes of signal transduction utilized by the early microorganisms. The concept finds its best example in the phototropic bacterium Rhodospirillum rubrum, an ancient living organism that evolved and adapted to the primordial atmosphere. This prokaryote can survive in complete darkness under anaerobic conditions using CO gas as the sole carbon and energy source [17]. This is achieved through the catalytic oxidation of CO to carbon dioxide (CO2) and hydrogen by the enzyme CO-dehydrogenase (CODH), a protein containing Ni-Fe-S clusters that utilizes water to transfer an oxygen atom to CO [18]. Notably, Rhodospirillum rubrum responds to exogenous CO gas by activating CooA, a heme/Fe-containing transcription factor that specifically regulates the expression of CODH enzymes [19]. CO gas binds avidly to the heme/Fe moiety of CooA to form a stable six-coordinate CooA-CO complex that allows sequence-specific DNA binding and transcription of CODH [20]. Thus, CooA senses CO through its heme/Fe group and transduces the CO signal into its direct binding to DNA thereby promoting the expression of CODH enzymes [21]. This primitive intracellular mode of sensing has been somehow preserved throughout evolution since NPAS2, a heme-dependent transcription factor that resembles CooA in its mode of action and is crucial in the

Foresti and Motterlini

control of circadian rhythm, is highly and selectively responsive to CO gas in mammals [22]. Another important primitive enzyme that deserves mentioning here is acetyl coenzyme A synthase (ACS). When acting in concert, the CODH/ACS catalytic sites are key to organic carbon fixation from CO2 in some bacteria and archaea [23]. This reductive acetyl-CoA pathway is also known as the Wood Ljungdahl Pathway and intermediates to the formation of acetyl-CoA include CO and formic acid. From a biological view point, we now know that CO produced endogenously by heme oxygenase enzymes in mammalian cells acts as a pivotal and versatile signaling molecule being able to interact with a diverse array of Feand metal-containing targets [10]. CO, either generated intracellularly or delivered at concentrations that do not compromise cell survival, modulates important physiological processes that in several circumstances are manifested as therapeutic effects in animal models of disease [24-26]. Paradoxically, the multiple beneficial activities mediated by CO in pathophysiological conditions reported in the last decade are still overshadowed by the renowned poisoning effects of this gas, believed to be primarily caused by its interference with the function of hemoglobin [27]. That is, the binding affinity of ferrous iron (Fe2+) in hemoglobin for CO is much higher compared to molecular oxygen (≈220fold) and the consequent formation of carbon monoxy hemoglobin (HbCO) decreases the O2-carrying capacity of red blood cells thereby compromising O2 delivery to tissues [25]. As we will scrutinize in the following sections, this assertion commonly accepted by the scientific community is misleading for different reasons, not least the fact that the toxicity mediated by CO strictly depends on the amount of gas inhaled by a living organism, the time of exposure and the metabolic status of the tissue being considered. Thus, to what extent is the elevation of HbCO in red blood cells a reliable and relevant marker of CO poisoning? Can we quantify precisely the threatening doses of CO in mammals? Or should we start to identify and characterize new markers of CO activity in order to distinguish between the harmful effects of this gas from its therapeutic potentials? Addressing these important questions should clarify some of the misconceptions related to CO poisoning (see below) and may facilitate the effective design of CO-based pharmaceuticals. IS CARBONMONOXY HEMOGLOBIN (HbCO) A RELIABLE MARKER OF CO POISONING? We believe the answer to the above question come into sight from an eloquent editorial comment written by Thomas Poulton in 1985 [28]. The letter was sent to the journal Chest in response to an article published by Aronow et al. who stated that banked blood should be strictly tested for the HbCO content and that blood containing relatively high levels of HbCO ought not to be given to patients suffering of significant cardiac or pulmonary disease [29]. In the introductory sentence of his commentary, Poulton vividly remarked that Aronow and colleagues identified and proposed a solution to a “problem which does not exist”. Poulton then continues his letter strongly arguing the data presented by Aronow: “Implicit in the authors’ concern, and in many of the papers they cite, is the mistaken notion that organ dysfunction associated with exposure to carbon

Interaction of Carbon Monoxide with Transition Metals

monoxide (CO) is a result of elevated levels of HbCO. This traditional view was laid to rest by Goldbaum et al. in an elegantly simple study published in 1975”. Indeed, Goldbaum and colleagues conducted a series of welldesigned experiments in vivo to address this important question: “What is the mechanism of carbon monoxide toxicity?” [30]. The authors studied 15 dogs that were divided into three groups of five dogs each. In Group 1, dogs were administered 13% CO gas by inhalation from a re-breathing bag. In Group 2, dogs were bled to a severe anemic state such that the hemoglobin content was reduced by approximately 70%. In this group dogs were then transfused with 1:1 Ringer’s lactate solution to adjust the volume. In Group 3, dogs were bled to the same anemic state as those in Group 2 but were then transfused with red blood cells which 80% hemoglobin was saturated with CO. The authors found that: dogs breathing CO gas (Group 1) died within 1 h with an average HbCO levels of 65%; dogs bled and kept in anemic state with blood hemoglobin reduced by 70% (Group 2) survived indefinitely; surprisingly, dogs bled and then transfused with red blood cells containing 80% HbCO from a donor dog (Group 3) also survived indefinitely with an average HbCO level of 60%. It is important to emphasize how the different treatments on dogs were perfectly matched to achieve the same degree of reduction in O2 supply and how well the experiments were planned in order to address the specific question under analysis. From the results, one can observe that a dramatic decrease in the O2 carrying capacity of hemoglobin to 30-40% can be achieved in two ways: either by reducing the amount of blood (Group 2) or by replacing an equivalent amount of O2 with CO bound to hemoglobin (Group 3). The results show, perhaps surprisingly, that in both cases this severe treatment is not lethal. The most striking result, however, emerges when comparing Group 1 and 3 in which dogs after treatment have nearly the same amount of HbCO in blood (65 and 60%), an effect that was again achieved in two different ways but with completely opposite outcomes. While dogs inhaling “free” CO gas died, the animals receiving CO “carried” by hemoglobin survived. The cross analysis of the three groups clearly indicate that CO strictly bound to hemoglobin does not compromise the O2 carrying capacity of this protein, even when 60% of the hemes are occupied by CO. On the other hand, administering CO gas becomes detrimental notwithstanding hemoglobin retains its capacity to deliver enough O2 to the organism. Thus, it is not the CO bound to hemoglobin that is toxic, rather the fraction that escapes it. We deduce that the level of HbCO in blood should not be taken as a reliable marker of CO poisoning which is in any case a poorly defined pathophysiological event. In fact, the quality of health outcomes in CO-poisoned patients usually does not correlate with HbCO levels. There is an evident lack of proper control groups in the studies and the results are often confounded by different times of exposure to CO, the amount of CO being inhaled and most importantly the auto-regulatory mechanisms that differ significantly among individuals [31, 32]. That a high level of HbCO is not harmful to mammalian organisms finds strong support in two more recently published papers. Cabrales et al. investigated the effect of CO in a model of hemorrhagic shock and resuscitation in hamsters. Shock was initially induced by 50% withdrawal of

Current Drug Targets, 2010, Vol. 11, No. 11

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blood and after 1 h blood volume was partially restored (25%) by a single infusion with fresh red blood cells (RBCs) saturated or unsaturated with CO. The authors found that systemic and microvascular parameters such as mean arterial pressure, functional capillary density and microvascular blood flow were not different after resuscitation with RBCs carrying O2 or CO; if anything, CO saturated blood partially mitigated cell injury after resuscitation [33]. Similarly, Sakai and co-workers studied how CO bound to either RBCs (CORBCs) or haemoglobin encapsulated into lipid vesicles (COHbV) affects resuscitation after hemorrhagic shock in rats and compared this with the effect of oxygenated blood (O2RBCs). The volume of CO-RBCs or CO-HbV used in the experiments was such that HbCO levels was about 40% during the first hour of resuscitation and then gradually returned to basal levels (20%) is detected [25, 32, 36]. For obvious reasons, and for the technical limitations highlighted above, the effect of smaller amounts of CO on mitochondrial activity in vivo under normal pO2 has never been a topic of interest in the medical field. Only recently, however, scientists started to question whether mitochondrial heme-dependent proteins other than cytochrome c oxidase could specifically interact with CO and began to investigate how mitochondrial function could be affected by concentrations of CO that are more physiologically relevant. A recent study on the interaction of cytochrome c with CO places this important issue under the right perspective as it reveals why scientists should not rely solely on CO/O2 binding affinity data in vitro when referring to the entire cellular scenario. As partly anticipated above, cytochrome c from eukaryotic cells is a water-soluble 13 kDa heme-containing protein that is encoded by a nuclear gene and normally resides on the intermembrane space side of the cristae within the inner mitochondrial membrane [37]. Its major function is to shuttle electrons from complex III to complex IV (cytochrome c oxidase) in the respiratory chain but at the same time it plays an important role in apoptosis. When a cell receives an apoptotic stimulus, cytochrome c is released into the cytosol where it engages the apoptotic proteaseactivating factor-1 to bind procaspase-9 which in turns activates caspase-3 and -7, the executioners of programmed cell death [37]. It is important to point out that in its native form, the heme iron in cytochrome c is coordinated by two internal axial ligands, His-18 and Met-80, and cannot bind CO or O2. However, studies with mutants in which a chemical modification of Met-80 precludes the interaction of the heme iron with this aminoacid allows external ligands like CO to bind and increase the effective redox potential [38]. It is also known that within the mitochondria cytochrome c forms a complex with cardiolipin, an anionic phospholipid that accounts for 25% of all phospholipids present in the


inner mitochondrial membrane [39]. Intriguingly, upon interaction with cardiolipin, isolated cytochrome c changes its tertiary structure, disrupts the heme-Met bond and binds CO with an affinity that is one order of magnitude higher than the affinity in myoglobin [39]. Notably, oxidation of cytochrome c also stimulates caspase activation, whereas reduction of cytochrome blocks caspase activation [37]. Together these results suggest that, despite the binding affinities for CO and O2 in vitro disfavor an interaction with reduced cytochrome c, within its native environment in mitochondria this protein can be highly responsive to CO. Thus, is it possible that the cytoprotective and anti-apoptotic activities observed in cells treated with CO gas or COreleasing molecules may well be related to blockade of this crucial apoptotic trigger and/or other heme-dependent proteins in mitochondria [40-42]? We need to remind ourselves that mitochondria are also a major source of reactive oxygen species (ROS) which are markedly increased during pathological conditions and represent the common denominator of aging and neurodegenerative diseases [43]. Although a direct link between the binding of CO to cytochrome c, inhibition of apoptosis and modulation of ROSderived mitochondria has not been demonstrated, circumstantial data from published literature in the last few years point to these organelles as a one of the targets for the therapeutic actions of CO. For instance, vascular smooth muscle cells treated with inflammatory cytokines to stimulate apoptosis and simultaneously exposed to low concentrations of CO gas (50–200 ppm) results in a significant decrease in positive annexin V staining and caspase-3 activation as well as reduced translocation of cytochrome c from the mitochondria into the cytosol [44]. Mitochondrial respiration and ROS production from these organelles are markedly modulated when CO is delivered as a gas or via CO-releasing molecules in human airway smooth muscle cells [45], cardiomyocytes [46], hepatocytes and endothelial cells [47, 48]. Surprisingly, under many circumstances a controlled delivery of CO to these tissues in vivo appears to improve rather than compromise respiration and energy metabolism [49-55]. For instance, administration of a COreleasing molecule has been shown to rescue mice from lethal sepsis through unidentified mechanisms that lead to restoration of mitochondrial membrane potential, increased mitochondrial respiratory function and energetic as well as enhanced mitochondrial biogenesis [53]. We must also emphasize that the ability to increase mitochondrial activities seems to be a peculiarity of CO gas since nitric oxide (NO), an analogous signaling molecule, inhibits mitochondrial and cellular respiration selectively by binding to cytochrome c oxidase [56] with an apparent affinity in vitro that surpasses both O2 and CO. Thus, the original codes of belief on CO being exclusively a competitor of O2 transport by hemoglobin and a mere inhibitor of mitochondrial respiration are strongly challenged. Other sensitive targets for CO must be searched within the cell (see below), knowing that mitochondria might play an important role in CO-mediated protective effects. In line with the evolutionary insights elaborated at the beginning, we have learned that preferential targets for CO in cells are still and remain transition metals. The demonstration that the toxicity of CO can be minimized by its binding to iron in hemoglobin offers new, albeit counterintuitive,


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opportunities to exploit this feature of CO for drug development [57]. From a chemical perspective, it is informative to know that the ferrous (Fe2+)-CO moiety within hemoglobin is, by definition, a “transition metal carbonyl ”. Interestingly, it is on this type of complexes that the first proposition of a prototypic class of CO-based pharmaceuticals was made. CO-METAL COMPLEXES: A LOOK INTO THE DEVELOPMENT OF CO-RELEASING MOLECULES (CO-RMs) AS THERAPEUTICS Transition metal carbonyls are a class of compounds primarily known in the organometallic chemistry field for their versatile use in industrial catalysis and purification processes [58]. These complexes contain a transition metal, such as Ni, Fe, manganese (Mn) or cobalt (Co), surrounded by a certain number of carbonyl (CO) groups. One of the main characteristics of these chemicals is that, under certain conditions, they can undergo dissociative loss of CO [59]. It is based on this peculiar feature that our group used the scaffold of metal carbonyls to demonstrate that some of these compounds can indeed liberate CO in biological systems. We coined the term “CO-releasing molecules” (CO-RMs) to describe those carbonyl complexes that as consequence of CO loss were capable of exerting a given pharmacological effect in cell systems therefore resembling the one promoted by heme oxygenase-derived CO [60]. Several reviews on this topic have been published in recent years and readers should refer to them for the full details of this discovery [61-64]. Here we just want to briefly reiterate that the development of CO-RMs was mainly motivated by a practical requirement of having a ‘solid form’ of CO. We thought this would greatly facilitate the use of CO in laboratory experiments since at the time CO gas was the only other option available when scientists wanted to study the role of CO in cellular function. The discovery of transition metal carbonyls as carriers of CO that can deliver the molecule to biological tissue identified for the first time a class of chemicals that fulfilled our needs [60]. Of course, dealing with CO produced by heme oxygenases is different from dealing with CO gas and transition metal carbonyls added an extra layer of complexity: the presence of the metal and the carbonyl moieties necessitated a deep understanding of transition metal carbonyl complexes chemistry and a completely novel approach for studying the behaviour of the compounds in biological systems [65, 66]. We established a series of biochemical and biological assays that helped us to compose a profile for all the substances that we tested over time, from the initial commercially available non “watersoluble” CORM-1 (Mn-based) and CORM-2 (Ru-based) [60, 66] to the first synthesized water-soluble CORM-3 (Rubased) [41] and all the other CO-RMs containing different metal centers subsequently produced by our chemist collaborators [67-70]. In alternative to transition metal carbonyls, a boranocarbonate (CORM-A1) was discovered as another type of CO-RM that does not contain a transition metal but is based on boron [71]. Therefore, a myoglobin assay for the detection of carbon monoxy myoglobin (MbCO) and a CO sensitive electrode were initially utilized to determine the rate of CO release from these compounds [60, 71]. In parallel, lactate dehydro-


genase and Alamar blue assays were employed in various cell types to assess thresholds of cell viability upon exposure for different times to varying concentrations of CO-RMs [60]. Vascular relaxation induced by CO-RMs in pre-contracted aortic rings [60, 71, 72] and their ability to lower nitrite production in lipopolysaccharide-stimulated macrophages [73, 74] were also used to define preliminary pharmacological activities, namely vasodilatation and antiinflammatory action. We then tested the compounds in models of disease, ranging from cardiac ischemia-reperfusion and infarction [41, 75, 76], cardiomyopathy [77], graft rejection and organ preservation [41, 78-80], collageninduced arthritis [81] to cysplatin-induced nephrotoxicity [42], bacterial infection [82, 83] and inflammatory disorders [53, 84, 85]. In addition, several collaborators who participated in the use of CORM-3 and other groups utilizing commercially available CORM-2 or CORM-3 synthesized in-house [84, 86-89] generated a wealth of information on the properties and functions of CO-RMs, enabling us to draw more conclusions on the characteristics of these compounds. An encouraging result from these studies is that the majority of activities elicited by CO-RMs appear to be caused by CO delivered to cells and tissues, as the CO-depleted compounds (negative controls) lack of the pharmacological effect [60, 73]. A second indication is that CO-RMs possess antiinflammatory activities and this property is appreciable in fast CO releasers such as CORM-2 and CORM-3 [73]. For example, our investigations highlighted that CORM-A1, the boron-based substance that was mostly studied, is a watersoluble compound that releases CO with a slower kinetic than the majority of transition metal complexes we examined. Furthermore, CO liberation by CORM-A1 is pHdependent, with faster release at acidic pH [71]. When the effect of CORM-A1 was compared to CORM-3 and other transition metal carbonyls in experimental settings using physiological pH it emerged that the latter could diminish NO production by activated macrophage in a concentrationdependent manner while CORM-A1 was ineffective (unpublished results). A similar profile has been obtained when we looked at the antibacterial effects of CO-RMs: while CORM3 and other metal containing CO-RMs are bactericidal, CORM-A1 has only a temporary bacteriostatic effect [83] (unpublished results). This does not seem to depend on the rate of CO release as metal-containing CO-RMs with a halflife similar to CORM-A1 were still effective in preventing bacterial growth (unpublished results). In addition, and perhaps most importantly, the cytotoxic effects of CO is less pronounced in the case of CORM-3 compared to CORM-A1 [61]. Thus, it appears that the metal center in CO-RMs can facilitate the action of CO and control its potential cellular toxicity. If this concept is not convincing enough, one should still refer to the lack of toxicity by HbCO (effectively an Fecarbonyl) corroborated above. It is important to emphasize that some of the transition metal carbonyls, such as CORM-3 and CORM-2 for example, do not spontaneously release CO but require first a vacant ligand to enter the metal center to favour the liberation of CO and at the same time a receptor to accept CO gas. In fact, while we observe an overlapping of data when measuring the rate of CO release from CORMA1 using both the myoglobin assay and the CO electrode [71], CO liberation from CORM-3 and CORM-2 can be detected only by using myoglobin as an acceptor of CO. The


need of a “metal acceptor” as target for CO has been confirmed by others suggesting that certain CO-RMs are stable in solution and dissociative loss of CO can be fully achieved if these conditions are met [90]. Within a series of newly synthesized CO-RMs containing molybdenum (Mo) and tungsten (W), it has been reported that in these complexes the rate of CO-release is not controlled by the metal–CO bond strengths but crucially by the rate of hydrolysis of the ligand [91]. Therefore, CO release in such systems is not a simple loss of CO from the coordination sphere of the metal and it is the nature of the interaction between the CO-RM and water which appears to control CO-release. More studies are needed to understand the exact mechanisms by which metal-CO carriers liberate CO and which factors control this release within the intracellular milieu. However, metal carbonyls represent the first example of compounds that can be optimized to control CO-mediated cellular toxicity and maximize its beneficial effects as results published so far in the literature confirm that CO-RMs can be finely tuned to the requirements needed and can be safely used within a range of doses (10-40 mg/kg) in pre-clinical models of diseases [53, 84, 88]. These promising features are encouraging as the right choice of transition metal and the ideal kinetic of CO release will enable the synthesis of safer compounds for a given therapeutic indication. CO-METAL SIGNALLING: WHERE IS THE INTRACELLULAR TARGET? As anticipated above, NO is another important signalling gas that shares some physiological effects with CO. However, unlike NO, there is not a well characterized intracellular binding protein or receptor generally accepted as the putative target of CO. In fact, activation of guanylate cyclase and S-nitrosylation of specific proteins explain a wide variety of cellular actions exerted by NO [92, 93], whereas for CO there is still an ongoing search for the appropriate cellular effector. For example, although CO activates guanylate cyclase and an increase in cGMP levels has been clearly detected in tissues over-expressing heme oxygenase-1 (HO1) or following exogenous CO administration [94-96], it is also known that the affinity of CO for this enzyme is much lower than for NO [97]. Guanylate cyclase is normally highly responsive to NO: for example, in vitro studies showed a 100-200-fold activation by NO compared to 4-fold activation by CO [97]. However, there might be circumstances such as NO inactivation and/or overproduction of CO whereby the enzyme can also be activated by CO. In order to identify which preferential targets could be hit by CO in the biological environment, it is worthy to briefly consider the chemical reactivity of this gas. Because of its chemical structure, where four of the ten electrons form a lone pair on each of the carbon and oxygen atoms, CO coordinates only to elements in the periodic table that are strong electron acceptors [11]. There are only few elements that at room temperature possess this property and they are boron and transition metals in low oxidation state. When CO forms a bond with a transition metal, the distribution of electrons in the orbitals of the metal and CO complex is such that the bond is linear and stable [11], although the coordination with CO makes the metal more reactive. These principles apply to the chemistry regulating transition metal complexes forma-


tion, but possibly extend to the behaviour of CO generated intracellularly by heme oxygenase. From this assumption it is envisaged that the interaction of CO with any potential cellular target should involve a metal, thus relatively restricting the number of molecular candidates. One obvious target is heme, containing iron as the metal center in the protoporphyrin ring, which is the prostetic group of many proteins and enzymes, including guanylate cyclase, and thus ubiquitously distributed in the cell. The binding of CO to heme is well documented as proteins such as hemoglobin, myoglobin, cytochrome P450 family and cytochrome c oxidase avidly sequester CO when the gas is present at high, non-physiological levels. As discussed above, usually this binding is associated with impaired protein/enzyme activity, since CO at concentrations that strongly compete with O2 will interfere with its transport/storage by hemoglobin and myoglobin and inhibit the catalytic activity of cytochrome P450 and cytochrome c oxidase. By inference, it is predicted that other heme-containing proteins may bind CO and therefore constitute a target for CO-mediated actions, with hemedependent mitochondrial enzymes as major participants because of the dense population of mitochondria in tissues, especially skeletal and heart muscle. The task here would be to examine briefly but systematically all the known cellular heme-containing proteins and, leaving aside the mitochondrial ones (see above), establish whether CO affects their activity. There is an interesting enzyme, NADPH oxidase, which encloses two heme groups in the enzyme complex and is affected by CO. The studies have demonstrated that the spectra of the heme moieties in the enzyme change when cells are exposed to CO (from CORM-2) [45] and that its activity is inhibited by CO [98]. As a consequence, it appears that CO exerts anti-proliferative effects in human airway smooth muscle [45] and decreases migration of vascular smooth muscle in models in vitro [98] via binding to NADPH oxidase. The inhibition of NADPH oxidase activity results in a decreased production of superoxide anion, indicating that one of the functions elicited by CO is regulation of the intracellular redox status. The case of potassium channels is also worth mentioning, as the mechanisms underlying channels activation by CO have not been fully elucidated and possibly involve the participation of heme. It was initially observed that CO increased the open probability of big conductance calcium-activated potassium channels and that a direct interaction of CO with histidine residues mediated this action [99]. However, another group showed that CO activated these channels by interacting with heme associated to a heme-binding domain of the channel [100]. The authors also demonstrated that activation by CO would occur only in the presence of heme, which binds CO, but not hemin, the oxidized form which does not interact with CO [100]. Despite additional studies [101, 102], the matter has not been resolved and clear evidence of the cofactors needed for CO to affect potassium channels activity is still lacking. There are proteins that do not contain heme but present aminoacidic residues on the surface or in the catalytic site that allow for binding of metals [103]. In particular, the aminoacids cysteine, hystidine and tyrosine are excellent binding sites for coordination with metals and plenty of examples exist in nature of metal-containing enzymes, such as xanthine oxidase (molybdenum), superoxide dismutases


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(copper, zinc, manganese, iron and nickel in some bacterial superoxide dismutases), alcohol dehydrogenase (zinc) and poly (ADP-ribose) polymerase 1 (zinc). These non-covalently bound metals are often involved in the catalytic action of the enzymes and could constitute the bridge for binding of CO to proteins; a reversible bond with CO would conesquently regulate enzyme activity. Recently, the active site of the [NiFe] hydrogenase from Desulfovibrio vulgaris has been shown to bind CO with resulting inhibition of enzyme function [104], suggesting that similar mechanisms might take place also in mammalian cells. Whether CO may regulate other enzymatic pathways in the absence of heme/metal mediators needs to be further investigated and the potential chemical reactions explaining such an interaction has not been described. In this respect, CO was shown to modulate cellular functions via p38 mitogen-activated protein kinase [105], but this might not be a direct activation and rather represents part of a signalling cascade. CO (from CORM-2) also appears to directly activate purinergic ionootropic P2X2 receptors [106]; however, the transition metal in CORM-2 (ruthenium) may be the intermediary factor assisting the coordination of CO to the channels and experiments using CO gas and metal chelators could help to elucidate this possibility. As a final note, we would like to point out that most experiments where the interaction of CO with different enzymes has been investtigated were conducted at high, non-physiological levels of CO. This suggests that the results have significance in the context of CO poisoning, but might be less relevant for physiological or pathophysiological scenarios where CO derives primarily from the enzymatic activity of HO-2 and HO-1. Indeed, it is tempting to speculate that in normal conditions and/or following over-expression of HO-1 the cellular role of CO is more akin to that of a signalling/modulating small molecule. That would indicate that regulation of the redox status or energy production (see above interaction of CO with mitochondria), fundamental processes associated with many disease states, might rely also on the participation and cellular sensing of CO. CONCLUSIONS The toxicity of CO as much as its potential therapeutic effects are manifested through selective transduction signals involving transition metals. This specificity of action makes CO a rather unique and peculiar molecule in biology and a true competitor of molecular oxygen. The design of more physiologically relevant models that better simulate the competition of CO and O2 with metal centers within the intracellular compartments will help to identify targets that may be of values in the discovery of new drugs. ACKNOWLEDGEMENTS The authors thank Dr. Nick Lane for critical reading of the manuscript and constructive advice. ABBREVIATIONS ACS

= Acetyl-CoA synthase

Co

= Cobalt


CO

= Carbon monoxide

CODH

= Carbon monoxide dehydrogenase


[19]

CO-RMs = Carbon monoxide-releasing molecules Fe

= Iron

HbCO

= Carbonmonoxy hemoglobin

HO-1

= Heme oxygenase-1

HO-2

= Heme oxygenase-2

Mn

= Manganese

Mo

= Molybdenum

Ni

= Nickel

NO

= Nitric oxide

RBCs

= Red blood cells

ROS

= Reactive oxygen species

Ru

= Ruthenium

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Received: January 28, 2010

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Revised: March 18, 2010

Accepted: May 31, 2010