Biomedical subjects

B S Masters

Publications and source records attributed to B S Masters.

At least 37 records · Page 2Linked to original sources

PubMed1999

ENDOR spectroscopic evidence for the position and structure of NG-hydroxy-L-arginine bound to holo-neuronal nitric oxide synthase.

Recently, we used 35 GHz pulsed 15N ENDOR spectroscopy to determine the position of the reactive guanidino nitrogen of substrate L-arginine relative to the high-spin ferriheme iron of holo-neuronal nitric oxide synthase (nNOS) [Tierney, D. L., et al. (1998) J. Am. Chem. Soc. 120, 2983-2984]. Analogous studies of the enzyme-bound reaction intermediate, NG-hydroxy-L-arginine (NOHA), singly labeled with 15N at the hydroxylated nitrogen (denoted NR), show that NR is held 3.8 A from the Fe, closer than the corresponding guanidino N of L-Arg (4.05 A). 1,2H ENDOR of NOHA bound to holo-nNOS in H2O and D2O discloses the presence of a single resolved exchangeable proton (H1) 4.8 A from Fe and very near the heme normal. The ENDOR data indicate that NOHA does not bind as the resonance-stabilized cation in which the terminal nitrogens share a positive charge. ENDOR-determined structural constraints permit two alternate structural models for the interaction of NOHA with the high-spin heme iron. In one model, H1 is assigned to the O-H proton; in the other, it is the NR-H proton. However, the alternatives differ in the placement of the N-O bond relative to the heme iron. Thus, a combination of the ENDOR data with appropriate diffraction studies can achieve a definitive determination of the protonation state of NR and thus of the tautomeric form that is present in the enzyme-NOHA complex. The mechanistic implications of this result are further discussed.

Arginine↗

PubMed1999

The stimulatory effects of Hofmeister ions on the activities of neuronal nitric-oxide synthase. Apparent substrate inhibition by l-arginine is overcome in the presence of protein-destabilizing agents.

A variety of monovalent anions and cations were effective in stimulating both calcium ion/calmodulin (Ca2+/CaM)-independent NADPH-cytochrome c reductase activity of, and Ca2+/CaM-dependent nitric oxide (NO.) synthesis by, neuronal nitric oxide synthase (nNOS). The efficacy of the ions in stimulating both activities could be correlated, in general, with their efficacy in precipitating or stabilizing certain proteins, an order referred to as the Hofmeister ion series. In the hemoglobin capture assay, used for measurement of NO. production, apparent substrate inhibition by L-arginine was almost completely reversed by the addition of sodium perchlorate (NaClO4), one of the more effective protein-destabilizing agents tested. Examination of this phenomenon by the assay of L-arginine conversion to L-citrulline revealed that the stimulatory effect of NaClO4 on the reaction was observed only in the presence of oxyhemoglobin or superoxide anion (generated by xanthine and xanthine oxidase), both scavengers of NO. Spectrophotometric examination of nNOS revealed that the addition of NaClO4 and a superoxide-generating system, but neither alone, prevented the increase of heme absorption at 436 nm, which has been attributed to the nitrosyl complex. The data are consistent with the release of autoinhibitory NO. coordinated to the prosthetic group of nNOS, which, in conjunction with an NO. scavenger, causes stimulation of the reaction.

Anions↗

PubMed1999

Assay of isoforms of Escherichia coli-expressed nitric oxide synthase.

The techniques described herein have added to our repertoire of experimental approaches for the characterization of the NOSs. These procedures have reinforced our conviction that the NOSs are structurally suited to perform unique functions in their cellular milieux and that these differences have physiological consequences.

Bacterial Proteins↗

PubMed1999

Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase.

NOS is a ubiquitous enzyme that has an oxygenase and reductase activity. NOS reduces electron acceptors, at the reductase domain, by a one-electron mechanism that is not inhibited by SOD. One example of this activity is the direct reduction of ferricytochrome c by nNOS. Redox cycling electron acceptors (EA in Scheme 1), such as lucigenin and NBT, are reduced by NOS to generate an intermediate radical (EAred). This radical can then be reoxidized to the parent compound by oxygen, and in the process generate superoxide. Consequently, both NBT and lucigenin will enhance NADPH-dependent superoxide generation in the presence of flavoprotein reductases such as NOS. The artificial generation of superoxide from lucigenin and NBT is a major pitfall in the use of these compounds as superoxide probes. We conclude that the use of ESR spin-trapping techniques, although not free of problems, is a viable technique for the detection and quantification of superoxide in systems containing nNOS.

Acridines↗

PubMed1999

Conformationally-restricted arginine analogues as alternative substrates and inhibitors of nitric oxide synthases.

Conformationally restricted arginine analogues (1-5) were synthesized and found to be alternative substrates or inhibitors of the three isozymes of nitric oxide synthase (NOS). A comparison of k(cat)/Km values shows that (E)-3,4-didehydro-D,L-arginine (1) is a much better substrate than the corresponding (Z)-isomer (2) and 3-guanidino-D,L-phenylglycine (3), although none is as good a substrate as is arginine; 5-keto-D,L-arginine (4) is not a substrate, but is an inhibitor of the three isozymes. Therefore, it appears that arginine binds to all of the NOS isozymes in an extended (E-like) conformation. None of the compounds exhibits time-dependent inhibition of NOS, but they are competitive reversible inhibitors. Based on the earlier report that N(omega)-propyl-L-arginine is a highly selective nNOS inhibitor (Zhang, H. Q.; Fast, W.; Marletta, M.; Martasek, P.; Silverman, R. B. J. Med. Chem. 1997, 40, 3869), (E)-N(omega)-propyl-3,4-didehydro-D,L-arginine (5) was synthesized, but it was shown to be weakly potent and only a mildly selective inhibitor of NOS. Imposing conformational rigidity on an arginine backbone does not appear to be a favorable approach for selective NOS inhibition.

Animals↗

PubMed1999

Imidazole-containing amino acids as selective inhibitors of nitric oxide synthases.

Two series of imidazole-containing amino acids (1a-e and 2a-c), all larger homologues and analogues of L-histidine, were prepared. Since imidazole and phenyl substituted imidazoles have been reported to be inhibitors of NOS and the mode of action of these compounds as heme ligands is a potential mechanism of inhibitory action, we designed imidazole-containing amino acids as combined inhibitors at both the amino acid as well as heme binding sites. To study the influence of the distance between the amino acid moiety and the imidazole moiety on inhibitory potency, the number of carbons between these two functional groups was varied from two to six. The structure-activity relationships of this class of inhibitors can be correlated with the distance between the heme and the amino acid binding sites of the enzyme. Two of the compounds (1b and 1d) with three and five methylenes between the imidazole and amino acid functional groups, respectively, were found to be potent and selective inhibitors for nNOS and iNOS over eNOS. When phenyl was substituted on the nitrogen of the imidazole, both the potency and isoform selectivity diminished.

Amino Acids↗

PubMed1998

The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding.

It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [3H]Nomega-nitro-L-arginine or [3H]BH4, but [3H]BH4 binding was reinstated after extended incubation with excess L-arginine. On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2, 6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated Kd for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 microM versus 2-3 microM). Here we propose that Cys331 plays an important role in stabilizing L-arginine binding to nNOS. Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH4 binding and nitric oxide synthetic activity.

Animals↗

PubMed1998

Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center.

Nitric oxide, a key signaling molecule, is produced by a family of enzymes collectively called nitric oxide synthases (NOS). Here, we report the crystal structure of the heme domain of endothelial NOS in tetrahydrobiopterin (H4B)-free and -bound forms at 1.95 A and 1.9 A resolution, respectively. In both structures a zinc ion is tetrahedrally coordinated to pairs of symmetry-related cysteine residues at the dimer interface. The phylogenetically conserved Cys-(X)4-Cys motif and its strategic location establish a structural role for the metal center in maintaining the integrity of the H4B-binding site. The unexpected recognition of the substrate, L-arginine, at the H4B site indicates that this site is poised to stabilize a positively charged pterin ring and suggests a model involving a cationic pterin radical in the catalytic cycle.

Amino Acid Sequence↗

PubMed1998

Inducible nitric-oxide synthase generates superoxide from the reductase domain.

In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O-2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O-2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O-2 production does not occur. Moreover, the mechanism and enzyme site responsible for O-2 generation is unknown. To determine whether iNOS produces O-2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. In the presence of NADPH, prominent O-2 adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this O-2 formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O-2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O-2 generation. Furthermore, strong O-2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O-2, and this mainly occurs at the flavin-binding sites of the reductase domain.

Animals↗

PubMed1998

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors.

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Animals↗

PubMed1997

Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production.

Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.

Adrenochrome↗

PubMed1997

An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase.

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

Amino Acid Sequence↗

PubMed1997

Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo.

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

Amino Acid Sequence↗

PubMed1997

Substrate and substrate analog binding to endothelial nitric oxide synthase: electron paramagnetic resonance as an isoform-specific probe of the binding mode of substrate analogs.

The binding of arginine analogs to endothelial nitric oxide synthase (eNOS, NOSIII) perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Using electron paramagnetic resonance as a probe of heme ligation geometry, four categories of high-spin complex could be distinguished. These are analogous to the four classes of high-spin complexes, stabilized individually by the binding of L-arginine, N-hydroxy-L-arginine (NHA), N-methyl-L-arginine (NMA), and N-nitro-L-arginine (NNA), which we have previously reported for the other two isoforms. Each of these species is five-coordinate and retains the axial thiolate ligand but each differs in its ligation geometry. N-Methyl-L-arginine is a relatively poor inhibitor of eNOS, and the NMA complex of eNOS differs from the N-methyl-L-arginine complexes of inducible nitric oxide synthase (iNOS, NOSII) and neuronal nitric oxide synthase (nNOS, NOSI) in that it is of lower rhombicity. We previously showed that inducible nitric oxide synthase, which binds NNA less tightly than eNOS and nNOS, could not form the lower rhombicity NNA complex characteristic of nNOS. Endothelial nitric oxide synthase readily forms such lower rhombicity complexes, which correlates with the tight binding of NNA to this isoform. Arginine and tetrahydrobiopterin promote loss of the flavin free radical EPR signal, while arginine analog inhibitors stabilize the radical; this suggests that the residual flavin radicals can serve as a source of reducing equivalents for slow turnover in the absence of endogenous reductant.

Animals↗

PubMed1997

Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin.

Adriamycin (or doxorubicin) is an active and broad spectrum chemotherapeutic agent. Unfortunately, its clinical use is severely restricted by a dose-limiting cardiotoxicity which has been linked to the formation of superoxide. Enzymatic one-electron reduction of adriamycin forms adriamycin semiquinone radical, which rapidly reacts with oxygen to form superoxide and adriamycin. In this way, adriamycin provides a kinetic mechanism for the one-electron reduction of oxygen by flavoenzymes such as NADPH-cytochrome P450 reductase and mitochondrial NADH dehydrogenase. We demonstrate here that the endothelial isoform of nitric oxide synthase (eNOS) reduces adriamycin to the semiquinone radical. As a consequence, superoxide formation is enhanced and nitric oxide production is decreased. Adriamycin binds to eNOS with a Km of approximately 5 microM, as calculated from both eNOS-dependent NADPH consumption and superoxide generation. Adriamycin stimulated superoxide formation is not affected by calcium/calmodulin and is abolished by the flavoenzyme inhibitor, diphenyleneiodonium. This strongly suggests that adriamycin undergoes reduction at the reductase domain of eNOS. A consequence of eNOS-mediated reductive activation of adriamycin is the disruption of the balance between nitric oxide and superoxide. This may lead eNOS to generate peroxynitrite and hydrogen peroxide, potent oxidants implicated in several vascular pathologies.

Doxorubicin↗

PubMed1997

Substrate binding-induced changes in the EPR spectra of the ferrous nitric oxide complexes of neuronal nitric oxide synthase.

A versatile diatomic physiological messenger, nitric oxide (NO), is biosynthesized by a group of flavo-heme enzymes, the nitric oxide synthases. We have examined the active site of the neuronal isoform by EPR spectroscopy of the ferrous nitric oxide complex. The nitric oxide complex of the substrate-free enzyme exhibits a cytochrome P450-type EPR spectrum typical of a hexacoordinate NO-heme complex with a non-nitrogenous proximal axial heme ligand. The NO complex of the substrate-free enzyme is rather unstable and spontaneously converts to a cytochrome P420 type pentacoordinate denatured form. Binding of L-arginine (l-Arg) enhances the stability of the hexacoordinate NO form. The EPR spectrum of the NO adduct of the enzyme-substrate complex has an increased g-anisotropy and well-resolved hyperfine couplings due to the 14N of nitric oxide. Significant perturbations in the NO EPR spectrum were observed upon Nomega-monomethyl-L-Arg and Nomega-hydroxy-L-Arg binding. The perturbations in the EPR spectrum indicate that L-Arg and its derivatives bind on the distal site of the heme in very close proximity to the bound NO to cause alterations in the heme-NO coordination structure. Interactions between the bound NO and the substrate or its analogues appear to affect the Fe-NO geometry, resulting in the observed spectral changes. We infer that analogous interactions with oxygen might be involved in the hydroxylation events during enzyme catalysis of nitric oxide synthase.

Animals↗

PubMed1997

Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes.

Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 A resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin-NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 A. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Amino Acid Sequence↗

PubMed1997

Kinetics of CO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry.

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce .NO and citrulline from L-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2-3 x 10(6) M-1 s-1) and another slow (2-4 x 10(4) M-1 s-1). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate except in the presence of both tetrahydrobiopterin and Arg. In the holoenzyme, slow forms dominate except when both reagents are absent. Geminate recombination is substantial, approximately 50%, only when fast forms predominate. Stopped-flow mixing found dissociation constants near 0.3 s-1. These data imply an equilibrium constant such that very little CO should bind at physiological conditions unless large CO concentrations are present locally.

Animals↗