Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.     

Ligand binding properties of bacterial hemoglobins and flavohemoglobins

Bacterial hemoglobins and flavohemoglobins share a common globin fold but differ otherwise in structural and functional aspects. The bases of these differences were investigated through kinetic studies on oxygen, carbon monoxide, and nitric oxide binding. The novel bacterial hemoglobins from Clostridium perfringens and Campylobacter jejuni and the flavohemoglobins from Bacillus subtilis and Salmonella enterica serovar Typhi have been analyzed. Examination of the biochemical and ligand binding properties of these proteins shows a clear distinction between the two groups. Flavohemoglobins show a much greater tendency to autoxidation compared to bacterial hemoglobins. The differences in affinity for oxygen, carbon monoxide, and nitric oxide between bacterial hemoglobins and flavohemoglobins are mainly due to differences in the association rate constants. The second-order rate constants for oxygen and carbon monoxide binding to bacterial hemoglobins are severalfold higher than those for flavohemoglobins. A similar trend is observed for NO association with the oxidized iron(III) form of the proteins. No major differences are observed among the values obtained for the dissociation rate constants for the two groups of bacterial proteins studied, and these constants are all rather similar to those for myoglobin. Taken together, our data suggest that differences exist between the mechanisms of ligand binding to bacterial hemoglobins and flavohemoglobins, suggesting different functions in the cell.     

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin.

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M.     

Kinetic studies of the reactions of O(2) and NO with reduced Thermus thermophilus ba(3) and bovine aa(3) using photolabile carriers

The reactions of molecular oxygen (O(2)) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba(3) and bovine heart aa(3) were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O(2) and NO complexes. The binding of O(2)/NO to reduced ba(3) in the absence of CO occurs with a second-order rate constant of 1×10(9)M(-1)s(-1). This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O(2)/NO binding in ba(3) is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O(2) and NO access to the active site in Tt ba(3), and that traditional CO flow-flash experiments do not accurately reflect the O(2) and NO binding kinetics in ba(3). We suggest that in ba(3) the binding of O(2) (NO) to heme a(3)(2+) causes rapid dissociation of CO from Cu(B)(+) through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu(B)(+). These findings indicate that structural differences between Tt ba(3) and the bovine aa(3) enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.     

Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.     

Conformation, co-operativity and ligand binding in human hemoglobin.

There does not appear to be any co-operativity manifest in the four combination rate constants for the binding of nitric oxide to deoxyhemoglobin. The time-course of the observed reaction is best fitted by statistically related rates, and the numerical relation between the rate constants for the binding of the fourth molecule of carbon monoxide and the fourth molecule of nitric oxide, which can be obtained independently, also argues for a statistical relation between the nitric oxide binding rate constants.In spite of the absence of co-operativity, the normal T → R transition occurs on nitric oxide binding, as demonstrated by the release of 8-hydroxy-1,3,6-pyrene trisulfonate, and the R-state shows the normal enhancement of reactivity towards carbon monoxide as compared with the T-state (30-fold).Competition experiments between carbon monoxide and nitric oxide in which the two ligands react simultaneously with deoxyhemoglobin suggest that the switching point (T → R) occurs on the average after 2.7 molecules of nitric oxide have been bound (in 0.05 m-2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol, pH 7) and after 3 molecules of carbon monoxide (in 0.05 m-phosphate, PH 7).     

The kinetics of binding of oxygen and carbon monoxide to Gastrophilus haemoglobin

The dimeric haemoglobin in the tracheal cells of the Gastrophilus larva was extracted and purified, and the spectral properties of its oxy- and carbon monoxide adducts are recorded. In dilute solutions the kinetic parameters of binding of oxygen and carbon monoxide were determined. In solutions between 0.1 and 50mum for oxygen k(on) is 1x10(7)m(-1).s(-1) and k(off) is 1s(-1); for carbon monoxide l(on) is 6.5x10(5)m(-1).s(-1) and l(off) is 0.14s(-1). These values are in agreement with previous equilibrium results on oxygen binding and carbon monoxide/oxygen partition. These results are discussed and compared with the known values for other monomeric protohaem proteins.     

Thiyl radicals react with nitric oxide to form S-nitrosothiols with rate constants near the diffusion-controlled limit

A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.     

Kinetic and equilibrium studies of the reactions of heme-substituted horse heart myoglobins with oxygen and carbon monoxide.

In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.     

Analysis of the contribution of the globin and reductase domains to the ligand-binding properties of bacterial haemoglobins

Bacterial Hbs (haemoglobins), like VHb (Vitreoscilla sp. Hb), and flavoHbs (flavohaemoglobins), such as FHP (Ralstonia eutropha flavoHb), have different autoxidation and ligand-binding rates. To determine the influence of each domain of flavoHbs on ligand binding, we have studied the kinetic ligand-binding properties of oxygen, carbon monoxide and nitric oxide to the chimaeric proteins, FHPg (truncated form of FHP comprising the globin domain alone) and VHb-Red (fusion protein between VHb and the C-terminal reductase domain of FHP) and compared them with those of their natural counterparts, FHP and VHb. Moreover, we also analysed polarity and solvent accessibility to the haem pocket of these proteins. The rate constants for the engineered proteins, VHb-Red and FHPg, do not differ significantly from those of their natural counterparts, VHb and FHP respectively. Our results suggest that the globin domain structure controls the reactivity towards oxygen, carbon monoxide and nitric oxide. The presence or absence of a reductase domain does not affect the affinity to these ligands.     

Trematode hemoglobins show exceptionally high oxygen affinity.

Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.     

Evidence of nitric oxide and angiotensin II regulation of circulation and cutaneous drinking in Bufo marinus

The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (l-NAME) increased vascular resistance (VR) 10% above baseline of 3.08+/-0.08 (n=11) mmHg/mL/min at 10 mg/kg and 20% above 3.05+/-0.08 (n=9) at 50 mg/kg in anesthetized toads (Bufo marinus). Blood pressure was unaffected by either dose of L-NAME. Blood flow decreased at the higher dose of L-NAME. L-arginine (300 mg/kg) reversed the effects of L-NAME on VR and blood flow in toads treated with 10 mg/kg but not with 50 mg/kg. Injection of 50 mg/kg L-NAME into empty-bladder toads produced a 10% decrease in water uptake, J(v), resulting in a J(v) of 1,267+/-11 cm(3)/cm(2)/s x 10(-7) (n=9) compared to 1,385+/-12 (n=8) for controls. Injection of 10 microg/kg angiotensin II (ANG II) increased J(v) 15% across the pelvic patch (J(v), cm(3)/cm(2)/s x 10(-7)), resulting in a J(v) of 1,723+/-12 cm(3)/cm(2)/s x 10(-7) (n=8) compared to 1,471+/-12 (n=8) for controls. It is hypothesized that during cutaneous drinking blood flow into the capillary bed of the pelvic patch is regulated by nitric oxide and ANG II.     

Nitric oxide and peroxynitrite cause irreversible increases in the Km for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K_m for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K_m. These include: treatment of the purified enzyme with peroxynitrite or high (μM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.     

Reaction mechanism between nitric oxide and glutathione mediated by Fe(III) myoglobin.

Ferrimyoglobin at pH 7.4 binds nitric oxide to yield nitric oxide adducts. In the presence of glutathione (GSH), nitrosoadducts of Mb(III) react with it to give nitrosoglutathione, whose concentration has been determined with an apparatus based on a specific and sensitive solid-state amperometric gas sensor. The reaction constant between the adduct and glutathione, kGSH = (47 +/- 1) M(-1) x s(-1), obtained by UV-Vis spectroscopy kinetic measurements, is about one-eighth of the constant with OH- determined by other authors. We can explain this fact with the higher nucleophilicity of OH- compared to GSH, due to the bulkiness and charge of the species. It is known that the formation of nitrosothiols starting from nitrite or NO (nitrogen monoxide) and glutathione, in the absence of oxygen, is impossible. Thus, from a biological point of view, it is important to point out that GSH reacts with NO in the presence of ferrimyoglobin, even at physiological pH, to form nitrosoglutathione.     

Functional properties of the hemoglobin from the South American snake Mastigodryas bifossatus

The hemolysate of Mastigodryas bifossatus shows two major hemoglobins with very close isoelectric points, and four different globin chains. The stripped hemolysate exhibits a low alkaline Bohr effect (Δlog P₅₀/ΔpH = −0.30 between pH7 and 8) and a decrease of the co-operativity from 2.3 to unity when the pH increases from 6.15 to 8.5. In the presence of ATP, large changes in the oxygen affinity and co-operativity are observed. The Bohr effect rises to −0.46 and the n₅₀ values stay at around 3 in the pH range 6–9. An increase in temperature induces a large decrease in the oxygen affinity for the stripped hemolysate. In the pH range between 7.5 and 8.5, the values of AH in kcal/M are around 10 fold larger for the stripped protein than for the protein in the presence of ATP. Measurements of rapid kinetics of oxygen dissociation and carbon monoxide binding reflect the ATP sensitivity observed in equilibrium experiments.     

Mechanism and biological role of nitric oxide binding to cytochrome c'

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.     

Reversible oxygenation of protoheme-imidazole complex in aqueous solution (1,2)

Solutions of protohemin in aqueous buffer containing imidazole were reduced and exposed to carbon monoxide forming the carbon monoxide-imidazole complex similar to that in carboxyhemoglobin. This complex is stable for long periods in the presence of low pressures of oxygen and thus the standard flash photolysis methods can be used to determine rates of combination of the heme-imidazole complex with oxygen. Combination rates for both carbon monoxide and oxygen are faster than any on rates for hemoglobin and oxygen dissociation rates are also faster. But the equilibrium constant for binding of this isolated site is larger than that for hemoglobin.     

Recent advances in heme-protein sensors

In recent years, an increasing number of proteins have been discovered which utilize heme cofactors to sense oxygen, carbon monoxide and nitric oxide. The identification and characterization of these proteins are revising our understanding of heme-mediated allostery first established in the early 1960s. Biochemical and structural studies are revealing new mechanisms for heme-driven conformational changes distinct from the classical hemoglobin model.     

In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum

Carbon monoxide induces delayed neurological and neuropathological alterations, including memory loss and cognitive impairment. The bases for the delay remain unknown. Activation of soluble guanylate cyclase by nitric oxide modulates some forms of learning and memory. Carbon monoxide binds to soluble guanylate cyclase, activating it but interfering with its activation by nitric oxide. The aim of this work was to assess whether exposure of rats to carbon monoxide alters the activity of soluble guanylate cyclase or its modulation by nitric oxide in cerebellum or cerebral cortex. Rats exposed chronically or acutely to carbon monoxide were killed 24 h or 7 days later. Acute carbon monoxide exposure decreased cyclic guanosine monophosphate (cGMP) content and reduced activation of soluble guanylate cyclase by nitric oxide. Cortex was more sensitive than cerebellum to chronic exposure, which reduced activation of soluble guanylate cyclase by nitric oxide in cortex. In cerebellum, chronic exposure induced delayed impairment of soluble guanylate cyclase activation by nitric oxide. Acute exposure effects were also stronger at 7 days than at 24 h after exposure. This delayed impaired modulation of soluble guanylate cyclase by nitric oxide may contribute to delayed memory loss and cognitive impairment in humans exposed to carbon monoxide.