Kinetic and mechanistic studies of the reactions of nitrogen monoxide and nitrite with ferryl myoglobin

Nitrogen monoxide (nitric oxide) generated endogenously has a variety of different properties. Among others it regulates blood pressure and transmission of nerve impulses, and has been shown to exert specific toxic effects, but also, paradoxically, to protect against various toxic substances. Recent studies suggest that NO* can serve as an antioxidant of the highly oxidizing ferryl myoglobin (MbFe(IV)=O), which has been proposed to be at least in part responsible for the oxidative damage caused by the reperfusion of ischemic tissues. In the present work we have determined the rate constant for the reaction between MbFe(IV)=O and NO* [(17.9+/-0.5)x10(6)M(-1)s(-1) at pH 7.5 and 20 degrees C] and we have shown that this reaction proceeds via the intermediate nitrito-metmyoglobin complex MbFe(III)ONO. Our results imply that this reaction is very likely to take place in vivo and might indeed represent a detoxifying pathway for both MbFe(IV)=O as well as NO*. Moreover, we have found that the rate of reaction of MbFe(IV)=O with nitrite is significantly lower (16+/-1 M(-1) s(-1) at pH 7.5 and 20 degrees C). Thus, this reaction probably plays a role only when NO* has been consumed completely and large concentrations of nitrite are still present. In contrast to the protecting role of NO*, the reaction with nitrite generates nitrogen dioxide which can contribute to tyrosine nitration. Indeed, we have demonstrated that nitrite can nitrate added tyrosine in the presence of iron(III) myoglobin and hydrogen peroxide.     

Interaction of nitrogen monoxide with hemoglobin and the artefactual production of S-nitroso-hemoglobin

Hemoglobin (Hb) is probably the most thoroughly studied protein in the human body. However, it has recently been proposed that in addition to the well known function of dioxygen and carbon dioxide transporter, one of the main roles of hemoglobin is to store and transport nitrogen monoxide. This hypothesis is highly disputed and is in contrast to the proposal that hemoglobin serves as an NO. scavenger in the blood. In this short review, I have presented the current status of research on the much-debated mechanism of the reaction between circulating hemoglobin and NO.. Despite the fact that oxyHb is extremely rapidly oxidized by NO., under basal physiological conditions the biological activity of NO. in the blood vessels is not completely lost. It has been shown that three factors reduce the efficiency of hemoglobin to scavenge NO.: a so-called red blood cell-free zone created close to the vessel wall by intravascular flow, an undisturbed layer around the red blood cells--where the NO. concentration is much smaller than the bulk concentration--and/or the red blood cell membrane. Alternatively, it has been proposed that NO. binds to Cys beta 93 of oxyHb, is liberated after deoxygenation of Hb, and consequently allows for a more effective delivery of O2 to peripheral tissues. However, because of the extremely fast rate of the reaction between NO. and oxyHb, experiments in vitro lead to artefactual production of large amounts of S-nitroso-hemoglobin. These results, together with other data, which challenge most steps of the NO.-transporter hypothesis, are discussed.     

Kinetics of reactions between hemoglobin and carbon monoxide. II. Analysis of kinetic schemes in linear approximation]

The kinetics of hemoglobin (Hb) reactions with carbon monoxide were calculated for a number of classical schemes: with one, two, four binding sites and an allosteric scheme with T- and R-conformers. The developed theory predicts the existence of only one relaxation time for every scheme the values of which depends on the velocity constants, saturation degree and the Hb concentration.     

The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.     

Kinetics of the reactions of trioxodinitrate and nitrite ions with cytochrome d in Escherichia coli.

The rate of reaction of trioxodinitrate with reduced cytochrome oxidase d in membrane particles from Escherichia coli at pH 7 and 25 degrees C depends linearly upon [HN2O3-] over the concentration range studied (up to 0.05 mM) and is also first-order in cytochrome d. The known rate of decomposition of trioxodinitrate to give NO- and NO2- is about 4.5-times faster than the rate of reaction of reduced cytochrome d with trioxodinitrate, implying that cytochrome d reacts directly with NO-, with a trapping ratio of between 0.20 and 0.25, rather than with trioxodinitrate. The implications of the facile formation of the NO(-)-nitrosyl complex of cytochrome d for the mechanism of denitrification are discussed with particular reference to the mechanism of N-N bond formation. The reaction of reduced cytochrome d with nitrite (a decomposition product of trioxodinitrate) under these conditions is much slower than that with trioxodinitrate. The kinetics show a biphasic dependence of initial rate upon nitrite concentration. The rate data at low [NO2-] are consistent with saturation of a high affinity site for nitrite, having Vmax = 4.29.10(-9) M s-1 and Km = 0.034 mM. The existence of two binding sites for nitrite is consistent with the suggestion that the cytochrome bd complex contains two cytochrome d haems.     

Mechanistic studies of the oxidation of pyridoxalated hemoglobin polyoxyethylene conjugate by nitrogen monoxide

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), a modified human-derived hemoglobin, is currently in clinical trials as a nitrogen monoxide scavenger for the treatment of shock. Stopped-flow spectroscopy studies of the reaction between nitrogen monoxide and PHP indicate that at pH 7 the second-order rate constant is (88 +/- 3) x 10(6) M(-1) s(-1), a value very similar to that for the unmodified human hemoglobin. At alkaline pH the reaction proceeds via the intermediate peroxynitrito complex PHP-Fe(III)OONO, which rapidly decomposes to nitrate and the iron(III) form of PHP. The rate of decay of PHP-Fe(III)OONO increases significantly with decreasing pH such that it does not accumulate in concentrations large enough to be observed spectroscopically under neutral or acidic conditions. Ion chromatographic analysis of the nitrogen-containing products of the NO(*)-mediated reaction of PHP shows that nitrate is formed quantitatively at both pH 7 and pH 9.     

Involvement of ferryl in the reaction between nitrite and the oxy forms of globins

The reaction between nitrite and the oxy forms of globins has complex autocatalytic kinetics with several branching steps and evolves through chain reactions mediated by reactive species (including radicals) such as hydrogen peroxide, ferryl and nitrogen dioxide, starting with a lag phase, after which it proceeds onto an autocatalytic phase. Reported here are UV–Vis spectra collected upon stopped-flow mixing of myoglobin with a supraphysiological excess of nitrite. The best fit to the experimental data follows an A → B → C reaction scheme involving the formation of a short-lived intermediate identified as ferryl. This is consistent with a mechanism where nitrite binds to oxy myoglobin to generate an undetectable ferrous-peroxynitrate intermediate, whose decay leads to nitrate and ferryl. The ferryl is then reduced to met by the excess nitrite. DFT calculations reveal an essentially barrierless reaction between nitrite and the oxy heme, with a notable outer-sphere component; the resulting metastable ferrous-peroxynitrate adduct is found to feature a very low barrier towards nitrate liberation, with ferryl as a final product—in good agreement with experiment.     

Kinetics and thermodynamics of oxygen and carbon monoxide binding to the T-state hemoglobin of Urechis caupo

The tetrameric hemoglobin from Urechis caupo is nearly ideal for studying ligation to the T-state. Our previous EXAFS study had shown that the Fe is displaced 0.35 A from the mean plane of the porphyrin in the HbCO derivative. We have carried out detailed kinetic studies of oxygen and CO ligation as a function of temperature in order to characterize both the kinetics and thermodynamics of ligation in this hemoglobin. The entropy change associated with ligation essentially corresponds to simple immobilization of the ligand and is virtually the same as that we have determined for leghemoglobin, an extreme R-state-type hemoglobin. The low ligand affinities thus derive from small enthalpies of ligation, which can be correlated with the large out of plane displacement of the Fe. Only oxygen pulse measurements revealed kinetic evidence for cooperative oxygen binding, but a direct measurement of oxygen binding gave a Hill number of 1.3. An allosteric analysis gave L = 2.6 and c = 0.048 (oxygen) and c = 0.77 (CO). The higher affinity state in this weakly cooperative hemoglobin is denoted T*, and it is for this state that thermodynamic quantities have been determined. The small differences between T and T* in CO binding were nevertheless sufficient to allow us to measure by flash photolysis the rate of the T*----T conformational change in terms of an allosteric model. The half-time for this transition was calculated to be 8-14 ms at 20 degrees C.     

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.     

Effect of pH on the kinetics of oxygen and carbon monoxide reactions with hemoglobin from the air-breathing fish,Loricariichthys

• 1.Loricariichthys sp., an air-breathing fish from the Amazon River has one major hemoglobin component.
• 2. Quantitative studies on the kinetics of O₂ dissociation and CO combination to the protein were performed by stopped-flow experiments at different pH values and a constant ATP concentration of 1.25 mM.
• 3. The oxygen dissociation shows a simple first order behavior and a strong pH dependence.
• 4. The CO combination kinetics, on the other hand, were homogeneous and fast at higher pH values and slow and clearly autocatalytic at pH values below 7.0.

     

Kinetics of carbon monoxide binding to fully and partially reduced human hemoglobin valency hybrids.

The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated.     

Inhibitors of the nitrite oxidation of hemoglobin

Different types of active inhibitors of the reaction of nitrite hemoglobin oxidation have been revealed and studied. The dependence of inhibition of methemoglobin formation, on concentration of inhibitors at pH 5.9 and 7.17 has been determined. Differential absorption spectra of the inhibitors in the presence sodium nitrite in UV and visual light has been studied. The values of oxidation-reduction potentials have been estimated. Possible mechanism of action of the inhibitors has been discussed.     

Oxidation of thiamine on reaction with nitrogen dioxide generated by ferric myoglobin and hemoglobin in the presence of nitrite and hydrogen peroxide

It is shown that nitrogen dioxide oxidizes thiamine to thiamine disulfide, thiochrome, and oxodihydrothiochrome (ODTch). The latter is formed during oxidation of thiochrome by nitrogen dioxide. Nitrogen dioxide was produced by incubation of nitrite with horse ferric myoglobin and human hemoglobin in the presence of hydrogen peroxide. After addition of tyrosine or phenol to aqueous solutions containing oxoferryl forms of the hemoproteins, thiamine, and nitrite, the yield of thiochrome greatly increased, whereas the yield of ODTch decreased. In the presence of high concentrations of tyrosine or phenol compounds ODTch was not formed at all. The neutral form of thiamine with the closed thiazole cycle and minor tricyclic form of thiamine do not enter the heme pocket of the protein and do not interact with the oxoferryl heme complex Fe(IV=O) or porphyrin radical. The tricyclic form of thiamine is oxidized to thiochrome by tyrosyl radicals located on the surface of the hemoprotein. The thiol form of thiamine is oxidized to thiamine disulfide by both hemoprotein tyrosyl radicals and oxoferryl heme complexes. Nitrite and also tyrosine, tyramine, and phenol readily penetrate into the heme pocket of the protein and reduce the oxyferryl complex to ferric cation. These reactions yield nitrogen dioxide as well as tyrosyl and phenoxyl radicals of tyrosine molecules and phenol compounds, respectively. Tyrosyl and phenoxyl radicals of low molecular weight compounds oxidize thiamine only to thiochrome and thiamine disulfide. The effect of oxoferryl forms of myoglobin and hemoglobin, nitrogen dioxide, and phenol on thiamine oxidative transformation as well as antioxidant properties of the hydrophobic thiamine metabolites thiochrome and ODTch are discussed.     

Inactivation of alcohol dehydrogenase (ADH) by ferryl derivatives of human hemoglobin

In this paper, inactivation of alcohol dehydrogenase (ADH) by products of reactions of H2O2 with metHb has been studied. Inactivation of the enzyme was studied in two systems corresponding to two kinetic stages of the reaction. In the first system H2O2 was added to the mixture of metHb and ADH [the (metHb+ADH)+H2O2] system (ADH was present in the system since the moment of addition of H2O2 i. e. since the very beginning of the reaction of metHb with H2O2). In the second system ADH was added to the system 5 min after the initiation of the reaction of H2O2 with metHb [the (metHb+H2O2)5 min+ADH] system. In the first case all the products of reaction of H2O2 with metHb (non-peroxyl and peroxyl radicals and non-radical products, viz. hydroperoxides and *HbFe(IV)=O) could react with the enzyme causing its inactivation. In the second system, enzyme reacted almost exclusively with non-radical products (though a small contribution of reactions with peroxyl radicals cannot be excluded). ADH inactivation was observed in both system. Hydrogen peroxide alone did not inactivate ADH at the concentrations employed evidencing that enzyme inactivation was due exclusively to products of reaction of H2O2 with metHb. The rate and extent of ADH inactivation were much higher in the first than in the second system. The dependence of ADH activity on the time of incubation with ferryl derivatives of Hb can be described by a sum of three exponentials in the first system and two exponentials in the second system. Reactions of appropriate forms of the ferryl derivatives of hemoglobin have been tentatively ascribed to these exponentials. The extent of the enzyme inactivation in the second system was dependent on the proton concentration, being at the highest at pH 7.4 and negligible at pH 6.0. The reaction of H2O2 with metHb resulted in the formation of cross-links of Hb subunits (dimers and trimers). The amount of the dimers formed was much lower in the first system i. e. when the radical forms dominated the reaction of inactivation.     

Kinetics and mechanistic studies of the reactions of metleghemoglobin, ferrylleghemoglobin, and nitrosylleghemoglobin with reactive nitrogen species

It is now established that nitrogen monoxide is produced not only in animals but also in plants. However, much less is known about the pathways of generation and the functions of in planta. One of the possible targets of is leghemoglobin (Lb), the hemoprotein found in high concentrations in the root nodules of legumes that establish a symbiosis with nitrogen-fixing bacteria. In analogy to hemoglobin and myoglobin, we have shown that different forms of Lb react not only with , but also with so-called reactive nitrogen species derived from it, among others peroxynitrite and nitrite. Because of the wider active-site pocket, the rate constants measured in this work for and for nitrite binding to metLb are 1 order of magnitude larger than the corresponding values for binding of these species to metmyoglobin and methemoglobin. Moreover, we showed that reactive nitrogen species are able to react with two forms of Lb that are produced in vivo but that cannot bind oxygen: ferrylLb is reduced by and nitrite, and nitrosylLb is oxidized by peroxynitrite. The second-order rate constants of these reactions are on the order of 10², 10⁶, and 10⁵ M⁻¹ s⁻¹, respectively. In all cases, the final reaction product is metLb, a further Lb form that has been detected in vivo. Since a specific reductase is active in nodules, which reduces metLb, reactive nitrogen species could contribute to the recycling of these inactive forms to regenerate deoxyLb, the oxygen-binding form of Lb.