Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD.

The objective of this study was to elucidate the nitric oxide-forming reactions of the iron-N-methyl-D-glucamine dithiocarbamate (Fe-MGD) complex from the nitrogen-containing compound hydroxyurea. The Fe2+(MGD)2 complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. The reaction of Fe2+(MGD)2 with NO yields the resultant NO-Fe2+(DETC)2 complex, which has a characteristic triplet EPR signal. It is widely believed that only NO reacts with Fe2+(MGD)2 to form the NO-Fe2+(MGD)2 complex. In this report, the mechanism leading to the formation of NO-Fe2+(MGD)2 was investigated using oxygen-uptake studies in conjunction with the EPR spin-trapping technique. We found that the air oxidation of Fe2+(MGD)2 complex results in the formation of the Fe3+(MGD)3 complex, presumably concomitantly with superoxide (O3*-). Dismutation of superoxide forms hydrogen peroxide, which can subsequently reduce Fe3+(MGD)3 back to Fe2+(MGD)2. The addition of NO to the Fe3+(MGD)3 complex resulted in the formation of the NO-Fe2+(MGD)2 complex. Hydroxyurea is not considered to be a spontaneous NO donor, but has to be oxidized in order to form NO. We present data showing that in the presence of oxygen, Fe2+(MGD)2 can oxidize hydroxyurea to yield the stable NO-Fe2+(MGD)2 complex. These results imply that hydroxyurea can be oxidized by reactive oxygen species that are formed from the air oxidation of the Fe2+(MGD)2 complex. Formation of the NO-Fe2+(MGD)2 complex in this case could erroneously be interpreted as spontaneous formation of NO from hydroxyurea. The chemistry of the Fe2+(MGD)2 complexes in aerobic conditions must be taken into account in order to avoid erroneous conclusions. In addition, the use of these complexes may contribute to the overall oxidative stress of the system under investigation.     

Electron paramagnetic resonance study of nitrosylprotoheme dimethyl ester complexes with aliphatic nitrogenous bases: characterization of the axial ligand trans to the nitrosyl group in nitrosylhemoproteins

The interaction of nitrosyl(protoporphyrin IX dimethyl ester) iron (II) (Fe(PPDME)(NO)) with aliphatic amines, anilines, and cyclic imines has been studied by electron paramagnetic resonance (EPR) measurements at room temperature and at 77K. At room temperature, the bases studied here were divided into two groups according to the exchange rate between two EPR-positive species, Fe(PPDME)(NO) and Fe(PPDME)(NO)B, in equilibrium; Fe(PPDME)(NO) + B

Fe(PPDME)-(NO)B. The Fe(PPDME)(NO)-base system with a fast exchange rate on the EPR time scale had a smaller equilibrium constant (K) than that with a slow rate. The EPR spectra of the Fe(PPDME)(NO)-base system both at room temperature and at 77K were markedly influenced by the steric interaction of the base with the porphyrin core.     

In vivo spin trapping of nitric oxide from animal tumors.

Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.     

Nitric oxide-forming reaction between the iron-N-methyl-D-glucamine dithiocarbamate complex and nitrite

The objective of this study was to elucidate the origin of the nitric oxide-forming reactions from nitrite in the presence of the iron-N-methyl-D-glucamine dithiocarbamate complex ((MGD)(2)Fe(2+)). The (MGD)(2)Fe(2+) complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. Although it is widely believed that only NO can react with (MGD)(2)Fe(2+) complex to form the (MGD)(2)Fe(2+).NO complex, a recent article reported that the (MGD)(2)Fe(2+) complex can react not only with NO, but also with nitrite to produce the characteristic triplet EPR signal of (MGD)(2)Fe(2+).NO (Hiramoto, K., Tomiyama, S., and Kikugawa, K. (1997) Free Radical Res. 27, 505-509). However, no detailed reaction mechanisms were given. Alternatively, nitrite is considered to be a spontaneous NO donor, especially at acidic pH values (Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998) Arch Biochem. Biophys. 357, 1-7). However, its production of nitric oxide at physiological pH is unclear. In this report, we demonstrate that the (MGD)(2)Fe(2+) complex and nitrite reacted to form NO as follows: 1) (MGD)(2)Fe(2).NO complex was produced at pH 7.4; 2) concomitantly, the (MGD)(3)Fe(3+) complex, which is the oxidized form of (MGD)(2)Fe(2+), was formed; 3) the rate of formation of the (MGD)(2)Fe(2+).NO complex was a function of the concentration of [Fe(2+)](2), [MGD], [H(+)] and [nitrite].     

Spin trapping of vascular nitric oxide using colloid Fe(II)-diethyldithiocarbamate

Currently available EPR spin-trapping techniques are not sensitive enough for quantification of basal vascular nitric oxide (NO) production from isolated vessels. Here we demonstrate that this goal can be achieved by the use of colloid Fe(DETC)(2). Rabbit aortic or venous strips incubated with 250 microM colloid Fe(DETC)(2) exhibited a linear increase in tissue-associated NO-Fe(DETC)(2) EPR signal during 1 h. Removal of endothelium or addition of 3 mM N(G)-nitro-l-arginine methyl ester (L-NAME) inhibited the signal. The basal NO production was estimated as 5.9 +/- 0.5 and 8.3 +/- 2.1 pmol/min/cm(2) in thoracic aorta and vena cava, respectively. Adding sodium nitrite (10 microM) or xanthine/xanthine oxidase in the incubation medium did not modify the intensity of the basal NO-Fe(DETC)(2) EPR signal. Reducing agents were not required with this method and superoxide dismutase activity was unchanged by the Fe(DETC)(2) complex. We conclude that colloid Fe(DETC)(2) may be a useful tool for direct detection of low amounts of NO in vascular tissue.     

Antioxidant capacity of mononitrosyl-iron-dithiocarbamate complexes: implications for NO trapping

Using EPR spectroscopy, we show that the water-soluble mononitrosyl iron complexes with N-methyl-D-glucamine dithiocarbamate (MNIC-MGD) ligands can easily react with superoxide and with peroxynitrite. The reaction with superoxide transforms the paramagnetic MNIC-MGD complex into an EPR silent complex with a reaction rate of 3 x 10(7) (M.s)(-1). Suppletion of ascorbate partially restores the complexes to their original paramagnetic state. We propose that the reaction of MNIC-MGD with either superoxide or peroxynitrite leads to identical EPR silent complexes. Our results have important implications for the technique of NO trapping in biosystems with Fe-dithiocarbamate complexes, where mononitrosyl-iron complexes (hydrophilic as well as hydrophobic) are formed as adducts in the trapping reaction. This principle is illustrated by NO trapping experiments on viable cultured endothelial cells. We find that MNIC-MGD acts as a very potent and water-soluble antioxidant with an efficiency exceeding most SOD mimics. Moreover, by accounting for the EPR silent fraction of iron complexes, the sensitivity of NO trapping can be enhanced considerably. The method was demonstrated for hydrophobic iron-dithiocarbamate complexes in endothelial cell cultures, where sensitivity for NO detection was enhanced by a factor of 5.     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a2+(3)-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a2+(3)-NO and recombination of NO with cytochrome a2+(3) (in the 30-70 K region) revealed no differences in structure between cytochrome a2+(3) in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a2+(3)-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a2+(3)-NO. Photodissociation of cytochrome a2+(3)-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a2+(3) to cytochrome a3+. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20-40 K region) which differ completely from those observed in cytochrome a2+(3)-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a2+(3)-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu2+B-NO compound. The triplet species with delta ms = 2 EPR signals at g 4 and delta ms = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

Thiolate coordination to Fe(II)-porphyrin NO centers

The interaction of the Fe(II)-porphyrin NO model complex [Fe(TPP)(NO)] (1, TPP=tetraphenylporphyrin) with thiophenolate ligands and tetrahydrothiophene is explored both computationally and experimentally. Complex 1 is reacted with substituted thiophenolates and the obtained six-coordinate adducts of type [Fe(TPP)(SR)(NO)](-) are investigated in solution using electron paramagnetic resonance (EPR) spectroscopy. From the obtained g values and (14)N hyperfine pattern of the NO ligand it is concluded that the interaction of the thiophenolates with the Fe(II) center is weak in comparison to the corresponding 1-methylimidazole adduct. The strength of the Fe-S bond is increased when alkylthiolates are used as evidenced by comparison with the published EPR spectra of ferrous NO adducts in cytochromes P450 and P450nor, which have an axial cysteinate ligand. These results are further evaluated by density functional (DFT) calculations. The six-coordinate model complex [Fe(P)(SMe)(NO)](-) (1-SMe; P=porphine ligand used for the calculations) has an interesting electronic structure where NO acts as a medium strong sigma donor and pi acceptor ligand. Compared to the N-donor adducts with 1-methylimidazole (1-MeIm), etc., donation from the pi(h)( *) orbital of NO to Fe(II) is reduced due to the stronger trans effect of the alkylthiolate ligand. This is reflected by the predicted longer Fe-NO bond length and smaller Fe-NO force constant for 1-SMe compared to the 1-MeIm adduct. Therefore, the Fe(II)-porphyrin NO adducts with trans alkylthiolate coordination have to be described as Fe(II)-NO(radical) systems. The N-O stretching frequency of these complexes is predicted below 1600cm(-1) in agreement with the available experimental data. In addition, 1-SMe has a unique spin density distribution where Fe has a negative spin density of -0.26 from the calculations. The implications of this unusual electronic structure for the reactivity of the Fe(II)-NO alkylthiolate adducts as they occur in cytochrome P450nor are discussed.     

Spin trapping nitric oxide from neuronal nitric oxide synthase: A look at several iron-dithiocarbamate complexes

The free radical, nitric oxide ( radicalNO), is responsible for a myriad of physiological functions. The ability to verify and study radicalNO in vivo is required to provide insight into the events taking place upon its generation and in particular the flux of radicalNO at relevant cellular sites. With this in mind, several iron-chelates (Fe2+(L)2) have been developed, which have provided a useful tool for the study and identification of radicalNO through spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. However, the effectiveness of radicalNO detection is dependent on the Fe2+(L)2 complex. The development of more efficient and stable Fe2+(L)2 chelates may help to better understand the role of radicalNO in vivo. In this paper, we present data comparing several proline derived iron-dithiocarbamate complexes with the more commonly used spin traps for radicalNO, Fe2+-di(N-methyl-D-glutamine-dithiocarbamate) (Fe2+(MGD)2) and Fe2+-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2). We evaluate the apparent rate constant (kapp) for the reaction of radicalNO with these Fe2+(L)2complexes and the stability of the corresponding Fe2+(NO)(L)2 in presence of NOS I.     

X-band and S-band EPR detection of nitric oxide in murine endotoxaemia using spin trapping by ferro-di(N-(dithiocarboxy)sarcosine)

Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of (.)NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)(2) were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of (.)NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of (.)NO, but also the redox properties of liver and lung might determine the formation of complexes of (.)NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for (.)NO-metry at room temperature using Fe(DTCS)(2) as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of (.)NO production in a whole organ, such as murine liver.     

Electronic spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) and its complexes with nitrogenous bases as model systems for nitrosylhemoproteins

Soret and visible absorption spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) (Fe(PPIXDME)(NO] and its complexes with nitrogenous bases (imidazoles, pyridines, aliphatic amines, and cyclic secondary amines) as model systems for nitrosylhemoproteins have been measured in various solvents. As the solvent polarity increases, the Soret and visible absorption bands for the five-coordinate Fe(PPIXDME) (NO) were shifted to shorter wavelengths. Accompanying the coordination of a nitrogenous base to the vacant axial position of Fe(PPIXDME)(NO), the Soret band becomes sharp and the band maximum is shifted to longer wavelengths. The band positions for the six-coordinate Fe(PPIXDME)(NO)(Base) complex are not sensitive to the pi-bonding ability of the axial ligand trans to NO group. The electronic spectra of five-coordinate Fe(PPIXDME)(NO) and six-coordinate Fe(PPIXDME)(NO)(Base) complexes are interpreted in relation to the structural information. The comparison of the spectra for model systems with those for nitrosylhemoproteins is discussed.     

Electron paramagnetic resonance imaging of nitric oxide organ distribution in lipopolysuccaride treated mice

The recent development of electron paramagnetic resonance (EPR) permits its application for in vivo studies of nitric oxide (NO). In this study, we tried to obtain 3D EPR images of endogenous NO in the abdominal organs of lipopolysuccaride (LPS) treated mice. Male ICR mice, each weighing about 30 g, received 10 mg/kg of LPS intraperitoneally. Six hours later, a spin trapping reagent comprised of iron and an N-dithiocarboxy sarcosine complex (Fe(DTCS)₂, Fe 200 mM, DTCS/Fe = 3) were injected subcutaneously. Two hours after this treatment, the mice were fixed in a plastic holder and set in the EPR system, equipped with a loop-gap resonator and a 1 GHz microwave. NO was detected as an NO-Fe(DTCS)₂ complex, which had a characteristic 3-line EPR spectrum. NO-Fe(DTCS)₂ complexes in organ homogenates were also measured using a conventional X-band EPR system. NO-Fe(DTCS)₂ spectra were obtained in the upper abdominal area of LPS treated mice at 8 h after the LPS injection. 3D EPR tiled and stereoscopic images of the NO distribution in the hepatic and renal areas were obtained at the same time. The NO-Fe(DTCS)₂ distribution in abdominal organs was confirmed in each organ homogenate using conventional X-band EPR. This is the first known EPR image of NO in live mice kidneys.     

17O]Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.     

Imidazole,imidazolate, and hydroxide complexes of (protoporphyrin IX)iron(III) and its dimethyl ester as model systems for ferric hemoproteins: Electron paramagnetic resonance and electronic spectral study

The EPR and electronic spectral changes upon titration of systems consisting of (protoporphyrin IX)iron(III) chloride (Fe(PPIX)Cl) or its dimethyl ester (Fe-(PPIXDME)Cl) and imidazole derivatives with tetrabutylammonium hydroxide solution have been measured at 77 and 298 °K in various solvents. The EPR and electronic spectra of the melt of Fe(PPIXDME)Cl in imidazole derivatives have been also measured. The imidazole derivatives studied here were imidazole and 4-methyl-, 4-phenyl-, 2-methyl-, 2,4-dimethyl-, 1-methyl-, and 1-acetylimidazole. The spectral changes upon addition of hydroxide were markedly different between the systems containing NH imidazoles (BH), with a dissociable proton, and those containing NR imidazoles (BR), without it. In the former systems, five spectral species were successively formed at 77 °K and were assigned to following complexes: [Fe(P)(BH)₂]⁺, Fe(P)(BH)(B), [Fe(P)(B)₂]^?, Fe(P)(BH)(OH), and [Fe(P)(B)(OH)]^?, where P is PPIX or PPIXDME. In the latter systems, initial complex, [Fe(P)(BR)₂]⁺, was found to be changed to final complex, Fe(P)(BR)(OH), through an intermediate at 77 °K. At 298 °K, both systems were found to react with hydroxide to finally form Fe(P)(OH). The crystal field parameters were evaluated using the EPR g values in low-spin complexes studied here and in hemoproteins. The five regions corresponding to five low-spin complexes could be distinguished in crystal field diagrams.     

Kinetics of increased generation of (.)NO in endotoxaemic rats as measured by EPR

Ferrous-diethyldithiocarbamate (Fe(DETC)(2)) chelate is a lipophilic spin trap developed for (.)NO detection by electron paramagnetic resonance (EPR) spectroscopy. Using this spin trap we investigated the kinetics of (.)NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg). The NO-Fe(DETC)(2) complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of (.)NO. We found that in blood, kidney, liver, heart and lung (.)NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h. Interestingly, in the eye bulb the maximum of (.)NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS. In brief, the highly lipophilic exogenous spin trap, Fe(DETC)(2) is well suited for assessment of (.)NO production in endotoxaemia. We demonstrated that the kinetics of increased production of (.)NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions. Accordingly, only in early endotoxaemia a high level of (.)NO is detected, while in late endotoxaemia (.)NO detectability is diminished most probably due to concomitant oxidant stress.     

Reductive nitrosylation of water-soluble iron porphyrins by S-nitroso-N-acetylpenicillamine: rate constants and EPR characterization

Reductive nitrosylation of the water-soluble iron derivatives of the cationic Fe(III)(TMPyP) and anionic Fe(III)(TPPS) porphyrins [where TMPyP=tetra-meso-(4-N-methylpyridiniumyl)porphinate and TPPS=tetra-meso-(4-sulfonatophenyl)porphinate] by the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was studied using optical absorption spectroscopy and electron paramagnetic resonance. Nitrosylation rates were obtained, the reaction was found to be first order in the SNAP concentration and the stoichiometry of the reaction was one to one. The similarity between the obtained second-order rate constants for both porphyrins, k(TMPyP)=0.84 x 10(3)M(-1)s(-1) and k(TPPS)=0.97 x 10(3)M(-1)s(-1), suggested that the reaction mechanism is approximately independent of the nature of the porphyrin meso-substituents. A mechanism was proposed involving the hydrolysis of SNAP by an out of plane liganded H(2)O yielding the sulfenic acid of N-acetylpenicillamine and the transfer of NO(-) to Fe(III). The EPR (electron paramagnetic resonance) spectra of the SNAP- and gaseous NO-treated porphyrins were obtained and compared. The difference between the spectra of the cationic and anionic porphyrins indicates different local symmetry and Fe-N-O bond angle. SNAP-treatment produced much more resolved hyperfine structures than gaseous NO-treatment.     

Dinitrosyl-iron complexes with thiol-containing ligands: spatial and electronic structures.

Parameters of the EPR signals of monomeric dinitrosyl-iron complexes with 1H-1,2,4-triazole-3-thiol (DNIC-MT), obtained by treating MT+ferrous iron in DMSO solution with gaseous NO, have been compared with those of the crystalline monomeric DNIC-MT with tetrahedral structure. Dissolved DNIC-MT were characterized by the isotropic EPR signal centered at g=2.03 with half-width of 0.7 mT and quintet hyperfine structure when recorded at ambient temperature or the anisotropic EPR signal with g( perpendicular)=2.045, g( parallel)=2.014 from frozen solution at 77 kappa, Cyrillic. DNIC-MT in crystalline state showed the structure-less symmetrical singlet EPR signal centered at g=2.03 and half-width of 1.7 mT at both room and liquid nitrogen temperature. The Lorentz shape of this signal indicates the strong exchange interaction between these complexes in the DNIC-MT crystal. Being dissolved in DMSO the crystalline sample of DNIC-MT demonstrated the EPR signal typical for DNIC-MT, obtained by treating MT+ferrous iron in DMSO solution with gaseous NO. Low spin (S=1/2) d(9) electron configuration of DNIC-MT with tetrahedral structure (formula [(MT-S(.))(2)Fe(-1)(NO(+))(2)](+)) was suggested to be responsible for the signal of DNIC-MT in crystalline state. Dissolving of the crystals of DNIC-MT may result in the change of their spatial and electronic structure, namely, tetrahedral structure of the complexes characterized by low spin d(9) electronic configuration transforms into a plane-square structure with d(7) electronic configuration and low spin S=1/2 state (formula [(MT- S(-))(2)Fe(+)(NO(+))(2)](+)). The latter was suggested to be characteristic of other DNICs with various thiol-containing ligands in the solutions. The proposed mechanism of these DNICs formation from ferrous iron, thiol and NO shows that the process could be accompanied by the ionization of NO molecules to NO(+) and NO(-) ions in the complexes. Detailed analysis of the shape of the EPR signals of these complexes provided additional information about the exchange interaction typical for DNIC-MT in crystals.     

Effect of hydrophobic structure on the catalysis of nitric oxide release from zwitterionic diazeniumdiolates in surfactant and liposome media.

The effect of phospholipid liposomes and surfactant micelles on the rate of nitric oxide release from zwitterionic diazeniumdiolates, R1R2N[N(O)NO]-, with significant hydrophobic structure, has been explored. The acid-catalyzed dissociation of NO has been examined in phosphate-buffered solutions of sodium dodecylsulfate (SDS) micelles and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-(1-glycerol)] sodium salt (DPPG) phospholipid liposomes. The reaction behavior of dibenzylamine-, monobenzylamine-, and dibutylamine-derived substrates [1]: R1 = C6H5CH2, R2 = C6H5CH2 NH2+(CH2)2, 2: R1 = C6H5CH2, R2 = NH3+(CH2)2, and 3: R1 = n-butyl, R2 = n-butyl-NH2+(CH2)6] has been compared with that of SPER/NO, 4: R1 = H2N(CH2)3, R2 = H2N(CH2) 3NH2+(CH2)4]. Catalysis of NO release is observed in both micellar and liposome media. Hydrophobic interactions contribute to micellar binding for 1-3 and appear to be the main factor facilitating catalysis by charge neutral DPPC liposomes. Binding constants for the association of 1 and 3 with SDS micelles were 3-fold larger than those previously obtained with comparable zwitterionic substrates lacking their hydrophobic structure. Anionic DPPG liposomes were much more effective in catalyzing NO release than either DPPC liposomes or SDS micelles. DPPG liposomes (at 10 mM total lipid) induced a 30-fold increase in the NO dissociation rate of SPER/NO compared to 12- and 14-fold increases in that of 1 and 3.     

EPR and M?ssbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K M?ssbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The M?ssbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and M?ssbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the M?ssbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.     

Spectroscopic characterization of mononitrosyl complexes in heme--nonheme diiron centers within the myoglobin scaffold (Fe(B)Mbs): relevance to denitrifying NO reductase

Denitrifying NO reductases are evolutionarily related to the superfamily of heme--copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N(2)O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines and Val-68 to a glutamic acid to create a nonheme Fe(B) site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV--vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and Cu(I)- or Zn(II)-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}(7) species that can be observed at liquid nitrogen temperature, the Fe(II)-loaded proteins are EPR silent at ≥30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using (15)NO and (15)N(18)O. The apo and Cu(I)-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of Fe(II) at the Fe(B) site shifts the heme ν(FeNO) by 17 cm(-1) and the ν(NO) by -50 cm(-1) to 1549 cm(-1). This low ν(NO) is without precedent for a six-coordinate heme {FeNO}(7) species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme Fe(II). Detection of a similarly low ν(NO) in the Zn(II)-loaded protein supports this interpretation.