Equivalent Nitrogens Research Articles

Mononuclear Transition Metal Complexes of 7‐Nitro‐1,3,5‐Triazaadamantane

AbstractComplexes of the type [MCl2(7‐nitro‐1,3,5‐triaza‐adamantane)2] (M = Zn(II), Pd(II), Pt(II)) and [MCl2(H2O)2(7‐nitro‐1,3,5‐triazaadamantane)2] (M = Mn (II), Co(II), Ni(II)) have been prepared and their structures have been analysed by X‐ray crystallography, elemental analysis, IR and solid state 13C and 15N NMR spectroscopy, supported by density functional theory/gauge independent atomic orbital (DFT/GIAO) calculations. In each case, 7‐nitro‐1,3,5‐triazaadamantane acts as a mono‐dentate ligand and binds to one metal centre only, in spite of the presence of three equivalent amino nitrogens. In the Co(II) and Ni(II) complexes, a two‐dimensional intermolecular hydrogen bonding network between the aqua‐ and the chloro ligands is established. The uncoordinated amines of the 7‐nitro‐1,3,5‐triaza‐adamantane are not involved in any H‐bonding, as a result of the exceptionally low basicity of this compound.

EPR Study of 5-Chlorosalicylate-Cu(II)-N,N-Diethylnicotinamide Ternary Complex Systems in Frozen Water–Methanol Solutions

As a continuation of our previous study of Cu(II) ternary complexes, two new systems containing 5-chlorosalicylic acid (5-ClsalH) and different Cu(II) salts, with varying N,N-diethylnicotinamide (denia) concentrations, system III (CuSO4(aq) ? 2(5-ClsalH(solv)) ? xdenia(l)) and system IV (Cu(ac)2(aq) ? 2 (5-ClsalH(solv)) ? xdenia(l)), where x = 0, 2, 4, 6 and 8, were prepared. The effects of Cu(II) salts containing anions of different basicity and N-donor ligand (denia) with varying ligand-to-metal ratio (x) on the formation of resulting com- plexes were studied by electron paramagnetic resonance (EPR) spectroscopy in frozen water-methanol solutions. Well-resolved 14 N superhyperfine splitting lines (nonet) in the perpendicular region of the second-derivative axially symmetric Cu(II) EPR spectra indicated that for x ( 4 in system III and for x C 2 in system IV, four equivalent nitrogens could be coordinated to the central Cu(II) ions in the equatorial plane. Such information cannot be obtained by EPR spectroscopy of powder samples of the given Cu(II) complex systems. The differences between denia and ronicol containing Cu(II) complexes are discussed.

Electronic Structure of a Binuclear Nickel Complex of Relevance to [NiFe] Hydrogenase

The binuclear complex [Ni(2)(L)(MeCN)(2)](3+) (L(2-) = compartmental macrocycle incorporating imine N and thiolate S donors) has a Ni(III) center bridged via two thiolate S-donors to a diamagnetic Ni(II) center. The ground-state has dominant 3d(z)(1)(2) character similar to that observed for [NiFe] hydrogenases in which Ni(III) is bridged via two thiolate donors to a diamagnetic center (Fe(II)). The system has been studied by X-ray crystallography and pulse EPR, ESEEM, and ENDOR spectroscopy in order to determine the extent of spin-delocalization onto the macrocycle L(2-). The hyperfine coupling constants of six nitrogen atoms have been identified and divided into three sets of two equivalent nitrogens. The most strongly coupled nitrogen atoms (a(iso) approximately 53 MHz) stem from axially bound solvent acetonitrile molecules. The two macrocycle nitrogens on the Ni(III) side have a coupling of a(iso) approximately 11 MHz, and those on the Ni(II) side have a coupling of a(iso) approximately 1-2 MHz. Density functional theory (DFT) calculations confirm this assignment, while comparison of the calculated and experimental (14)N hyperfine coupling constants yields a complete picture of the electron-spin density distribution. In total, 91% spin density is found at the Ni(III) of which 72% is in the 3d(z)(2) orbital and 16% in the 3d(xy) orbital. The Ni(II) contains -3.5% spin density, and 7.5% spin density is found at the axial MeCN ligands. In analogy to hydrogenases, it becomes apparent that binding of a substrate to Ni at the axial positions causes a redistribution of the electron charge and spin density, and this redistribution polarizes the chemical bonds of the axial ligand. For [NiFe] hydrogenases this implies that the H(2) bond becomes polarized upon binding of the substrate, which may facilitate its heterolytic splitting.

Isocyanurate Anion Radicals via Electron-Initiated Cycloaddition of Isocyanates

Room temperature sodium metal reductions of alkyl isocyanates lead to the rapid electron-initiated formation of alkyl isocyanurate anion radicals, which exhibit EPR coupling to only two equivalent nitrogens. Reduction of (13)C-enriched ethyl isocyanate reveals that the odd electron localizes in the pi system of one carbonyl in the isocyanurate ring. EPR line-width alternation effects indicate that at least two stable conformers are in rapid equilibrium undergoing fast exchange.

A continuous-wave electron-nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine beta-mono-oxygenase of the adrenal medulla.

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu(2+) centre with the following EPR parameters: g(z) 2.24, g(x,y) 2.06, A(Cu)(z) 19.0 mT and A(Cu)(x,y) 1.0 mT. Simulation of the Cu(2+) spectrum yielded a best match by using four equivalent nitrogens (A(N)=1.5 mT, 42 MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu(2+), in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron-nuclear double resonance (CW ENDOR; 9.4 GHz, 5-20 K) experiments at g( perpendicular) of the Cu(II) spectrum show a weak coupling to protons with an A(H) of 2.9 MHz that corresponds to a distance of 3.8 A (1 A identical with 0.1 nm), assuming that it is a purely dipolar coupling. Incubation in (2)H(2)O leads to a significant decrease in these (1)H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu(2+) by diethyldithiocarbamic acid. The (1)H and (14)N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu(2+) enzyme, dopamine beta-mono-oxygenase (DbetaM). For DbetaM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and (2)H(2)O-exchangeable protons (2.9 MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40 MHz) from the co-ordinating N of the histidines in DbetaM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

A continuous-wave electron–nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine β-mono-oxygenase of the adrenal medulla

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu2+ centre with the following EPR parameters: gz 2.24, gx,y 2.06, ACuz 19.0mT and ACux,y 1.0mT. Simulation of the Cu2+ spectrum yielded a best match by using four equivalent nitrogens (AN = 1.5mT, 42MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu2+, in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron—nuclear double resonance (CW ENDOR; 9.4GHz, 5–20K) experiments at g⊥ of the Cu(II) spectrum show a weak coupling to protons with an AH of 2.9MHz that corresponds to a distance of 3.8Å (1Å≡0.1nm), assuming that it is a purely dipolar coupling. Incubation in 2H2O leads to a significant decrease in these 1H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu2+ by diethyldithiocarbamic acid. The 1H and 14N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu2+ enzyme, dopamine β-mono-oxygenase (DβM). For DβM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and 2H2O-exchangeable protons (2.9MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40MHz) from the co-ordinating N of the histidines in DβM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

Five-coordinate nitrosyl iron(II) tetraphenylporphyrin exhibits porphyrin ring 14N symmetry about the Fe–N–O plane: a hyperfine sublevel correlation spectroscopy study

Hyperfine sublevel correlation experiments were performed on five-coordinate nitrosyl iron(II) tetraphenylporphyrin (ON–FeTPP) in frozen toluene solution. The spectrum exhibits pairs of equivalent pyrrole nitrogens N 1,2 and N 3,4. The quadrupole parameters agree with literature values for other pyrrole nitrogens. N 2s spin densities of −0.0021 to −0.0022 and −0.00096 on N 1,2 and N 3,4, respectively, are found. The anisotropic hyperfine interaction results from magnetic dipole interactions between the pyrrole 14 N nuclei and electron spin located in the Fe 3d z 2 -type orbital and in the N 2p orbitals in the ( yz) plane bisecting the pyrrole C–N–C angles. The spin density in the Fe 3d z 2 -type orbital along the normal to the porphyrin plane ( y axis) is ∼0.7. The two pyrrole N 2p orbitals are quantized along axes y ′ and z ′, 9–10° from y and z, respectively. The N 2p y ′ and N 2p z ′ spin densities range from −0.005 to −0.009 and +0.007 to +0.010, respectively.

Crystal Structures of Substrate and Inhibitor Complexes with AmpC β-Lactamase: Possible Implications for Substrate-Assisted Catalysis

Group I β-lactamases are major resistance determinants to β-lactam antibiotics. Despite intense study, the identity of the catalytic base, the direction of hydrolytic attack, and the functional difference between β-lactam substrates and β-lactam inhibitors remain controversial. To explore these questions, we determined the X-ray crystal structures of several representative β-lactams in their acyl−adduct complexes with the group I β-lactamase AmpC. A complex with the substrate loracarbef and a deacylation-deficient mutant enzyme reveals an ordered water molecule that is consistent with β-face attack of the catalytic water in group I β-lactamases. The ring nitrogen of the substrate is placed to hydrogen-bond with this water in the position it is thought to adopt in the deacylation transition state. In complexes with the inhibitors cloxacillin and moxalactam, conformational restrictions displace the equivalent ring nitrogens, sterically blocking the formation of the presumed deacylation transition-state stru...

The role of the NO pathway in the control of cardiac function

Nitric oxide (NO) acts as an autocrine- and paracrine-acting signaling autacoid that, among other functions, has been shown to regulate cardiac contractile responsiveness to beta-adrenergic and muscarinic cholinergic agonists. Nitric oxide (NO) is formed by the oxidation of one of two equivalent guanidino nitrogens in L-arginine by O 2 to form NO and L-citrulline. This reaction is catalyzed by a family of enzymes termed NO synthases. Three distinct isoforms of NOS have been identified, each the product of a separate gene. Cellular constituents of cardiac muscle, including ventricular myocytes as well as microvascular endothelial cells, have been shown to express the “endothelial constitutive” isoform of NO synthase (ecNOS or NOS3) in vivo, and both cell types also express the NO synthase isoform induced by specific inflammatory cytokines (iNOS or NOS2) in vivo and in vitro. While NO-dependent intracellular signalling in cardiac myocytes clearly involves the activation of guanylate cyclase and downstream signalling by cGMP, there is accumulating evidence that non-cGMP-dependent regulatory signalling events are also initiated by NO. In addition, decreased contractile responsiveness of cardiac myocytes to beta-adrenergic agonists, following induction of NOS2 by inflammatory cytokines, requires the presence of insulin and the co-induction of enzymes responsible for production of tetrahydrobiopterin, a NOS co-factor. Inappropriate or excessive production of NO by cardiac myocytes and by microvascular endothelial cells likely contributes to the cardiac contractile dysfunction characteristic of the systemic inflammatory response syndrome and cardiac allograft rejection.

Structural characterization of mononuclear Cu(II) and its nitrite complex in the active site of Carcinus maenas hemocyanin

The preparation of a mononuclear Cu(II) derivative of Carcinus maenas hemocyanin (Cu(II)-Hc) and a nitrite complex of the derivative (Cu(II)-Hc-NO2-) are described. Several techniques have been used in their characterization, including X-ray absorption, continuous wave (cw) EPR, and electron spin-echo envelope modulation (ESEEM) spectroscopies. EXAFS results for Cu(II)-Hc indicate the presence of three ligands at 1.99 +/- 0.01 A and a fourth one at 2.26 +/- 0.01 A from the copper. The same coordination number and very similar bond lengths were obtained for Cu(II)-Hc-NO2-. On the basis of simulations of three-pulse ESEEM spectra, three equivalent imidazole nitrogens coupled to Cu(II) were identified in Cu(II)-Hc. Upon the binding of nitrite, a decrease in the hyperfine interaction for two of the three imidazole nitrogens was observed by ESSEM. Further, the results of a two-pulse ESEEM experiment are consistent with the assignment of the protons of a water ligand to Cu(II), which is displaced when nitrite is added. An analysis of X-ray absorption K-edge spectra suggests a coordination geometry intermediate between square-planar and tetrahedral for the metal centers in Cu(II)-Hc and Cu(II)-Hc-NO2-, in agreement with the g and ACu values determined by cw-EPR. On the basis of these results, an equivalent structure is suggested for Cu(II)-Hc-NO2- and the Cu(II) site in green half-methemocyanin, a partially oxidized binuclear derivative formed in the reaction of the native protein with nitrite.

Spectroscopic parameters, electrode potentials, acid ionization constants, and electron exchange rates of the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radicals and ions

The characteristics of the colorless ABTS[sup 2[minus]] ion [ABTS = 2,2[prime]-azinobis(3-ethylbenzothiazoline-6-sulfonate)] and of the persistent, intensely-colored radical ABTS[sup [sm bullet][minus]] have been examined since some reported values are in disagreement. The standard reduction potentials of ABTS[sup [sm bullet][minus]]/ABTS[sup 2[minus]] and ABTS[sup [sm bullet][minus]]/HABTS[sup [minus]] are 0.68 and 0.81 V vs NHE, respectively. A second wave in the CV is associated with ABTS[sup 0]/ABTS[sup [sm bullet][minus]], E[sub 1/2] = 1.09 V. The pH dependence of E[degrees] leads to pK[sub a](HABTS[sup [minus]]) = 2.2 [+-] 0.3; a spectrophotometric pH titration gives 2.08 [+-] 0.02 at [mu] = 1.0 M. The protonated radical HABTS[sup [sm bullet]] was not detected (pK[sub a] < 0), consistent with the higher acidity of the radical relative to its reduced precursor. The EPR spectrum of ABTS[sup [sm bullet][minus]] shows a multiline spectrum centered at g = 2.0036 [+-] 0.0004 from the hyperfine coupling to two sets of two equivalent nitrogens as well as six aromatic hydrogens. Line broadening of the methyl and aromatic resonances of ABTS[sup 2[minus]] in the presence of the radical gives an electron exchange rate constant of (4 [+-] 1) [times] 10[sup 7] L mol[sup [minus]] s[sup [minus]] for ABTS[sup 2[minus]]/ABTS[sup [sm bullet][minus]] in neutralmore » aqueous solution. In 1.0 M perchloric acid, Fe[sup 3+] oxidizes HABTS[sup [minus]] (k = 1.30 [times] 10[sup 2] L mol[sup [minus]1] s[sup [minus]1]) and Fe[sup 2+] reduces ABTS[sup [sm bullet][minus]] (k = 7.5 [times] 10[sup 2] L mol[sup [minus]1] s[sup [minus]1]). Although the equilibrium constant K = k[sub f]/k[sub r] for the reaction agrees with that predicted by the measured potentials, the individual rate constants are much lower than those one calculates from the Marcus cross-relation, which is typical for reactions of Fe(H[sub 2]O)[sub 6][sup 3+] and Fe(H[sub 2]O)[sub 6][sup 2+]. 28 refs., 6 figs., 2 tabs.« less

Inducible nitric oxide synthase activity in hepatocytes is dependent on the coinduction of tetrahydrobiopterin synthesis.

Nitric oxide (NO) is a short-lived radical derived from the oxidation of one of the two chemically equivalent quanido nitrogens of L-arginine1. Three isoforms of the enzyme which produces NO, NO synthase (NOS), have been identified thus far. Two NOS isoforms, endothelial and neuronal are expressed constitutively (cNOS) and release relatively small amounts of NO immediately upon stimulation. A third isoform has been termed inducible NOS (iNOS) because it is not present in resting cells but is expressed if cells are exposed to inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) and/or cytokines. Upon purification all three isoforms are known to be dependent on tetrahydrobiopterin (BH4) for maximal activity2–5. This observation adds to the list of four enzymes previously known to be dependent on BH4. Although the precise role of BH4 in the five-electroh oxidation of L-arginine to NO and citrulline remains uncertain, recent reports using intact cells in culture indicate that both endothelial cNOS6 and smooth muscle cell7 isoforms are dependent on BH4 availability.

Solution and powder X‐ and Q‐band ESR spectra of the 1,3,5‐ trithia‐2,4, 6‐triazapentalenyl radical

AbstractFrom the ESR spectra of the stable 1,3,5‐trithia‐2,4,6‐triazapentalenyl free radical, obtained at two microwave frequencies in both the solution and the solid state, it was shown to be a π radical. From the spectra and their computer simulations were derived an unpaired s‐spin population of 1.7% for the unique nitrogen and 0.1% for the two equivalent nitrogens. The corresponding px‐spin populations were found to be 43.5% and 1.8%, respectively. The unpaired s‐spin population of the two equivalent sulphurs was estimated to be 0.3%.

Inactivation of macrophage nitric oxide synthase activity by NG-Methyl-L-arginine

·N=O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (·N=O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of ·N=O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by ·N=O. NG-Methyl-L-arginine was a competitive inhibitor of ·N=O synthase activity derived from activated murine macrophages with a Ki of 6.2 μM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37° C the kinact was 0.050 min−1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine asts as a mechanism-based enzyme inactivator of murine macrophage ·N=O synthase.

Metabolic fate of L-arginine in relation to microbiostatic capability of murine macrophages.

L-arginine is required for the fungistatic action of murine macrophages in vitro. To further investigate this requirement, L-arginine metabolism by macrophages was measured under conditions where fungistasis either succeeded or failed. Macrophage fungistasis correlated with metabolism of L-arginine to citrulline, nitrite, and nitrate. The metabolic rate was dependent on extracellular L-arginine concentration, reaching a maximum of 67 nmol nitrite/h per mg protein. It accounted for one-third of arginine consumed by fungistatic macrophages. Equimolar amounts of citrulline and total nitrite plus nitrate accumulated in medium. This was consistent with the hypothesis that one of the equivalent guanidino nitrogens of L-arginine was oxidized to both nitrite and nitrate leaving L-citrulline as the amino acid reaction product. The analogue, NG-mono-methyl-L-arginine, selectively inhibited nitrogen oxidation and it was shown previously that it inhibited fungistatic capability. Resident macrophages were not fungistatic and their nitrogen oxidation was low. Once macrophages began producing nitrite/nitrate, protein synthesis was not required during the next 8 h for either fungistasis or nitrogen oxidation. Two-thirds of L-arginine consumption was due to macrophage arginase yielding L-ornithine and urea, which accumulated in medium. This activity was dissociated from macrophage fungistasis. Nitrogen oxidation metabolism by macrophages is linked to a mechanism that inhibits proliferation of fungi. This may involve synthesis of an intermediate compound(s) that has antimicrobial properties.

Spectrochemical and electrochemical studies of 21-thiatetra( p-tolyl)porphyrin and its copper(II) complexes

Electrochemistry, NMR, ESR and UV-Vis spectroscopy of 21-thiatetra( p-tolyl)porphyrin (STpTPH) and respective copper(II) (21-tiatetra( p-tolyl)porphyrin) X Cu(STpTP)X (X=Cl −, Br −, HCO 3 −, ClO 4 −, PPh 3) complexes have been investigated. Monothiaporphyrin is an analogue of widely studied tetraphenylporphyrin, where one of the pyrrole rings has been replaced by a thiophene moiety. The impact of the replacement and the coordinating abilities of thiophene sulfur have been studied. STpTPH undergoes a two step proton addition in dichloromethane. Mono- and dication formations result in distorting the planar thiaporphyrin structure as confirmed by NMR and UV-Vis data. The electronic spectra of Cu(STpTP)X have a similar pattern to the monocation form but the pronounced dependence of the band positions on the ligand character has been established. An ESR spectrum pattern is typical for quasi-tetragonal Cu(II)systems. Parallel features show well resolved seven lines associated with coordination of three nearly equivalent nitrogens. The ESR parameters, particularly hyperfine coupling constant A, have been correlated with the geometry of Cu(II) porphyrins or porphyrin- like ligands. The influence of the axial ligand on the ESR parameters has also been discussed. Cyclic voltammetry studies show that Cu(STpTP)X exhibits a well defined Cu(II)/Cu(I) reduction ( E 1/2= −0.14 ±0.03 V depending on the axial ligand), reported previously only for Cu(II) N-substituted porphyrins but not for Cu(II) porphyrins. The second reduction step results in demetalation. The physicochemcial properties of Cu(STpTP)X are considerably different from Cu(TTP) and closely resemble those of Cu(II) N-substituted porphyrin. Comparison between the electrochemical and spectroscopical properties has been discussed in terms of the general stability of a Cu(II)-axial ligand bond.

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate.

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].

Total Reconstitution of Copper-Zinc Superoxide Dismutase

Abstract The total reconstitution of copper-zinc superoxide dismutase by stoichiometric addition of the metals to completely metal-free apoprotein is described. Reconstitution has been monitored by electron paramagnetic resonance (EPR) and absorption spectroscopy, enzymic activity, and acrylamide gel electrophoresis. Under the same conditions, it has been possible to prepare highly active enzymes with cobalt, mercury, or cadmium in place of zinc. The properties of these derivatives demonstrate that the replacement is exact, that copper has the major influence on the spectral properties of the enzyme, and that occupation of the zinc site is necessary in order to attain fully native properties. A technique is described which allows the extraction of superhyperfine patterns from the bulk of magnetic resonance spectra. Use of this technique shows that the superhyperfine patterns of superoxide dismutase from animal sources differ from those of fungal enzymes. In addition, the technique of shows that the superhyperfine patterns in the EPR spectra reconstituted Cu2-, Cu2, Cd2-, Cu2, Hg2-, and Cu2, Zn2-derivatives are very similar to that in the EPR spectrum of native enzyme. Reconstitution with 63Cu and treatment with cyanide demonstrates that three magnetically equivalent nitrogens are the source of these patterns. The results of this study are shown to be consistent with the copper binding site being a distorted square planar arrangement of ligands and neighboring the zinc-binding site.

Electron Paramagnetic Resonance of Magnetically Dilute Cupric (3d9, 2D) Ion in Single Crystals of Zinc 3-Pyridine Sulfonate

Single crystals of cupric 3-pyridine sulfonate diluted in the diamagnetic zinc compound were prepared and observed for paramagnetic resonance. The symmetry of the magnetic complex is orthorhombic, with two of the g values only slightly different. The ground state of the ion is dz2 which exhibits practically no orbital angular momentum along z. An analysis of the cupric hyperfine structure at room temperature has been performed, arriving at a value of the polarization of the core in very good agreement with the theoretical estimate. No nitrogen superhyperfine structure is resolved at room temperature, although experiments at 77°K show conclusively interaction with two equivalent nitrogens, which is interpreted in detail.

Electron spin resonance of Cu(II) in copper-hemoglobin complexes

Electron spin resonance data indicate that two cupric ions bind to a molecule of hemoglobin at pH 5.4 to 8.9. The binding sites of both ions are identical and are characterized by the presence of resolved superhyperfine structure indicating a considerably covalent bond between the metal and most probably four equivalent nitrogens. Introduction of more than two Cu(II) ions produces precipitation of the protein accompanied by changes in the ESR spectra. The ESR spectra of Cu(II) are identical in oxygenated and methemoglobin. Fe(II) of the former oxidizes partly to Fe(III). Comparison with copper-myoglobin complexes indicates that the metal sites are different in the two proteins.