Fe(II)-Bleomycin is activated in air to form an electron paramagnetic resonance (EPR)-active spec... more Fe(II)-Bleomycin is activated in air to form an electron paramagnetic resonance (EPR)-active species, termed "activated bleomycin", that cleaves DNA, when present. When DNA is absent, the potential DNA cleavage activity is lost and the drug becomes self inactivated. A method is described for the preparation and purification of this self-inactivated product from bleomycin A2, together with some of its physical properties. It is shown that the loss of DNA cleavage activity parallels an alteration of bithiazole fluorescence, attributed to chemical change at this residue. EPR evidence is brought forth that the Cu(II) binding site of inactivated bleomycin in not altered, nor is the ability to form a species with Fe(II) and O2 having the identical spectroscopic signature as activated bleomycin.
Flavodoxins, the FMN-containing electron carriers present in a variety of rriicroorganisms, have ... more Flavodoxins, the FMN-containing electron carriers present in a variety of rriicroorganisms, have lower oxidation-reduction potentials than other members of the flavoprotein family. Crystal strucfure analyses of these proteins, with FMN in the oxidized, semiquinone, and reduced forms, have been carried out in an attempt to understand how the interaction with proteins modulates the oxidation-reduction potential of flavins. Initial studies with flavodoxin from Clostridium MP have suggested that the oxidized/semiquinone potential depends on formatign of a hydrogen bond between the polypeptide backbone and the "(5) of the flavin semiquinone, while the semiquinone/reduced potential may depend on constraints on the bending and motion of the reduced isoalloxazine ring. These'proposals have been explored by structural comparisons with other species of flavodoxins, especially the protein from Anacystis nidulans, which has a higher potential for the oxidized/semiquinqne equilibrium than most other flavodoxins.
Electron spin echo envelope modulation spectroscopy identified two ligand 14N interactions with t... more Electron spin echo envelope modulation spectroscopy identified two ligand 14N interactions with the mixedvalence, Fe(IVII1) diiron center of methane monoogygenase hydroxylase from Methylococcus capsulatus (Bath). Characteristic features of the spectra obtained at 9 and 10 GHz were analyzed and fit by simulation. One of the nitrogens possessedo superhyperfine parameters (Aiso = 0.8 MHz, re, = 3.2 A, e2Qq = 1.8 MHz, q = 0.35) consistent with a non-coordinating amino nitrogen of a histidine imidazole ligand to a Fe(II1). The second, mo5e strongly interacting nitrogen (Aiso = 5.0 MHz, re, = 2.2 A, e2Qq = 3.0 MHz, q = 0.3) corresponds to the N, directly bound to the effective Fe(I1). These findings extend the previous electron nuclear double resonance results on the Methylosinus trichosporium hydroxylase (Hendrich, M. P., Fox, B. G., Andersson, K. K., Debrunner, P. G., and Lipscomb, J. D. (1992) J. Biol. Chem. 267,261-269), which identified the N,-Fe(I1) interaction but failed to quantify its magnitude. Measurement of the linear electric field g shift of this mixed-valence species indicated that the site is charge-polarized on to one of the iron atoms, and its symmetry suggests that either charge is shifted away from the Fe-Fe axis (if g,, is defined by the Fe-Fe axis) or that gmia and g,, are perpendicular to the Fe-Fe axis (charge strongly localized at Fe(II1) and axis taken as g m 3 .
The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using ... more The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using electrospray ionization mass spectrometry (ESI-MS) and EPR spectroscopy. A rapid reaction occurs that ultimately yields several cleavage products of the drug, the structures of which have been deduced from their masses. The intermediates observed by ESI-MS suggest a mechanism for the reaction of FemBLM with PhIO that does not involve hypervalent iron or activated oxygen but, instead, hypervalent iodine I(II1) as the oxidant. Indeed, we could not detect the production of an iron oxene complex of BLM (O=FeVBLM) or hydroperoxyferric BLM (HOO-Fern-BLM). Furthermore, we show that the products formed when FemBLM is reacted with PhIO are identical to those formed when FemBLM is activated in air or when FemBLM is reacted with H202. Surprisingly, the same products are obtained when ZnnBLM or metal-free BLM is reacted with PhIO, indicating that a metal ion is not required for the oxidation of BLM by PhIO.
The requirements of a cloned macrophage-like cell line, J774.16, for oxygen metabolism, and the n... more The requirements of a cloned macrophage-like cell line, J774.16, for oxygen metabolism, and the nature of the defect in oxidative metabolism in a variant clone derived from it, J774.C3C, were studied. Upon stimulation with phorbol myristate acetate (PMA), the parental clone produced approximately 1 nmol O2-/min/10(6) cells, whereas the variant clone produced no detectable O2- under the same conditions. Sustained O2- production by J774.16 was totally dependent on extracellular glucose; in glucose-free medium, the cells initiated O2- production but could not sustain it. When cells were stimulated with PMA, glucose-C-1 oxidation of J774.16 cells increased 20-fold while that of J774.C3C remained at resting levels. O2- production in J774.16 cells was inhibited by some agents known to block mitochondrial electron transport before coenzyme Q, such as rotenone and tetrathiafulvalene, whereas antimycin A enhanced O2- production. A dissociation between O2- production and glucose-C-1 oxidation was observed when J774.16 was treated with certain metabolic inhibitors. Quinacrine, 2,4-dinitrophenol, chlorpromazine, and trifluoperazine inhibited O2- production completely under conditions in which glucose-C-1 oxidation was reduced only by 30%. Rotenone inhibited O2- production with no effect on glucose-C-1 oxidation whereas antimycin A augmented O2- production 50% but inhibited glucose oxidation by 20%. Glucose transport studies, with 2-deoxy-D-glucose, showed that the Km for glucose transport of both clones was about 1 mM, indicating that cells could effectively transport glucose even at low concentrations. The Vmax for glucose transport in both J774.16 and variant J774.C3C cells doubled after PMA stimulation, indicating that the variant was effectively stimulated by PMA, even though O2- was not produced. Similarly, PMA induced protein phosphorylation in both clones. No differences between clones J774.16 and J774.C3C in hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, or glutathione peroxidase activities could be found. When dithionite-reduced and -oxidized difference spectra of plasma membranes of these clones were compared, comparable levels of b-type cytochrome were found in both clones. However, CO difference spectra indicated that CO was bound to a b-type cytochrome (presumed to be b-245) in clone J774.16 but not in J774.C3C.(ABSTRACT TRUNCATED AT 400 WORDS)
The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using ... more The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using electrospray ionization mass spectrometry (ESI-MS) and EPR spectroscopy. A rapid reaction occurs that ultimately yields several cleavage products of the drug, the structures of which have been deduced from their masses. The intermediates observed by ESI-MS suggest a mechanism for the reaction of FemBLM with PhIO that does not involve hypervalent iron or activated oxygen but, instead, hypervalent iodine I(II1) as the oxidant. Indeed, we could not detect the production of an iron oxene complex of BLM (O=FeVBLM) or hydroperoxyferric BLM (HOO-Fern-BLM). Furthermore, we show that the products formed when FemBLM is reacted with PhIO are identical to those formed when FemBLM is activated in air or when FemBLM is reacted with H202. Surprisingly, the same products are obtained when ZnnBLM or metal-free BLM is reacted with PhIO, indicating that a metal ion is not required for the oxidation of BLM by PhIO.
Electron spin echo envelope modulation (ESEEM) spectroscopic examination of Cu(II)-proteins and -... more Electron spin echo envelope modulation (ESEEM) spectroscopic examination of Cu(II)-proteins and -enzymes has been used to identify endogenous ligands to the metal and their number, the presence or absence of coordinated water both in the vicinity of and ligated to the metal, and the mode of binding of inhibitory anions and catalytic substrates. For Cu(I1) centers, such as in stellacyanin, imidazole is identified as a metal ligand based on ESEEM comparisons of the nuclear quadrupole interaction (nqi) of the remote, protonated 14N of the coordinated ligand with that for a Cu(II)-imid model.' For some Cu proteins, such as phenylalanine hydroxylase, galactose oxidase and amine oxidase, the nqi parameters are so different from those for the Cu(II)-imid model remote af4to suggest alterations in the local environment of the N of the coordinated heterocycle. For this reason ESEEM studies were carried out on $$(II)-substituted imid models and -doped single crystals ' in order to understand how structural changes in imid and substituted imid affect nqi parameters. In a model study,' it was shown that the largest changes in nqi, which can be related to external N-H or N-C bond polarization and N oQGta1 occupancy, occurs with Nalkylation of the remote N of metal coordinated imid; alkyl substitution on ring carbon has much less affect. For a Cu(II)-imid model, nqi parameters can be changed by altering the H-bonding capabilities of ring N-H such as with i&O or formate. From single crystal studies, differences in N orbital occupancy of imid a$ determined from the nqi, can be related to the N-H bond length . It is suggested that the variation of nqi observed for some Cu(II)-proteins arises from variations of Hbonding capabilities at the remote nitrogen of the coordinated histidine imidazole side chain. It is further suggested that these H-bonding differences may alter the properties of the metal centers.
Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrup... more Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrupole interactions of the remote nitrogen of pyrazole and pyridazine in CuII(dien)-heterocycle complexes. The quadrupole parameters were obtained by field-dependent ESEEM spectral simulations for the amino nitrogen of pyrazole (e2qQ = 2.86 MHz and 7 = 0.74) and for the imino nitrogen of pyridazine (e2qQ = 5.35 MHz and 7 = 0.02). The values obtained for pyridazine are comparable to those for metal-free solid pyridazine (Schempp, E.; Bray, P. J. J. Chem. Phys. 1967,46,1186. Since the quadrupole parameters for the amino nitrogen in metal-free solid pyrazole have not been reported, a comparison is made with the values obtained in the gas-phase measurement using microwave spectroscopy (e2qQ = 3.02 MHz and 7 = 0.52: Nygaard, L.; et al. J. Mol. Struct. 1974, 22, 401). Although the difference in quadrupole parameters for pyrazole in the Cu"(dien) complex as compared to values for pyrazole in the gas phase is small, a hydrogen-bonding effect can be attributed to the change observed. An explanation for the differences in quadrupole parameters between the amino nitrogen of imidazole and of pyrazole complexed to Cu"(dien) as a consequence of cross-substitution of nitrogen and carbon in the five-membered heterocycle is given.
Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrup... more Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrupole interactions of the remote nitrogen of pyrazole and pyridazine in CuII(dien)-heterocycle complexes. The quadrupole parameters were obtained by field-dependent ESEEM spectral simulations for the amino nitrogen of pyrazole (e2qQ = 2.86 MHz and 7 = 0.74) and for the imino nitrogen of pyridazine (e2qQ = 5.35 MHz and 7 = 0.02). The values obtained for pyridazine are comparable to those for metal-free solid pyridazine (Schempp, E.; Bray, P. J. J. Chem. Phys. 1967,46,1186. Since the quadrupole parameters for the amino nitrogen in metal-free solid pyrazole have not been reported, a comparison is made with the values obtained in the gas-phase measurement using microwave spectroscopy (e2qQ = 3.02 MHz and 7 = 0.52: Nygaard, L.; et al. J. Mol. Struct. 1974, 22, 401). Although the difference in quadrupole parameters for pyrazole in the Cu"(dien) complex as compared to values for pyrazole in the gas phase is small, a hydrogen-bonding effect can be attributed to the change observed. An explanation for the differences in quadrupole parameters between the amino nitrogen of imidazole and of pyrazole complexed to Cu"(dien) as a consequence of cross-substitution of nitrogen and carbon in the five-membered heterocycle is given.
The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface... more The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5 -8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-+Lys (M44K) azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pK, 9-10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR gll value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation. Fax: +31 71 274537. Abbreviations. Ches, 2-(N-cyclohexylamino)ethmesulfonic acid; Cu(1) azurin, reduced azurin; Cu(I1) azurin, oxidized azurin; Em, midpoint potential ; ESE, electron self-exchange ; ESEEM, electron spin-echo envelope modulation; M44K, Met44+Lys ; RR, resonance Raman; wt, wild type; FT, Fourier transform. due His117 is located in the centre of this patch. The replacement of Met44 by the protonated lysine residue hardly affects the spectroscopic properties of the Cu site, but causes a considerable decrease of the k,,, value of azurin. At pH >8, deprotonation of Lys44 produces a new type-] Cu site, whereas the magnitude of the k,,, value of the protein is largely restored. At pH 5 and pH 8, the midpoint potential, Em, of Met44+Lys (M44K) azurin is higher than the wildtype (wt) azurin Em by 60 mV. At pH > 8, deprotonation of Lys44 in M44K azurin reduces the En, difference from 60 mV to 40 mV. The pH dependence of the wt and M44K azurin En, values is analyzed in terms of the titration of His35, His83 and Lys44. In addition, the effects of the M44K mutation and the Cu-ion oxidation state (I or TI) on the pK, values of the titratable His35 and His83 of azurin are reported and analyzed. The structural and functional implications of the findings are discussed.
... Richard S. Magliozzo*,f and Jack Peisacht.8 Department of Molecular Pharmacology and Departme... more ... Richard S. Magliozzo*,f and Jack Peisacht.8 Department of Molecular Pharmacology and Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 Received June 17, 1991; Revised Manuscript Received ...
Electron spin echo envelope modulation (ESEEM) spectroscopy and angle-selective spectral simulati... more Electron spin echo envelope modulation (ESEEM) spectroscopy and angle-selective spectral simulation were used to study the electron-nuclear hyperfine and nuclear quadrupole interactions of substituted imidazoles axially coordinated in dZ2 ground state ...
Fe(II)-Bleomycin is activated in air to form an electron paramagnetic resonance (EPR)-active spec... more Fe(II)-Bleomycin is activated in air to form an electron paramagnetic resonance (EPR)-active species, termed "activated bleomycin", that cleaves DNA, when present. When DNA is absent, the potential DNA cleavage activity is lost and the drug becomes self inactivated. A method is described for the preparation and purification of this self-inactivated product from bleomycin A2, together with some of its physical properties. It is shown that the loss of DNA cleavage activity parallels an alteration of bithiazole fluorescence, attributed to chemical change at this residue. EPR evidence is brought forth that the Cu(II) binding site of inactivated bleomycin in not altered, nor is the ability to form a species with Fe(II) and O2 having the identical spectroscopic signature as activated bleomycin.
Flavodoxins, the FMN-containing electron carriers present in a variety of rriicroorganisms, have ... more Flavodoxins, the FMN-containing electron carriers present in a variety of rriicroorganisms, have lower oxidation-reduction potentials than other members of the flavoprotein family. Crystal strucfure analyses of these proteins, with FMN in the oxidized, semiquinone, and reduced forms, have been carried out in an attempt to understand how the interaction with proteins modulates the oxidation-reduction potential of flavins. Initial studies with flavodoxin from Clostridium MP have suggested that the oxidized/semiquinone potential depends on formatign of a hydrogen bond between the polypeptide backbone and the "(5) of the flavin semiquinone, while the semiquinone/reduced potential may depend on constraints on the bending and motion of the reduced isoalloxazine ring. These'proposals have been explored by structural comparisons with other species of flavodoxins, especially the protein from Anacystis nidulans, which has a higher potential for the oxidized/semiquinqne equilibrium than most other flavodoxins.
Electron spin echo envelope modulation spectroscopy identified two ligand 14N interactions with t... more Electron spin echo envelope modulation spectroscopy identified two ligand 14N interactions with the mixedvalence, Fe(IVII1) diiron center of methane monoogygenase hydroxylase from Methylococcus capsulatus (Bath). Characteristic features of the spectra obtained at 9 and 10 GHz were analyzed and fit by simulation. One of the nitrogens possessedo superhyperfine parameters (Aiso = 0.8 MHz, re, = 3.2 A, e2Qq = 1.8 MHz, q = 0.35) consistent with a non-coordinating amino nitrogen of a histidine imidazole ligand to a Fe(II1). The second, mo5e strongly interacting nitrogen (Aiso = 5.0 MHz, re, = 2.2 A, e2Qq = 3.0 MHz, q = 0.3) corresponds to the N, directly bound to the effective Fe(I1). These findings extend the previous electron nuclear double resonance results on the Methylosinus trichosporium hydroxylase (Hendrich, M. P., Fox, B. G., Andersson, K. K., Debrunner, P. G., and Lipscomb, J. D. (1992) J. Biol. Chem. 267,261-269), which identified the N,-Fe(I1) interaction but failed to quantify its magnitude. Measurement of the linear electric field g shift of this mixed-valence species indicated that the site is charge-polarized on to one of the iron atoms, and its symmetry suggests that either charge is shifted away from the Fe-Fe axis (if g,, is defined by the Fe-Fe axis) or that gmia and g,, are perpendicular to the Fe-Fe axis (charge strongly localized at Fe(II1) and axis taken as g m 3 .
The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using ... more The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using electrospray ionization mass spectrometry (ESI-MS) and EPR spectroscopy. A rapid reaction occurs that ultimately yields several cleavage products of the drug, the structures of which have been deduced from their masses. The intermediates observed by ESI-MS suggest a mechanism for the reaction of FemBLM with PhIO that does not involve hypervalent iron or activated oxygen but, instead, hypervalent iodine I(II1) as the oxidant. Indeed, we could not detect the production of an iron oxene complex of BLM (O=FeVBLM) or hydroperoxyferric BLM (HOO-Fern-BLM). Furthermore, we show that the products formed when FemBLM is reacted with PhIO are identical to those formed when FemBLM is activated in air or when FemBLM is reacted with H202. Surprisingly, the same products are obtained when ZnnBLM or metal-free BLM is reacted with PhIO, indicating that a metal ion is not required for the oxidation of BLM by PhIO.
The requirements of a cloned macrophage-like cell line, J774.16, for oxygen metabolism, and the n... more The requirements of a cloned macrophage-like cell line, J774.16, for oxygen metabolism, and the nature of the defect in oxidative metabolism in a variant clone derived from it, J774.C3C, were studied. Upon stimulation with phorbol myristate acetate (PMA), the parental clone produced approximately 1 nmol O2-/min/10(6) cells, whereas the variant clone produced no detectable O2- under the same conditions. Sustained O2- production by J774.16 was totally dependent on extracellular glucose; in glucose-free medium, the cells initiated O2- production but could not sustain it. When cells were stimulated with PMA, glucose-C-1 oxidation of J774.16 cells increased 20-fold while that of J774.C3C remained at resting levels. O2- production in J774.16 cells was inhibited by some agents known to block mitochondrial electron transport before coenzyme Q, such as rotenone and tetrathiafulvalene, whereas antimycin A enhanced O2- production. A dissociation between O2- production and glucose-C-1 oxidation was observed when J774.16 was treated with certain metabolic inhibitors. Quinacrine, 2,4-dinitrophenol, chlorpromazine, and trifluoperazine inhibited O2- production completely under conditions in which glucose-C-1 oxidation was reduced only by 30%. Rotenone inhibited O2- production with no effect on glucose-C-1 oxidation whereas antimycin A augmented O2- production 50% but inhibited glucose oxidation by 20%. Glucose transport studies, with 2-deoxy-D-glucose, showed that the Km for glucose transport of both clones was about 1 mM, indicating that cells could effectively transport glucose even at low concentrations. The Vmax for glucose transport in both J774.16 and variant J774.C3C cells doubled after PMA stimulation, indicating that the variant was effectively stimulated by PMA, even though O2- was not produced. Similarly, PMA induced protein phosphorylation in both clones. No differences between clones J774.16 and J774.C3C in hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, or glutathione peroxidase activities could be found. When dithionite-reduced and -oxidized difference spectra of plasma membranes of these clones were compared, comparable levels of b-type cytochrome were found in both clones. However, CO difference spectra indicated that CO was bound to a b-type cytochrome (presumed to be b-245) in clone J774.16 but not in J774.C3C.(ABSTRACT TRUNCATED AT 400 WORDS)
The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using ... more The reaction of ferric bleomycin (FemBLM) with iodosylbenzene (PhIO) has been investigated using electrospray ionization mass spectrometry (ESI-MS) and EPR spectroscopy. A rapid reaction occurs that ultimately yields several cleavage products of the drug, the structures of which have been deduced from their masses. The intermediates observed by ESI-MS suggest a mechanism for the reaction of FemBLM with PhIO that does not involve hypervalent iron or activated oxygen but, instead, hypervalent iodine I(II1) as the oxidant. Indeed, we could not detect the production of an iron oxene complex of BLM (O=FeVBLM) or hydroperoxyferric BLM (HOO-Fern-BLM). Furthermore, we show that the products formed when FemBLM is reacted with PhIO are identical to those formed when FemBLM is activated in air or when FemBLM is reacted with H202. Surprisingly, the same products are obtained when ZnnBLM or metal-free BLM is reacted with PhIO, indicating that a metal ion is not required for the oxidation of BLM by PhIO.
Electron spin echo envelope modulation (ESEEM) spectroscopic examination of Cu(II)-proteins and -... more Electron spin echo envelope modulation (ESEEM) spectroscopic examination of Cu(II)-proteins and -enzymes has been used to identify endogenous ligands to the metal and their number, the presence or absence of coordinated water both in the vicinity of and ligated to the metal, and the mode of binding of inhibitory anions and catalytic substrates. For Cu(I1) centers, such as in stellacyanin, imidazole is identified as a metal ligand based on ESEEM comparisons of the nuclear quadrupole interaction (nqi) of the remote, protonated 14N of the coordinated ligand with that for a Cu(II)-imid model.' For some Cu proteins, such as phenylalanine hydroxylase, galactose oxidase and amine oxidase, the nqi parameters are so different from those for the Cu(II)-imid model remote af4to suggest alterations in the local environment of the N of the coordinated heterocycle. For this reason ESEEM studies were carried out on $$(II)-substituted imid models and -doped single crystals ' in order to understand how structural changes in imid and substituted imid affect nqi parameters. In a model study,' it was shown that the largest changes in nqi, which can be related to external N-H or N-C bond polarization and N oQGta1 occupancy, occurs with Nalkylation of the remote N of metal coordinated imid; alkyl substitution on ring carbon has much less affect. For a Cu(II)-imid model, nqi parameters can be changed by altering the H-bonding capabilities of ring N-H such as with i&O or formate. From single crystal studies, differences in N orbital occupancy of imid a$ determined from the nqi, can be related to the N-H bond length . It is suggested that the variation of nqi observed for some Cu(II)-proteins arises from variations of Hbonding capabilities at the remote nitrogen of the coordinated histidine imidazole side chain. It is further suggested that these H-bonding differences may alter the properties of the metal centers.
Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrup... more Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrupole interactions of the remote nitrogen of pyrazole and pyridazine in CuII(dien)-heterocycle complexes. The quadrupole parameters were obtained by field-dependent ESEEM spectral simulations for the amino nitrogen of pyrazole (e2qQ = 2.86 MHz and 7 = 0.74) and for the imino nitrogen of pyridazine (e2qQ = 5.35 MHz and 7 = 0.02). The values obtained for pyridazine are comparable to those for metal-free solid pyridazine (Schempp, E.; Bray, P. J. J. Chem. Phys. 1967,46,1186. Since the quadrupole parameters for the amino nitrogen in metal-free solid pyrazole have not been reported, a comparison is made with the values obtained in the gas-phase measurement using microwave spectroscopy (e2qQ = 3.02 MHz and 7 = 0.52: Nygaard, L.; et al. J. Mol. Struct. 1974, 22, 401). Although the difference in quadrupole parameters for pyrazole in the Cu"(dien) complex as compared to values for pyrazole in the gas phase is small, a hydrogen-bonding effect can be attributed to the change observed. An explanation for the differences in quadrupole parameters between the amino nitrogen of imidazole and of pyrazole complexed to Cu"(dien) as a consequence of cross-substitution of nitrogen and carbon in the five-membered heterocycle is given.
Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrup... more Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the nuclear quadrupole interactions of the remote nitrogen of pyrazole and pyridazine in CuII(dien)-heterocycle complexes. The quadrupole parameters were obtained by field-dependent ESEEM spectral simulations for the amino nitrogen of pyrazole (e2qQ = 2.86 MHz and 7 = 0.74) and for the imino nitrogen of pyridazine (e2qQ = 5.35 MHz and 7 = 0.02). The values obtained for pyridazine are comparable to those for metal-free solid pyridazine (Schempp, E.; Bray, P. J. J. Chem. Phys. 1967,46,1186. Since the quadrupole parameters for the amino nitrogen in metal-free solid pyrazole have not been reported, a comparison is made with the values obtained in the gas-phase measurement using microwave spectroscopy (e2qQ = 3.02 MHz and 7 = 0.52: Nygaard, L.; et al. J. Mol. Struct. 1974, 22, 401). Although the difference in quadrupole parameters for pyrazole in the Cu"(dien) complex as compared to values for pyrazole in the gas phase is small, a hydrogen-bonding effect can be attributed to the change observed. An explanation for the differences in quadrupole parameters between the amino nitrogen of imidazole and of pyrazole complexed to Cu"(dien) as a consequence of cross-substitution of nitrogen and carbon in the five-membered heterocycle is given.
The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface... more The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5 -8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-+Lys (M44K) azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pK, 9-10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR gll value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation. Fax: +31 71 274537. Abbreviations. Ches, 2-(N-cyclohexylamino)ethmesulfonic acid; Cu(1) azurin, reduced azurin; Cu(I1) azurin, oxidized azurin; Em, midpoint potential ; ESE, electron self-exchange ; ESEEM, electron spin-echo envelope modulation; M44K, Met44+Lys ; RR, resonance Raman; wt, wild type; FT, Fourier transform. due His117 is located in the centre of this patch. The replacement of Met44 by the protonated lysine residue hardly affects the spectroscopic properties of the Cu site, but causes a considerable decrease of the k,,, value of azurin. At pH >8, deprotonation of Lys44 produces a new type-] Cu site, whereas the magnitude of the k,,, value of the protein is largely restored. At pH 5 and pH 8, the midpoint potential, Em, of Met44+Lys (M44K) azurin is higher than the wildtype (wt) azurin Em by 60 mV. At pH > 8, deprotonation of Lys44 in M44K azurin reduces the En, difference from 60 mV to 40 mV. The pH dependence of the wt and M44K azurin En, values is analyzed in terms of the titration of His35, His83 and Lys44. In addition, the effects of the M44K mutation and the Cu-ion oxidation state (I or TI) on the pK, values of the titratable His35 and His83 of azurin are reported and analyzed. The structural and functional implications of the findings are discussed.
... Richard S. Magliozzo*,f and Jack Peisacht.8 Department of Molecular Pharmacology and Departme... more ... Richard S. Magliozzo*,f and Jack Peisacht.8 Department of Molecular Pharmacology and Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 Received June 17, 1991; Revised Manuscript Received ...
Electron spin echo envelope modulation (ESEEM) spectroscopy and angle-selective spectral simulati... more Electron spin echo envelope modulation (ESEEM) spectroscopy and angle-selective spectral simulation were used to study the electron-nuclear hyperfine and nuclear quadrupole interactions of substituted imidazoles axially coordinated in dZ2 ground state ...
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