Hyperfine evidence of strong double exchange in multimetallic {[Fe4S4]-Fe} active center of Escherichia coli sulfite reductase

 The spin-coupling model of the {[Fe₄S₄]-Fe} center of the active site of sulfite reductase, which includes the Heisenberg exchange and double-exchange interactions, explains the peculiarities of the induced paramagnetism. The effective hyperfine constants of the SiR^–1-NO complex are explained in the model with maximal pair spins S₁₂=S₃₄=9/2 in the ground state formed by strong double exchange. Received: 12 January 1996 / Accepted: 23 January 1996     

Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized "ready" state

 Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized "ready" state, Ni_r, in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6 MHz, respectively. Their dipolar coupling is small (2.1 and 1.4 MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5 MHz and a larger anisotropic contribution of 3.5 MHz. Based on a comparison with structural data obtained from X-ray crystallography of single crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Ni_r, an assignment of the ¹H hyperfine couplings could be achieved. H1 and H2 were assigned to the β-CH₂ protons of the bridging cysteine Cys533 and H3 could belong to a β-CH₂ proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530. Received: 26 November 1998 / Accepted: 1 April 1999     

Heterometallic [AgFe3S4] ferredoxin variants: synthesis, characterization, and the first crystal structure of an engineered heterometallic iron–sulfur protein

Heterometallic [AgFe₃S₄] iron–sulfur clusters assembled in wild-type Pyrococcus furiosus ferredoxin and two variants, D14C and D14H, are characterized. The crystal structure of the [AgFe₃S₄] D14C variant shows that the silver(I) ion is indeed part of the cluster and is coordinated to the thiolate group of residue 14. Cyclic voltammetry shows one redox pair with a reduction potential of +220 mV versus the standard hydrogen electrode which is assigned to the [AgFe₃S₄]^2+/+ couple. The oxidized form of the [AgFe₃S₄] D14C variant is stable in the presence of dioxygen, whereas the oxidized forms of the [AgFe₃S₄] wild type and D14H variants convert to the [Fe₃S₄] ferredoxin form. The monovalent d ¹⁰ silver(I) ion stabilizes the [Fe₃S₄]^+/0 cluster fragment, as opposed to divalent d ¹⁰ metal ions, resulting in more than 0.4 V difference in reduction potentials between the silver(I) and, e.g., zinc(II) heterometallic [MFe₃S₄] ferredoxins. The trend in reduction potentials for the variants containing the [AgFe₃S₄] cluster is wild type ≤ D14C < D14H and shows the same trend as reported for the variants containing the [Fe₃S₄] cluster, but is different from the D14C < D14H < wild type trend reported for the [Fe₄S₄] ferredoxin. The similarity in the reduction potential trend for the variants containing the heterometallic [AgFe₃S₄] cluster and the [Fe₃S₄] cluster can be rationalized in terms of the electrostatic influence of the residue 14 side chains, rather than the dissociation constant of this residue, as is the case for [Fe₄S₄] ferredoxins. The trends in reduction potentials are in line with there being no electronic coupling between the silver(I) ion and the Fe₃S₄ fragment.     

Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I Ectothiorhodospira halophila

4 S₄]^3 + and the reduced [Fe₄S₄]^2 + clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and ⁵⁷Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe₄S₄]^3 + clusters, the M?ssbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe^3 + atoms couple to the “ferric-ferric” pair, and one Fe^2 + and one Fe^3 + atom give the “ferric-ferrous pair”. For the simulation of the M?ssbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by ⁵⁷Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of M?ssbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical M?ssbauer spectra. Spin-spin and spin-lattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the M?ssbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the M?ssbauer spectrum, indicating a mixed-valence state of Fe^2.5 + for each. Received: 15 February 1999 / Accepted: 31 August 1999     

Synthesis and characterization of [Fe(III)(qsal)2][M(III)(pds)2] (M = Cu, Au)

Salts of the Fe(III) spin crossover cation [Fe^III(qsal)₂]⁺ (qsalH = N-(8-quinolyl)salicylaldimine) and monoanions [M^III(pds)₂]⁻ (M = Cu, Au; pds = pirazine-2,3-diselenolate) with formula [Fe^III(qsal)₂][M^III(pds)₂] were prepared and characterized by single crystal X-ray diffraction and magnetic measurements. These two salts present magnetic properties essentially due to the Fe^III centres in the high-spin state (S = 5/2), and do not have any spin transition.     

Searching for switchable molecular conductors: Salts of [M(dcbdt)2] (M = Ni, Au) anions with [Fe(sal2-trien)] and [Fe(phen)3]

Salts of [Fe^III(sal₂-trien)]⁺and [Fe^II(phen)₃]²⁺ cations and M[(dcbdt)₂]⁻ anions with M = Ni and Au (dcbdt = dicyanobenzenedithiolate) with formula [Fe(sal₂-trien)] [M(dcbdt)₂] and [Fe(phen)₃] [M(dcbdt)₂]₂ were obtained and characterized by single X-ray diffraction and magnetic measurements. None of these salts shows a clear spin crossover behaviour and their magnetic properties are due essentially to the cations in a high spin S = 5/2 and low spin states for the Fe^III and Fe^II salts respectively. The magnetic Ni sublattices in both compounds appear to have a negligible direct contribution to the magnetization but enhance the AF interactions in the cation sublattice.     

Probing the structural, electronic and magnetic properties of multicenter Fe2S2 0/−, Fe3S4 0/− and Fe4S4 0/− clusters

The structural, electronic and magnetic properties of neutral and anion Fe₂S₂, Fe₃S₄ and Fe₄S₄ have been investigated with the aid of previous photoelectron spectroscopy and density functional theory calculations. Theoretical electron detachment energies (both vertical and adiabatic) of anion clusters for the lowest energy structure were computed and compared with the experimental results to verify the ground states. The optimized structures show that the ground state structures of Fe₂S₂ ^0/?, Fe₃S₄ ^0/? and Fe₄S₄ ^0/? favor high spin state and are similar to their structures in proteins. The electron delocalization pattern for all the clusters and the nature of bonding between Fe and S atoms were studied by analyzing molecular orbitals. Natural population analysis demonstrates that Fe atoms act as an electron donor in all clusters, and the electron density difference map clearly shows the direction of the electron flow over the whole complex. Furthermore, the investigated magnetism shows that the Fe atoms carried most of the magnetic moments, which is due mainly to the 3d state, while only very small magnetic moments are found on S atoms.     

Experimental data and calculated parameters in FeS polymetallic centers in proteins

 Covariance between J and B parameters and indetermination of the actual symmetry suggest caution in making general statements about their values in Fe_nS_n centers. The Fe₄S₄ ³⁺ case is further complicated by electronic isomerism and lack of information on the actual symmetry. Electronic ground states and relative J and B values are discussed in the light of hyperfine coupling values. Received: 12 January 1996 / Accepted: 26 January 1996     

FTIR, Raman, and UV-Vis spectroscopic and DFT investigations of the structure of iron-lead-tellurate glasses

In this work, the effects of iron ion intercalations on lead–tellurate glasses were investigated via FTIR, Raman and UV-Vis spectroscopies. This homogeneous glass system has compositions xFe₂O₃·(100−x)[4TeO₂·PbO₂], where x = 0–60 mol%. The presented observations in these mechanisms show that the lead ions have a pronounced affinity towards [TeO₃] structural units, resulting in the deformation of the Te–O–Te linkages, and leading to the intercalation of [PbO _n ] (n = 3, 4) and [FeO _n ] (n = 4, 6) entities in the [TeO₄] chain network. The formation of negatively charged [FeO₄]¹⁻ structural units implies the attraction of Pb²⁺ ions in order to compensate for this electrical charge. Upon increasing the Fe₂O₃ content to 60 mol%, the network can accommodate an excess of oxygen through the formation of [FeO₆] structural units and the conversion of [TeO₄] into [TeO₃] structural units. For even higher Fe₂O₃ contents, Raman spectra indicate a greater degree of depolymerization of the vitreous network than FTIR spectra do. The bands due to the Pb–O bond vibrations are very strongly polarized and the [TeO₄] structural units convert into [TeO₃] units via an intermediate coordination stage termed “[TeO₃₊₁]” structural units. Our UV-Vis spectroscopic data show two mechanisms: (i) the conversion of the Fe³⁺ to Fe²⁺ at the same time as the oxidation of Pb²⁺ to Pb⁺⁴ ions for samples with low Fe₂O₃ contents; (ii) when the Fe₂O₃ content is high (x ≥ 50 mol%), the Fe²⁺ ions capture positive holes and are transferred to Fe³⁺ ions through a photochemical reaction, while the Pb²⁺ ions are formed by the reduction of Pb⁴⁺ ions. DFT calculations show that the addition of Fe₂O₃ to lead–tellurate glasses seems to break the axial Te–O bonds, and the [TeO₄] structural units are gradually transformed into [TeO₃₊₁]- and [TeO₃]-type polyhedra. Analyzing these data further indicates a gradual conversion of the lead ions from covalent to ionic environment. There is then a charge transfer between the tri- and tetracoordinated tellurium atoms due to the capacity of the lead–tellurate network to form the appropriate coordination environments containing structural units of opposite charge, such as iron ions, [FeO₄]¹⁻.     

Determination of ligand conformation in reduced [2Fe-2S] ferredoxin from cysteine β-proton hyperfine couplings

 The isotropic hyperfine couplings of cysteine β-protons in iron-sulfur clusters of proteins provide information about the structure and conformation of the clusters if their magnetic resonance peaks can be resolved and assigned. The application of two-dimensional ESEEM (HYSCORE) spectroscopy to the reduced [2Fe-2S] cluster in ferredoxin from red marine algae Porfira umbilicalis is described. After deuterium substitution of the exchangeable protons, highly-resolved, orientationally-selected HYSCORE spectra show cross-peaks from strongly coupled, nonexchangeable protons. When cross-peaks from all the HYSCORE spectra are linearized and transformed to a common nuclear Zeeman frequency, they fall along five straight lines. Four of these sets of peaks are assigned to β-protons of the cysteine ligands. The isotropic and anisotropic hyperfine couplings for these protons are extracted from the slopes and intercepts of these lines. Two rescaling procedures are examined for the conversion of the experimentally measured isotropic couplings from different irons in [2Fe-2S] and [4Fe-4S] clusters. The couplings from P. umbilicalis appear to fit the same empirical dependence on Fe-S-C-H dihedral angle as do the couplings from a [4Fe-4S] model cluster. A method to assign protons for proteins of unknown structure is proposed that yields the correct assignment as derived from the crystal structure of the highly homologous protein from Spirulina platensis. The conformations of the cysteines in the reduced protein, derived without any adjustable parameters from this procedure and the empirical functions, are consistent with those reported for the latest refinement of the crystal structure of the oxidized protein. Received: 24 September 1997 / Accepted: 28 October 1997     

Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O₂ in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe³⁺ (g = 4.3) and aqueous Mn²⁺ ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe³⁺ hemes were observed, probably arising from cytochrome c peroxidase and the a₃:Cu_b site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe₂S₂]⁺ cluster of succinate dehydrogenase, the [Fe₂S₂]⁺ cluster of the Rieske protein of cytochrome bc ₁, and the [Fe₃S₄]⁺ cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g _ave = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe₄S₄ clusters (60–85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O₂, was a quadrupole doublet with ΔE _Q = 1.15 mm/s and δ = 0.45 mm/s, assigned to [Fe₄S₄]²⁺ clusters. Substantial high-spin non-heme Fe²⁺ (up to 20%) and Fe³⁺ (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.     

Probing the ground state of the purple mixed valence CuA center in nitrous oxide reductase: a CW ENDOR (X-band) study of the 65Cu, 15N-histidine labeled enzyme and interpretation of hyperfine couplings by molecular orbital calculations

 CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, Cu_A site in nitrous oxide reductase were obtained after insertion of ⁶⁵Cu or both ⁶⁵Cu and ¹⁵N-histidine. The ¹⁴N/¹⁵N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for ¹⁴N was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin density of 3–5% was estimated for the nitrogen donors to Cu_A, indicating that the ground state is ²B_3u. Proton hyperfine structure was detected from four C_β protons of coordinating cysteine residues. Their isotropic and anisotropic parts were deconvoluted by spectral simulation. From the anisotropic couplings a spin density of 16–24% was estimated for each of the cysteine thiolate donors of Cu_A. The [N_HisCu(RS)₂CuN_His]⁺ core structure of Cu_A in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallographically determined Cu_A* structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92 : 11955–11959), but distinct from the Cu_A structure of Paracoccus denitrificans cytochrome c oxidase (Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376 : 660–669). The angular dependence of the isotropic couplings as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gradient-corrected density functional method and the semiempirical INDO/S method were compared with experimentally derived spin populations, and good agreement between theory and experiment was found for both calculations. The ground state of Cu_A is best represented by the resonance structures of the form [Cu^IS^–S^–Cu^II↔ Cu^IS^•S^–Cu^I↔ Cu^IS^–S^•Cu^I↔ Cu^IIS^–S^–Cu^I]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization of this novel type of cluster. Received: 17 September 1997 / Accepted: 28 October 1997     

Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins

NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-¹³C, ¹⁵N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β-¹³C; α,β-²H₂] Cys and (2R, 3R)-[β-¹³C; α,β-²H₂] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ² and χ³, can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.     

1H NMR spectra of vertebrate [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

3 S₄]⁺, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T ₁, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2–4.2 K. The T ₁ values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g ₃ values and in ⁵⁷ Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T ₁ is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at ∼20 cm⁻¹ above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported ⁵⁷Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J ₁₃=J, J ₁₂=J(1+ε′), J ₂₃=J(1+ε), where J _ij is the isotropic exchange coupling between ferric ions i and j, and ε and ε′ are measures of coupling inequivalence. We have extended their theory to include the effects of ε′≠0 and thus derived an exact expression for the energy of the doublet excited state for any ε, ε′. This excited state energy corresponds roughly to ε J and is in the range 5–10 cm⁻¹ for each of these four 3Fe proteins. This magnitude of the product ε J, determined by our time-domain relaxation studies in the temperature range 2–4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5–50 K, NMR studies in the range 293–303 K, and static susceptibility measurements in the range 1.8–200 K. We suggest that an apparent disagreement as to the individual values of J and ε be resolved in favor of the values obtained by susceptibility and NMR (J≳200 cm⁻¹ and ε≳0.02 cm⁻¹ ), as opposed to a smaller J and larger ε as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters. Received: 23 December 1999 / Accepted: 8 March 2000     

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH₂ protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH⁻ bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Maurice van Gastel and Matthias Stein contributed equally to this work.     

β-Amyloid enhances intracellular calcium rises mediated by repeated activation of intracellular calcium stores and nicotinic receptors in acutely dissociated rat basal forebrain neurons

β-Amyloid, a 39–43 amino acid peptide, may exert its biological effects via neuronal nicotinic acetylcholine receptors. Using the ratiometric dye, fura-2, we examined the effect of soluble β-amyloid_1–42 on the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in acutely dissociated rat basal forebrain neurons. Focal applications of nicotine (0.5–20 mM), evoked dose-dependent increases in intracellular [Ca²⁺]_i that were mediated by the entry of extracellular Ca²⁺ via nicotinic acetylcholine receptors, and the release of intracellular Ca²⁺ from stores. With repeated nicotine challenges, the nicotinic responses were potentiated by 98 ± 12% (P < 0.05) while β-amyloid_1–42 (100 nM) was present for ∼5 min. This potentiation became larger during the subsequent washout of β-amyloid_1–42, which was associated with a gradual rise in baseline [Ca²⁺]_i. Application of β-amyloid_1–42 by itself did not alter [Ca²⁺]_i, and β-amyloid_1–42 also had no significant effect on the response to repeated KCl challenges. Therefore, β-amyloid_1–42 caused neither gross disturbance of cellular Ca²⁺ homeostasis nor enhancement of voltage-gated Ca²⁺ channels. Interestingly, β-amyloid_1–42 transiently potentiated the response to repeated caffeine challenges, which was also associated with a transient rise in baseline [Ca²⁺]_i. β-amyloid_1–42 potentiation of nicotine-evoked rises in [Ca²⁺]_i was reversed by the SERCA pump inhibitor, thapsigargin, and the mitochondrial Na⁺/Ca²⁺ exchanger inhibitor, CGP-37157. These results suggest that the dysregulation of [Ca²⁺]_i by β-amyloid_1–42 during multiple challenges with nicotine or caffeine involved the sensitization or overfilling of intracellular stores that are maintained by SERCA pump and Ca²⁺ efflux from the mitochondria.     

Dinitrosyl iron complexes [E5Fe(NO)2] (E = S, Se): A precursor of Roussin’s black salt [Fe4E3(NO)7]

[PPN][Se₅Fe(NO)₂] (1) and [K-18-crown-6-ether][S₅Fe(NO)₂] (2′) were synthesized and characterized by IR, UV-Vis, EPR spectroscopy, magnetic susceptibility, and X-ray structure. [PPN][Se₅Fe(NO)₂] easily undergoes ligand exchange with S₈ and (RS)₂ (R = C₇H₄SN (5), o-C₆H₄NHCOCH₃ (6), C₄H₃S (7)) to form [PPN][S₅Fe(NO)₂] and [PPN][(SR)₂Fe(NO)₂]. The reaction displays that [E₅Fe(NO)₂]⁻ (E = Se (3), S (4)) facilely converts to [Fe₄E₃(NO)₇]⁻ by adding acid HBF₄ or oxidant [Cp₂Fe][BF₄] in THF, respectively. Obviously, complexes 1 and 2′ serve as the precursors of the Roussin’s black salts 3 and 4. The electronic structure of {Fe(NO)₂}⁹ core of [Se₅Fe(NO)₂]⁻ is best described as a dynamic resonance hybrid of {Fe⁺¹(NO)₂}⁹ and {Fe⁻¹(NO⁺)₂}⁹ modulated by the coordinated ligands. The findings, EPR signal of g = 2.064 for 1 at 298 K, implicate that the low-molecular-weight DNICs and protein-bound DNICs may not exist with selenocysteine residues of proteins as ligands, since the existence of protein-bound DNICs and low-molecular-weight DNICs in vitro has been characterized with a characteristic EPR signal at g = 2.03. In addition, complex 2′ treated human erythroleukemia K562 cancer cells exposed to UV-A light greatly decreased the percentage survival of the cell cultures.     

Electrical bursting and luminal calcium oscillation in excitable cell models

 It is shown in this paper that electrical bursting and the oscillations in the intracellular calcium concentration, [Ca²⁺]_i, observed in excitable cells such as pancreatic β-cells and R-15 cells of the mollusk Aplysia may be driven by a slow oscillation of the calcium concentration in the lumen of the endoplasmic reticulum, [Ca²⁺]_lum. This hypothesis follows from the inclusion of the dynamic changes of [Ca²⁺]_lum in the Chay bursting model. This extended model provides answers to some puzzling phenomena, such as why isolated single pancreatic β-cells burst with a low frequency while intact β-cells in an islet burst with a much higher frequency. Verification of the model prediction that [Ca²⁺]_lum is a primary oscillator which drives electrical bursting and [Ca²⁺]_i oscillations in these cells awaits experimental testing. Experiments using fluorescent dyes such as mag-fura-2-AM or aequorin could provide relevant information. Received: 17 August 1995/Accepted in revised form: 10 July 1996