Low-temperature Electron Paramagnetic Resonance Research Articles

Reaction of formate with the fast form of cytochrome oxidase: a model for the fast to slow conversion.

The ability to isolate preparations of cytochrome oxidase which are highly homogeneous has facilitated a study of the effects of various reagents on the purified enzyme. The addition of either sodium formate, formamide, formaldehyde, or sodium nitrite to enzyme which reacts in a single rapid kinetic phase with cyanide causes a blue-shift of 4-6 nm of the net (cytochrome a + cytochrome a3) Soret maximum. Only the derivative prepared by adding sodium formate demonstrates measurable intensity in the g' = 12 region of the low-temperature electron paramagnetic resonance (EPR) spectrum. This g' = 12 resonance is characteristic of cytochrome oxidase which has undergone a modification at the binuclear center and thereby reacts sluggishly with cyanide. As the site of cyanide binding in resting enzyme as been demonstrated to be CuB [Yoshikawa, S., & Caughey, W.S. (1990) J. Biol. Chem. 265, 7945-7958], it is proposed that formate can bind to CuB and the fast to slow transition is rationalized by using this proposal. The g' = 12 signal is also produced upon the addition of sodium formate to mitochondrial preparations, suggesting that the species responsible for this behavior may have possible physiological relevance. Physical properties of the formate derivative and data for other reagents reacted with the fast-reacting enzyme preparation are presented.

Low temperature EPR and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate: quinone oxidoreductase indicate bis-histidine coordination of the heme iron

Bacillus subtilis cytochrome b-558 was expressed in high amounts in Escherichia coli, solubilized from membranes with detergent and purified free from other hemoproteins. The cytochrome possibly contains two heme groups. To determine the axial ligands to the low-spin heme and the heme rhombicity, the cytochrome was analyzed using low-temperature electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopy. The combined results exclude bis-methionine, bis-lysine and histidine-methionine coordination. Bis-histidine coordination of the heme(s) with a near perpendicular orientation of the imidazole planes is strongly suggested by the highly axial low-spin EPR signals and the intense near infrared MCD spectrum (Δϵ=380 M −1·cm −1 at 4.2 K and 5 T) of the charge-transfer band at 1600 nm.

Influence of polypeptides on the reactivity of thiols toward iron(III) complex ions

Oxidation of L-cysteine by quaterpyridineiron( III) complex ions (FeT) anchored to ordered poly(L-glutamate) or poly(D-glutamate) was studied in the pH range 7–8. Surprisingly, electron transfer from substrate to the central metal ion does not take place, despite the favorable thermodynamic driving force. Instead, a stable, polymer-supported Fe IIIT-cysteinate complex forms, to which no stereoselectivity is associated. This finding contrasts the known lability of Fe III-thiolate complexes, ultimately yielding disulfides and iron( II) species, and is relevant in view of the current interest in model compounds of cytochrome P-450, where cysteine is axially bound to ferric porphyrin. The formation of this complex was studied by kinetics and low-temperature electron paramagnetic resonance spectroscopy. The stereochemical features of the diastereomeric adducts were investigated by theoretical conformational analysis and compared with those already computed for the encounter complexes with some L-dihydroxy substrates. The results collectively emphasize the environmental control of the polypeptide matrix on the stability of cysteinate sulfur axial ligation.

Electron spin resonance studies of wild-type and mutant cytochromes P-450 d: effects of mutations at proximal, aromatic and distal sites on g values

Low-temperature (6–40 K) electron spin resonance (ESR) spectra of cytochrome P-450 d ( P-450 d) and its 17 mutants have been measured. The spectra of the wild-type and all mutant P-450 ds showed signals at around g = 8, 3.7 and 1.7, while they didn't show any signal at around g = 2 up to 40 K. It was thus suggested that all of these P-450 ds essentially take the ferric high-spin form. The g values of the proximal mutants were closer to those of the wild-type than those of the distal and aromatic mutants, suggesting that mutations at the distal and aromatic sites influence the electronic state of the heme more profoundly than those of the proximal site. The distal multiple mutants whose distal sequences are the same as those of the low-spin type P-450s such as rat P-450 c, mouse P 1-450 and P 3-450 showed only high-spin ESR signals. Thus the spin state of P-450 dS (the wild-type and all mutants) may not be solely due to specific characteristics of the distal site, but to the unique nature of the whole heme environment of P-450 d. It is also suggested that the amino acids at the distal region of P-450 d may be located close to the heme, so that the water molecule cannot bind to the heme, thus taking the high-spin state. Both the aromatic mutants showed rather large deviations of the g values from those of wild-type P-450 d, suggesting that the aromatic region somehow interacts with the heme.

The .Si identical to Si3 defect at various (111)Si/SiO2 and (111)Si/Si3N4 interfaces

The low-temperature (2<or=T<or=45 K) X- and K-band electron spin resonance (ESR) properties of the Si/SiO2 interfacial (111) PbO defect-identified with .Si identical to Si3 and with the unpaired sp3 hybrid along (111)-as localised at three 'different' (111)Si/SiO2 interfaces, are comparatively surveyed. It is observed that differences in the ESR details develop in moving from state-of-the-art thermally oxidised Si/SiO2 over a Si/SiO2 structure grown under strongly reduced O2 pressure to the Si/native oxide case, indicating increasing interfacial strain or disorder. Secondly, ESR data are presented on the first observation of the .Si identical to Si3 defect-now called (111)PbNO-in (111)Si/Si3N4, thermally grown at 1100+or-20 degrees C in NH3. The main ESR signatures of the PbO and PbNO centres are very similar, from which their common origin is concluded. Yet, the differences in ESR properties from state-of-the-art Si/SiO2 to the Si/native SiO2 structures are all exhibited by PbNO too; in fact, they show up more intensely and reveal the more strained nature of the Si/Si3N4 interface. The PbNO observation confirms the PbO(PbNO) defect as a prototype dangling-bond defect with the unpaired hybrid pointing out of the Si substrate. Generally, the PbO(PbNO) defect appears to be a highly sensitive probe apt to reflect variations in the physico-chemical nature of the interface through its low-temperature ESR features.

The axial ligands of heme in cytochromes: a near-infrared magnetic circular dichroism study of yeast cytochromes c, c1, and b and spinach cytochrome f

Room temperature near-infrared magnetic circular dichroism and low-temperature electron paramagnetic resonance measurements have been used to characterize the ligands of the heme iron in mitochondrial cytochromes c, c1, and b and in cytochrome f of the photosynthetic electron transport chain. The MCD data show that methionine is the sixth ligand of the heme of oxidized yeast cytochrome c1; the identify of this residue is inferred to be the single conserved methionine identified from a partial alignment of the available cytochrome c1 amino acid sequences. A different residue, which is most likely lysine, is the sixth heme ligand in oxidized spinach cytochrome f. The data for oxidized yeast cytochrome b are consistent with bis-histidine coordination of both hemes although the possibility that one of the hemes is ligated by histidine and lysine cannot be rigorously excluded. The neutral and alkaline forms of oxidized yeast cytochrome c have spectroscopic properties very similar to those of the horse heart proteins, and thus, by analogy, the sixth ligands are methionine and lysine, respectively.

A spectroelectrochemical cell designed for low temperature electron paramagnetic resonance titration of oxygen-sensitive proteins

In this paper we describe an anaerobic titrator made virtually from glass with a small amount of high vacuum epoxy mounted directly to a quartz EPR tube. A complete titration may be carried out with as little as 600 μl of sample. This cell features the anaerobic manipulation of an electrochemically poised solution from an electrochemical pouch to an EPR tube. The cell uses a gold foil working electrode and Ag AgCl reference and counter electrodes. The reference and counter electrodes are isolated from the sample by leached Vycor glass. In the work reported here, we used this cell to determine the equilibrium redox potential of methyl viologen in an EPR titration. With methyl viologen as an indicator we found that the cell has a residual oxygen level of 1.5 μ m with a leak rate of 0.005 nmol/min. After moving the solution into the EPR tube, freezing, performing EPR, and thawing, the potential of the methyl viologen solution drifted only 2 mV. During the titration, the poised potentials were stable, drifting only 1 mV/min. Formal potentials as low as −630 mV in a vitamin B 12-type protein have been determined with this cell (S. R. Harder, W.-P. Lu, B. A. Feinberg, and S. W. Ragsdale (1989) Biochemistry, in press) .

Superoxide anion scavenging effect and superoxide dismutase activity ofGinkgo biloba extract

Ginkgo biloba extract is known to be efficient in diseases associated with free radical generation. The purpose of this work was to study, under in vitro conditions, the action of Ginkgo biloba extract (Gbe) against superoxide anion (O2-.), which is directly or indirectly implicated in cell damage. Gbe appears to have both an O2-. scavenging effect and also a superoxide dismutase activity. Its antiradical effect was demonstrated by low temperature electron spin resonance and in a non-enzymatic system (phenazine methosulfate-NADH), and its enzymatic activity was shown by polarographic determination.

P-rich Si particles in separation by implanted oxygen structures revealed by low-temperature electron-spin resonance

Low-temperature X- and K-band electron-spin-resonance measurements on separation by implanted oxygen structures formed by implanting oxygen to a dose ≊1.7×1018 cm−2 on [001] c-Si wafers—both n and p type [dopant concentration ≊(9–28)x1014 cm−3 ]—reveal the presence of a signal due to submetallic Si:P effectively doped to [P]≊2.0×1018 cm−3 . The signal is identified as originating from the polyhedron-shaped c-Si precipitates known to remain in the buried SiO2 layer near the bulkside Si/SiO2 interface, even after high-temperature annealing. The capstone in this identification stems from the faceted structure of these Si islands, which, combined with the concomitant misfit-induced and plane-index related strain, accounts for the anisotropic g and linewidth—not observed as such in bulk Si:P. This result indicates an impurity effect as contributing to the persistence of these Si microcrystallites upon annealing.

Magnetic circular dichroism and electron paramagnetic resonance studies of iron(II)-metallothionein

The electronic and magnetic properties of the Fe(II)-thiolate centers in Fe(II)-metallothionein have been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies at various levels of Fe(II) incorporation. In agreement with previous results [Good, M., & Vasák, M. (1986) Biochemistry 25, 8353-8356], rabbit liver metallothionein was found to bind a maximum of seven Fe(II) ions, with cluster formation occurring when more than four Fe(II) ions are bound at pH 8.5. The results indicate that all the iron in fully loaded Fe(II)-metallothionein is accommodated in Fe(II)-thiolate clusters that have either S = 0 or S = 2 ground states as a result of antiferromagnetic coupling between high-spin Fe(II) ions. By analogy with the cluster composition and mechanism of assembly that have been established for other divalent metal ions, the clusters with S = 0 and S = 2 ground states are attributed to tetranuclear and trinuclear centers, respectively. EPR signals indicative of S = 2 species were observed for samples containing monomeric tetrathiolate-Fe(II) centers and trinuclear Fe(II)-thiolate clusters. However, the nature of the zero-field splitting of the S = 2 ground states that is indicated by the EPR signals is not consistent with that deduced from Mössbauer and magnetic circular dichroism studies, suggesting heterogeneity in both types of center.

Observation of the localized Si dangling-bond Pb defect at the Si/Si3N4 interface.

Low-temperature electron-spin resonance (ESR) reveals the presence of the ${P}_{b}$ defect (identified with Si\ensuremath{\equiv}${\mathrm{Si}}_{3}$) at the thermally grown (111)Si/${\mathrm{Si}}_{3}$${\mathrm{N}}_{4}$ interface. This constitutes the first observation of this defect (called ${P}_{\mathrm{bN}}$) at a natural Si/solid interface other than the Si/Si${\mathrm{O}}_{2}$ one. It is argued that ESR analysis of the ${P}_{\mathrm{bN}}$ center is a powerful tool for characterizing the Si/${\mathrm{Si}}_{3}$${\mathrm{N}}_{4}$ interface on an atomic scale. This observation again confirms ${P}_{b}$ as a prototype dangling-bond center, its main properties being determined by the backside Si matrix.

Effects of anion size and disorder on spin resonance in the BEDT-TTF salts

The effects of different linear trihalide anions on electron spin resonance (ESR) spectra have been studied for single crystals of four BEDT-TTF salts plus one crystal containing mixed anions. Structural disorder is reflected in an increased low-temperature (residual) ESR linewidth for both ß-(BEDT-TTF) 2I 3 and ß-(BEDT-TTF) 2I 2Br and for (nominally!) ß-(BEDT_TTF) 2 (AuI 2) 0.82(I 3) 0.18 Suppression of the disorder transition in ß-(BEDT-TTF) 2I 3 by application of shear stress to form the ß ∗- phase decreases the residual linewidth while increasing the onset of superconductivity from 1.5 to ≈8 K. Anion size (or equivalently unit cell size) has been shown to correlate with the superconducting transition temperature in these isomorphic salts, but we find no correlation between anion size and the ESR spin susceptibility, linewidth or microwave conductivity.

Electron paramagnetic resonance and magnetic circular dichroism studies of electron-transfer flavoprotein-ubiquinone oxidoreductase from pig liver

Pig liver electron-transfer flavoprotein-ubiquinone oxidoreductase has been investigated by room temperature UV-visible, low-temperature electron paramagnetic resonance and low-temperature magnetic circular dichroism spectroscopies. The results provide unambiguous evidence for the presence of a single [4Fe-4S] cluster that is diamagnetic in the isolated enzyme and becomes paramagnetic with an S = 1 2 , ground state on reduction with dithionite or enzymatically with the physiological electron donor. The EPR data for samples at pH 7.8 indicate that FAD is reduced by one electron to the anionic semiquinone form in the enzymatically reduced enzyme, and by two electrons to the hydroquinone form by excess dithionite. The possibility of weak spin-spin interaction between the FAD semiquinone and the [4Fe-4S] 1+ center is discussed in the light of the observation of a small increase in the linewidth of the Fe-S EPR in enzymatically reduced samples.

Electron paramagnetic resonance and magnetic susceptibility studies of dimanganese concanavalin A. Evidence for antiferromagnetic exchange coupling.

The double Mn2+ complex of concanavalin A with bound saccharide (SMMPL) was examined by electron paramagnetic resonance (EPR) spectroscopy and magnetic susceptibility measurements. A room temperature X-band (9 GHz) EPR spectrum of SMMPL revealed a relatively weak, broad resonance in contrast to the spectrum with a six-line hyperfine-split pattern observed for the mononuclear, high-spin Mn2+ complex found in Ca2+-Mn2+-concanavalin A with saccharide present (SCMPL). The EPR spectrum of SMMPL at 77 K, however, consisted of a series of overlapping patterns of 11 hyperfine-split lines near g = 2.0 with members of each pattern separated by 47 G, half the value of the hyperfine splitting of SCMPL. These 11-line patterns are preserved at Q-band (35 GHz), indicating that the manganese ions in SMMPL form a spin-coupled, binuclear center. As expected for an exchange-coupled system, the EPR signal of SMMPL at 77 K saturates at a higher microwave power than those for SCMPL or Mn2+ aquoion. There is also a marked loss of EPR signal intensity for SMMPL between 4.2 and 1.4 K, which supports the view that the pair of manganese ions is exchanged-coupled. The temperature dependence of both the magnetic susceptibility and the low-temperature EPR spectral intensity can be explained by a model in which the two high-spin Mn2+ ions of SMMPL are antierromagnetically exchanged-coupled with an isotropic coupling constant J = 1.8 cm-1 (for the spin Hamiltonian Hex = JS1.S2). Zero-field splitting D' was estimated to be 375 G from the EPR spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Precautions when interpreting EPR and dc magnetization measurements of high-Tc RBa2Cu3Oy-phase superconducting materials.

Many electron-paramagnetic-resonance (EPR) and magnetic susceptibility measurements for the high-${T}_{c}$ superconductors of the form $R{\mathrm{Ba}}_{2}{\mathrm{Cu}}_{3}{\mathrm{O}}_{9\ensuremath{-}x}$ ($R=\mathrm{Y}$ or a rare earth) have been reported. Excluding local moment resonances due solely to the $R$ atoms, we show that all of the EPR measurements known to us are not intrinsic to the superconducting phase, but rather are due to low concentrations of spurious phases typically present in the samples. BaCu${\mathrm{O}}_{2}$ is found to be the main source of the low-temperature EPR signal and of the magnetic susceptibility which has been reported for $\mathrm{Y}{\mathrm{Ba}}_{2}{\mathrm{Cu}}_{3}{\mathrm{O}}_{9\ensuremath{-}x}$ above ${T}_{c}$.

ESR of Er 3+, Gd 3+, Yb 3+ and Dy 3+ in the heavy-fermion compounds UPt 3 and UBe 13

Low temperature ( T>0.6K ) Electron Spin Resonance (ESR) experiments were carried out for the Heavy-Fermion compounds UBe 13 and UPt 3 doped with several Rare Earth ions. The measured Korringa rate for all Rare Earth were always within the same order of magnitude of a normal metal and no significant deviation from the linear behavior was observed. The experimental results lead to the conclusion that the coupling between the Uranium ion and the Rare Earth ion through the s-f electron interaction is very small, if any.

Low-temperature ESR study of P bO defects residing in the (111) Si/native oxide interface

A K-band electron spin resonance (ESR) study of the (111) Si/native SiO 2 structure has been carried out in the temperature range 2.4 ≲ T ≲30 K. The ESR signal of the interfacial [111] P bO center was observed, exhibiting in general similar properties to the well-documented [111] P bO signal observed on firmly thermally-oxidized (TO) Si. However, many details in the P bO spectrum differ like, e.g., the strain-induced inhomogeneous ( g-spread) part (♢ B G pp) of the linewidth. For B|[111] ( B being the externally applied magnetic field), the 29Si hyperfine structure of the [111] P bO center has been observed which is characterized by the hyperfine splitting a hf = 159±2 G and the hf signal width ♢ B hf pp = 14.8±0.5 G. Both the observed ♢ B G pp and ♢ B hf pp reveal the existence of a significantly larger strain-induced g distribution in Si/native SiO 2. Apart from the [111] P bO center, also the signals of the P bO defects with their dangling orbital parallel to [11 1 ], [1 1 1] or [ 1 11] have been observed; as yet, these signals have not been observed on TO structures. The saturation behaviour has been examined showing the P bO Si/native oxide interface center to be much less saturable than the P bO in TO structures. The observations are related to the particular physico-chemical interface structure.

Spectroscopic Properties of Siroheme Extracted from Sulfite Reductases

Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two p K values, 4.2 and 9.0, were determined by pH titration in the range 2–12. The first p K (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second p K (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with g XXX = 6.0 and g= 2.0, typical of high-spin ferric hemes (S = 5 2 ), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with g XXX = 2.38 and g = 1.76 that is indicative of a low-spin ferric heme (S = 1 2 ). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment.

Spectroelectrochemical properties of ruthenium(II) tris chelate complexes of 2,2'-bipyrimidine and 2,2'-bipyrazine

The electronic absorption and resonance Raman (RR) spectra of the reduction products of (Ru(bpm)/sub 3/)/sup 2-n/ and mixed-ligand complexes (Ru(bpz)/sub x/(bpy)/sub 3-x/)/sup 2-n/ (x = 1-3, n = 0-3) are discussed, as well as the low-temperature electron spin resonance (ESR) spectrum of (Ru(bpz)/sub 3/)/sup 0/ (bpm = 2.2'-bipyrimidine, bpz = 2,2'-bipyrazine, and bpy = 2,2'-bipyridine). The ultraviolet (UV) absorption and RR spectra for the reduction products of the tris(bipyrimidine) complex are analogous to those of the tris(bipyridine) complex and argue strongly for a localized redox orbital description. While the ESR and near-IR (near infrared) absorption spectra also argue for localized redox orbitals in the bpz complexes, the simple (bpz)/sub 3-n//(bpz/sup -/)/sub n/ ..pi.. ..-->.. ..pi..* chromophoric behavior typical in the UV absorption spectrum is not observed. Furthermore, there is a gradual shifting of the RR peaks as electrons are added, with no distinct vibrational pattern from reduced and unreduced bipyrazine ligands for the one- and two-electron-reduction products as seen in the complexes of (Ru(bpy)/sub 3/)/sup 2-n/ and (Ru(bpm)/sub 3/)/sup 2-n/.

Low-temperature electron paramagnetic resonance study of the ferricytochrome c-cardiolipin complex.

The electron paramagnetic resonance spectrum of the ferricytochrome c complex with cardiolipin was observed at temperatures below 20 K. For the low-spin iron(III) heme system complexed with the negatively charged lipid, the tetragonal and rhombic ligand field parameters (delta/lambda = 3.58, V/lambda = 1.82) differ significantly from those (delta/lambda = 2.53, V/lambda = 1.49) of the free ferricytochrome c sample. The g values of the complex (gx = 1.54 +/- 0.02, gy = 2.26 +/- 0.01, gz = 3.02 +/- 0.01) are compared to the values for free ferricytochrome c (gx = 1.25 +/- 0.02, gy = 2.25 +/- 0.01, gz = 3.04 +/- 0.01). Spectral alterations are interpreted in terms of the ligand field changes induced within the heme group by association with the negatively charged phosphoglyceride.