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X-band rapid-scan EPR was implemented on a commercially available Bruker ELEXSYS E580 spectrometer. Room temperature rapid-scan and continuous-wave EPR spectra were recorded for amorphous silicon powder samples. By comparing the resulting signal intensities the feasibility of performing quantitative rapid-scan EPR is demonstrated. For different hydrogenated amorphous silicon samples, rapid-scan EPR results in signal-to-noise improvements by factors between 10 and 50. Rapid-scan EPR is thus capable of improving the detection limit of quantitative EPR by at least one order of magnitude. In addition, we provide a recipe for setting up and calibrating a conventional pulsed and continuous-wave EPR spectrometer for rapid-scan EPR.

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Electron spin relaxation times of a Nycomed triarylmethyl radical (sym-trityl) in water, 1:1 water:glycerol, and 1:9 water:glycerol were measured at L-band, S-band, and X-band by pulsed EPR methods. In H(2)O solution, T(1) is 17+/-1 micros at X-band at ambient temperature, is nearly independent of microwave frequency, and exhibits little dependence on viscosity. The temperature dependence of T(1) in 1:1 water:glycerol is characteristic of domination by a Raman process between 20 and 80 K. The increased spin-lattice relaxation rates at higher temperatures, including room temperature, are attributed to a local vibrational mode that modulates spin-orbit coupling. In H(2)O solution, T(2) is 11+/-1 micros at X-band, increasing to 13+/-1 micros at L-band. For more viscous solvent mixtures, T(2) is much shorter than T(1) and weakly frequency dependent, which indicates that incomplete motional averaging of hyperfine anisotropy makes a significant contribution to T(2). In water and 1:1 water:glycerol solutions continuous wave EPR linewidths are not relaxation determined, but become relaxation determined in the higher viscosity 1:9 water:glycerol solutions. The Lorentzian component of the 250-MHz linewidths as a function of viscosity is in good agreement with T(2)-determined contributions to the linewidths at higher frequencies.

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Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.

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Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.

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Cigarette smoking causes profound suppression of pulmonary T cell responses, which has been associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. Exposure of human T cells to cigarette tar or its major phenolic components, hydroquinone and catechol, causes an immediate cessation of DNA synthesis without cytotoxicity. However, little is known of the mechanisms by which this phenomenon occurs. In this report we demonstrate that hydroquinone and catechol inhibit lymphocyte proliferation by quenching the essential tyrosyl radical in the M2 subunit of ribonucleotide reductase.

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Saturation recovery (SR) electron paramagnetic resonance was used to determine the distance between iron and nitroxyl for spin-labeled metmyoglobin variants in low-spin and high-spin states of the Fe(III). The interspin distances were measured by analyzing the effect of the heme iron on the spin-lattice relaxation rates of the nitroxyl spin label using the modified Bloembergen equation for low-spin species, and an analogue of the Bloembergen equation for high-spin species. Insight simulations of the spin-labeled protein structures also were used to determine the interspin distances. The distances obtained by SR for high-spin and low-spin complexes with 15-20 A interspin distances, for low-spin CN(-) and high-spin formate adducts at distances up to about 30 A, and results from Insight calculations were in good agreement. For variants with 25-30 A interspin distances, the distances obtained by SR for the fluoride adducts were shorter than observed for the CN(-) or formate adducts or predicted by Insight simulations. Of the heme axial ligands examined (CN(-), imidazole, F(-), and formate), CN(-) is the best choice for determination of iron-nitroxyl distances in the range of 15-30 A.

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X-band (9.1 GHz) and S-band (3.4 GHz) electron paramagnetic resonance (EPR) spectra for particulate methane monooxygenase (pMMO) in whole cells from Methylococcus capsulatus (Bath) grown on (63)Cu and (15)N were obtained and compared with previously reported spectra for pMMO from Methylomicrobium album BG8. For both M. capsulatus (Bath) and M. album BG8, two nearly identical Cu(2+) EPR signals with resolved hyperfine coupling to four nitrogens are observed. The EPR parameters for pMMO from M. capsulatus (Bath) (g( parallel) = 2.244, A( parallel) = 185 G, and A(N) = 19 G for signal one; g( parallel) = 2.246, A( parallel) = 180 G, and A(N) = 19 G for signal two) and for pMMO from M. album BG8 (g( parallel) = 2.243, A( parallel) = 180 G, and A(N) = 18 G for signal one; g( parallel) = 2. 251, A( parallel) = 180 G, and A(N) = 18 G for signal two) are very similar and are characteristic of type 2 Cu(2+) in a square planar or square pyramidal geometry. In three-pulse electron spin echo envelope modulation (ESEEM) data for natural-abundance samples, nitrogen quadrupolar frequencies due to the distant nitrogens of coordinated histidine imidazoles were observed. The intensities of the quadrupolar combination bands indicate that there are three or four coordinated imidazoles, which implies that most, if not all, of the coordinated nitrogens detected in the continuous wave spectra are from histidine imidazoles.

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The temperature dependence of spin-lattice relaxation rates was analyzed for four high-spin nonheme iron proteins between 5 and 20 K, for three high-spin iron porphyrins between 5 and 118 K, and for four high-spin heme proteins between 5 and 150 to 298 K. For the nonheme proteins the zero-field splittings, D, are less than 0.7 cm(-1), and the relaxation is dominated by the Orbach and Raman processes. For the iron porphyrins and heme proteins D is between 4 and 12 cm(-1) and the relaxation is dominated by the Orbach process between about 5 and 100 K and by a local mode at higher temperatures. The relaxation rates for the heme proteins in glassy matrices extrapolated to values at room temperature that are similar to values obtained by NMR relaxivity in fluid solution. This similarity suggests that for high-spin Fe(III) heme proteins with effective intramolecular spin-lattice relaxation processes, the additional motional freedom gained when a relatively large protein goes from glassy solid to liquid solution at room temperature has little impact on spin-lattice relaxation.

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The syntheses and the solid state structural and spectroscopic solution characterizations of VO(Me-acac)2 and VO(Et-acac)2 (where Me-acac is 3-methyl-2,4-pentanedionato and Et-acac is 3-ethyl-2,4-pentanedionato) have been conducted since both VO(acac)2 and VO(Et-acac)2 have long-term in vivo insulin-mimetic effects in streptozotocin-induced diabetic Wistar rats. X-ray structural characterizations of VO(Me-acac)2 and VO(Et-acac)2 show that both contain five-coordinate vanadium similar to the parent VO(acac)2. The unit cells for VO(Et-acac)2 and VO(Me-acac)2 are both triclinic, P1, with a = 9.29970(10) A, b = 13.6117(2) A, c = 13.6642(2) A, alpha = 94.1770(10) degrees, beta = 106.4770(10) degrees, gamma = 106.6350(10) degrees for VO(Et-acac)2 and a = 7.72969(4) A, b = 8.1856(5) A, c = 11.9029(6) A, alpha = 79.927(2) degrees, beta = 73.988(2)degrees, gamma = 65.1790(10)degrees for VO(Me-acac)2. The total concentration of EPR-observable vanadium(IV) species for VO(acac)2 and derivatives in water solution at 20 degreesC was determined by double integration of the EPR spectra and apportioned between individual species on the basis of computer simulations of the spectra. Three species were observed, and the concentrations were found to be time, pH, temperature, and salt dependent. The three complexes are assigned as the trans-VO(acac)2.H2O adduct, cis-VO(acac)2.H2O adduct, and a hydrolysis product containing one vanadium atom and one R-acac- group. The reaction rate for conversion of species was slower for VO(acac)2 than for VO(malto)2, VO(Et-acac)2, and VO(Me-acac)2; however, in aqueous solution the rates for all of these species are slow compared to those of other vanadium species. The concentration of vanadium(V) species was determined by 51V NMR. The visible spectra were time dependent, consistent with the changes in species concentrations that were observed in the EPR and NMR spectra. EPR and visible spectroscopic studies of solutions prepared as for administration to diabetic rats documented both a salt effect on speciation and formation of a new halogen-containing complex. Compound efficacy with respect to long-term lowering of plasma glucose levels in diabetic rats traces the concentration of the hydrolysis product in the administration solution.

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Inhibitory treatment by acetate, followed by illumination and rapid freezing, is known to trap the S(2)Y(Z)(*) state of the O(2)-evolving complex (OEC) in photosystem II (PS II). An EPR spectrum of this state exhibits broad split signals due to the interaction of the tyrosyl radical, Y(Z)(*), with the S = 1/2 S(2) state of the Mn(4) cluster. We present a novel approach to analyze S(2)Y(Z)(*) spectra of one-dimensionally (1-D) oriented acetate-inhibited PS II membranes to determine the magnitude and relative orientation of the S(2)Y(Z)(*) dipolar vector within the membrane. Although there exists a vast body of EPR data on isolated spins in oriented membrane sheets, the present study is the first of its kind on dipolar-coupled electron spin pairs in such systems. We demonstrate the feasibility of the technique and establish a rigorous treatment to account for the disorder present in partially oriented 1-D membrane preparations. We find that (i) the point-dipole distance between Y(Z)(*) and the Mn(4) cluster is 7.9 +/- 0.2 A, (ii) the angle between the interspin vector and the thylakoid membrane normal is 75 degrees, (iii) the g(z)()-axis of the Mn(4) cluster is 70 degrees away from the membrane normal and 35 degrees away from the interspin vector, and (iv) the exchange interaction between the two spins is -275 x 10(-)(4) cm(-)(1), which is antiferromagnetic. Due to the sensitivity of EPR line shapes of oriented spin-coupled pairs to the interspin distance, the present study imposes a tighter constraint on the Y(Z)-Mn(4) point-dipole distance than obtained from randomly oriented samples. The geometric constraints obtained from the 1-D oriented sample are combined with published models of the structure of Mn-depleted PS II to propose a location of the Mn(4) cluster. A structure in which Y(Z) is hydrogen bonded to a manganese-bound hydroxide ligand is consistent with available data and favors maximal orbital overlap between the two redox center that would facilitate direct electron- and proton-transfer steps.

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EPR signal and noise, calculated from first principles, are compared with measured values of signal and noise on an S-band (ca. 2.7 GHz) EPR spectrometer for which all relevant gains and losses have been measured. Agreement is within the uncertainty of the calculations and the measurements. The calculational model that provided the good agreement is used to suggest approaches to optimizing spectrometer design.

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Direct measurements of electron spin-echo signal and noise in well-characterized X-band and S-band spectrometers agree with predictions of frequency dependence based on first principles. For the particular spectrometers compared, the echo at 9.52 GHz was 9.5 times larger than the echo at 2.68 GHz, after scaling for differences in spectrometer gain. The calculated ratio was 7.6. This result contrasts with prior predictions that the frequency dependence would be much greater.

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The temperature dependence of X-band electron spin-lattice relaxation between about 10 and 300 K in magnetically dilute solids and up to the softening temperature in glassy solvents was analyzed for three organic radicals and 14 S = 12 transition metal complexes. Contributions from the direct, Raman, local vibrational mode, thermally activated, and Orbach processes were considered. For most samples it was necessary to include more than one process to fit the experimental data. Debye temperatures were between 50 and 135 K. For small molecules the Debye temperature required to fit the relaxation data was higher in 1:1 water:glycerol than in organic solvents. For larger molecules the Debye temperature was less dependent upon solvent and more dependent upon the characteristics of the molecule. The coefficients of the Raman process increased with increasing g anisotropy and decreasing rigidity of the molecule. For the transition metal complexes the data are consistent with major contributions from local modes with energies in the range of 185 to 350 K (130 to 240 cm-1). The coefficient for this contribution increases in the order 3d < 4d transition metal. For C-60 anions there is a major contribution from a thermally activated process with an activation energy of about 240 cm-1. For low-spin hemes the dominant contribution at higher temperatures is from a local mode or thermally activated process with a characteristic energy of about 175 cm-1.

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The solvent and temperature dependence of the rate constant for spin echo dephasing, 1/Tm, for 0.2 to 1.2 mM glassy solutions of chromyl bis(1-hydroxy-cyclohexanecarboxylic acid), CrO(HCA)-2; aquo vanadyl ion, VO2+ (aq), and vanadyl bis(trifluoroacetylacetonate), VO(tfac)2 were examined. At low temperatures where 1/T1 << 1/Tm, 1/Tm in 1:1 H2O:glycerol is dominated by solvent protons. At low temperature 1/Tm increases in the order 1:1 H2O:glycerol or 9:1 CF3CH2OH:ethyleneglycol (no methyl groups) < 9:1 i-PrOH:MeOH (hindered methyl groups) < 9:1 n-PrOH:MeOH (less hindered methyl groups). This solvent dependence of 1/Tm is similar to that observed for nitroxyl radicals, which indicates that the effect of solvent methyl groups on spin-echo dephasing at low temperature is quite general. At higher temperatures the echo dephasing is dominated by spin-lattice relaxation and is concentration dependent. As the glass softens, echo dephasing is dominated by the onset of molecular tumbling.

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A mutant of the ferric enterobactin receptor, FepA, containing a valine to cysteine (V338C) substitution was made and the purified protein selectively modified with a sulfhydryl-specific nitroxide spin label. In reconstituted liposomes, interaction of the attached spin label with a combination of water-soluble and lipid-soluble relaxation agents indicated that the V338C site was located in the polar headgroup region of the membrane, approximately 1.5-4.5 A above the phosphate groups of the lipids. Binding of the ligand, ferric enterobactin (FeEnt), to the purified spin-labeled protein produced a significant decrease in both the rotational freedom and accessibility of the nitroxide, indicating the formation of new structural contacts between the spin label and either the protein or the bound ligand. Electron spin-echo (ESE) measurements of the nitroxide phase-memory relaxation rate in the presence and absence of bound ligand showed substantial dipolar coupling between the Fe3+ of FeEnt and the spin label and provided an iron-nitroxide distance estimate in the range of 20-30 A. We conclude that the ligand-induced changes in spin label motion and accessibility are due to new tertiary contacts with the protein and not to direct contact with the ligand. These studies suggest that V338C may occupy a hinge region connecting the ligand binding surface loop to the beta-barrel and provide the strongest evidence to date of an in vitro ligand-induced conformational change in FepA.

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Continuous wave EPR spectra of the nitroxyl signals for four spin-labeled high-spin (h.s.) Fe(III) porphyrins showed partially resolved splittings at temperatures near 4 K. Axial ligands were fluoride, chloride, or bromide. As temperature was increased to 20 to 30 K the iron-nitroxyl splitting collapsed due to increasing rates of iron relaxation. Electron spin-echo (ESE) spectroscopy showed that above about 6 K collapse of the iron-nitroxyl spin-spin splitting caused a dramatic increase in the nitroxyl phase memory relaxation rates. Electron spin relaxation rates were determined for Fe(tetratolylporphyrin)X, X = F, Cl, Br, in toluene solution by ESE or inversion recovery at 4.5 to 6 K and by analysis of the temperature-dependent contributions to the continuous wave EPR linewidths between about 10 and 120 K. Above about 10 K iron relaxation rates increase in the order X = F < Cl < Br, which is the order of increasing zero-field splitting. Saturation recovery data for two spin-labeled h.s. iron(III) porphyrins between about 15 and 120 K and for two additional spin-labeled h.s. iron(III) porphyrins between about 85 and 120 K demonstrated that interaction with the h. s. iron enhanced the electron spin relaxation rate of the spin label. The saturation recovery curves for the nitroxyl were analyzed to determine interspin distances using a modified version of the Bloembergen equation and independently determined iron relaxation rates. Interspin distances were between 11.6 and 15.0 A, were independent of axial ligand, and were in good agreement with values obtained previously for low-spin Fe(III) and Cu(II) analogs.

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To optimize simulations of CW EPR spectra for high-spin Fe(III) with zero-field splitting comparable to the EPR quantum, information is needed on the factors that contribute to the line shapes and line widths. Continuous wave electron paramagnetic resonance (EPR) spectra obtained for iron transferrin carbonate from 4 to 150 K and for iron transferrin oxalate from 4 to 100 K did not exhibit significant temperature dependence of the line shape, which suggested that the line shapes were not relaxation determined. To obtain direct information concerning the electron spin relaxation rates, electron spin echo and inversion recovery EPR were used to measure T(1) and T(m) for the high-spin Fe(III) in iron transferrin carbonate and iron transferrin oxalate between 5 and 20-30 K. For comparison with the data for the transferrin complexes, relaxation times were obtained for tris(oxalato)ferrate(III). The relaxation rates are similar for the three complexes and do not exhibit a strong dependence on position in the spectrum. Extrapolation of the observed temperature dependence of the relaxation rates to higher temperatures gives values consistent with the conclusion that the CW line shapes are not relaxation determined up to 150 K.

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The radical generated by gamma-irradiation of crystalline L-alanine was examined by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) at 1.8, 3.2, 4.9, 9.1 and 19.4 GHz. The spin-flip satellite lines that make a prominent contribution to the saturated spectra at 9.1 GHz are less conspicuous at lower frequencies because of overlap with the allowed transitions. The spin-lattice relaxation times measured by long-pulse saturation recovery and phase memory times measured by electron spin echo increase with increasing microwave frequency.

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The production of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/.OH in aqueous solutions of menadione and DMPO is enhanced by fluorescent room light. The formation of DMPO/.OH requires oxygen and water, is enhanced by superoxide dismutase, and occurs to a much smaller extent for benzoquinone than for menadione. This process is assigned as photo-initiated redox cycling of the menadione, which causes oxidation of DMPO to DMPO+. and reduction of oxygen to superoxide. DMPO+. reacts with water to produce DMPO/.OH. Although DMPO/.OOH was not observed in the menadione solutions, the possibility that some of the DMPO/.OH was produced by decomposition of DMPO/.OOH cannot be ruled out. There is no evidence for participation of hydroxyl radicals. Because benzosemiquinone is less readily oxidized than the semiquinone of menadione, redox cycling is less favorable for benzoquinone than for menadione and smaller quantities of DMPO/.OH are produced by photoexcitation of benzoquinone than of menadione.

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Asp537 and Asp812 are essential in the catalytic mechanism of T7 RNA polymerase. The mutants D537N and D812N have no detectable activity whereas the mutants D537E and D812E have significantly reduced activity relative to the wild-type. The hypothesis that these two amino acids act as metal-binding ligands has been tested using EPR with Mn2+ as the metal ion. Mn2+ is able to substitute for Mg2+ in transcription by T7 RNAP on templates containing the T7 promoter. Mg2+ and Mn2+ compete for binding sites, with the former having lower affinity. Mn2+ binding to the wild-type enzyme and the mutants D537N, D812N, D537E, D812E, and Y649F was measured over the concentration range of 25 microM to 1.5 mM. The data were analyzed by nonlinear least-squares fits to the binding isotherms, and the analysis gave approximately two Mn(2+)-binding sites in all cases and a Kd for the wild-type of approximately 340 microM. The Kd value for the mutant Y639F, in which Asp537 and Asp 812 are not mutated, is comparable to the value for the wild-type. Mn2+ binding to the double mutants, D537N/D812N and D537E/D812E, appears to be nonspecific. The Kd values of the Asp-->Asn mutants are only 2-5 times larger than the value for the wild-type, in contrast to the drastic diminution of enzymatic activity in the mutants. The geometry of metal binding to these Asp residues may be crucial in determining the catalytic competence. Mn2+ binding to the wild-type enzyme in the presence of nucleotides, measured by flow dialysis, is characterized by two Mn(2+)-binding sites with a Kd value of ca. 150 microM. The similarity in values of Kd with and without nucleotide suggests that nucleotides do not have a drastic effect on Mn2+ binding. Our results indicate that monodentate carboxylate oxygens of both conserved Asp residues bridge the two metal ions.

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