Preliminary analysis of the integrated EPR signals of fingernails to validate the dosimetry method based on peak-to-peak amplitudes

NADH:quinone oxidoreductase (complex I) plays a central role in cellular energy metabolism, and its dysfunction is found in numerous human mitochondrial diseases. Although the understanding of its structure and function has been limited, the x-ray crystal structure of the hydrophilic part of Thermus thermophilus complex I recently became available. It revealed the localization of all redox centers, including 9 iron-sulfur clusters and their coordinating ligands, and confirmed the predictions mostly made by Ohnishi et al. (Ohnishi, T., and Nakamaru-Ogiso, E. (2008) Biochim. Biophys. Acta 1777, 703-710) based on various EPR studies. Recently, Yakovlev et al. (Yakovlev, G., Reda, T., and Hirst, J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12720-12725) claimed that the EPR signals from clusters N4, N5, and N6b were misassigned. Here we identified and characterized cluster N5 in the Escherichia coli complex I whose EPR signals had never been detected by any group. Using homologous recombination, we constructed mutant strains of H101A, H101C, H101A/C114A, and cluster N5 knock-out. Although mutant NuoEFG subcomplexes were dissociated from complex I, we successfully recovered these mutant NuoCDEFG subcomplexes by expressing the His-tagged NuoCD subunit, which had a high affinity to NuoG. The W221A mutant was used as a control subcomplex carrying wild-type clusters. By lowering temperatures to around 3 K, we finally succeeded in detecting cluster N5 signals in the control for the first time. However, no cluster N5 signals were found in any of the N5 mutants, whereas EPR signals from all other clusters were detected. These data confirmed that, contrary to the misassignment claim, cluster N5 has a unique coordination with His(Cys)(3) ligands in NuoG.

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

Unusual spin interactions in the 24 heme hydroxylamine oxidoreductase and diheme cytochrome c 554 from nitrosomonas

Gamma-irradiated dry fruits: An example of a wide variety of long-time dependent EPR spectra

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.

Spin trapping of .NO 2 radicals produced by uv photolysis of RDX, HMX, and nitroguanidine

The nitrogenase MoFe protein contains the active site metallocluster called FeMo-cofactor [7Fe-9S-Mo-homocitrate] that exhibits an S = 3/2 EPR signal in the resting state. No interaction with FeMo-cofactor is detected when either substrates or inhibitors are incubated with MoFe protein in the resting state. Rather, the detection of such interactions requires the incubation of the MoFe protein together with its obligate electron donor, called the Fe protein, and MgATP under turnover conditions. This indicates that a more reduced state of the MoFe protein is required to accommodate substrate or inhibitor interaction. In the present work, substitution of an arginine residue (alpha-96(Arg)) located next to the active site FeMo-cofactor in the MoFe protein by leucine, glutamine, alanine, or histidine is found to result in MoFe proteins that can interact with acetylene or cyanide in the as-isolated, resting state without the need for the Fe protein, or MgATP. The dithionite-reduced, resting states of the alpha-96(Leu)-, alpha-96(Gln)-, alpha-96(Ala)-, or alpha-96(His)-substituted MoFe proteins show an S = 3/2 EPR signal (g = 4.26, 3.67, 2.00) similar to that assigned to FeMo-cofactor in the wild-type MoFe protein. However, in contrast to the wild-type MoFe protein, the alpha-96-substituted MoFe proteins all exhibit changes in their EPR spectra upon incubation with acetylene or cyanide. The alpha-96(Leu)-substituted MoFe protein was representative of the other alpha-96-substituted MoFe proteins examined. The incubation of acetylene with the alpha-96(Leu) MoFe protein decreased the intensity of the normal FeMo-cofactor signal with the appearance of a new EPR signal having inflections at g = 4.50 and 3.50. Incubation of cyanide with the alpha-96(Leu) MoFe protein also decreased the FeMo-cofactor EPR signal with concomitant appearance of a new EPR signal having an inflection at g = 4.06. The acetylene- and cyanide-dependent EPR signals observed for the alpha-96(Leu)-substituted MoFe protein were found to follow Curie law 1/T dependence, consistent with a ground-state transition as observed for FeMo-cofactor. The microwave power dependence of the EPR signal intensity is shifted to higher power for the acetylene- and cyanide-dependent signals, consistent with a change in the relaxation properties of the spin system of FeMo-cofactor. Finally, the alpha-96(Leu)-substituted MoFe protein incubated with (13)C-labeled cyanide displays a (13)C ENDOR signal with an isotropic hyperfine coupling of 0.42 MHz in Q-band Mims pulsed ENDOR spectra. This indicates the existence of some spin density on the cyanide, and thus suggests that the new component of the cyanide-dependent EPR signals arise from the direct bonding of cyanide to the FeMo-cofactor. These data indicate that both acetylene and cyanide are able to interact with FeMo-cofactor contained within the alpha-96-substituted MoFe proteins in the resting state. These results support a model where effective interaction of substrates or inhibitors with FeMo-cofactor occurs as a consequence of both increased reactivity and accessibility of FeMo-cofactor under turnover conditions. We suggest that, for the wild-type MoFe protein, the alpha-96(Arg) side chain acts as a gatekeeper, moving during turnover in order to permit accessibility of acetylene or cyanide to a specific [4Fe-4S] face of FeMo-cofactor.

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.

Objective To explore the feasibility of EPR in vivo fingernail dosimetry to address the difficulty in separating mechanically induced signals from fingernail EPR dosimetry for need of nuclear medical emergency aid. Methods Using the specially designed EPR in vivo measurement system, uncut fingernails were measured to obtain the characteristics of EPR signal without mechanically induced signals. The in vivo fingernail experiment was carried out to evaluate the impact of in vivo condition on the spectra. Actual in vivo measurement experiment was conducted to evaluate the interference of the in vivo condition on EPR spectra. Results The background signal distribution of uncut fingernails was obtained and background signals had no significant difference between male and female(P>0.05). The dose response curve in the range of 2-10 Gy was established, and the half-life of the fingernail radiation-induced signal was approximately 5 d. The water treatment combined with temperature-changing was established for restoring the background signal. EPR signal obtained after restoring treatment has no significant difference with background signal(P>0.05). The EPR spectra of in vivo fingernails were obtained. Conclusions The EPR spectra without mechanically induced signals can be acquired by this method. The feasibility of the in vivo fingernail EPR dosimetry is preliminarily verified. Key words: Electron paramagnetic resonance; Fingernail; In vivo; Accident; Dosimetry

The investigated Ni doped forsterite was grown with the floating zone technique. The EPR spectra were taken at room temperature using both 9.5 and 35 GHz. All specimens show EPR signals resulting from Mn2+ at M2 and Fe3+ at M1, M2, and Si positions. Ni2+ EPR signals are observed at 35 GHz but not at 9.5 GHz. The Ni2+ spectra are described by the spin Hamiltonian $$H = \beta SgB + D\left( {S_{\text{z}}^{\text{2}} - \left( {S + 1} \right)S/3} \right) + E\left( {S_{\text{x}}^{\text{2}} - S_{\text{y}}^{\text{2}} } \right)$$ with S=1. The EPR parameters obtained are: D=−39.56 cm−1, E=−0.58 cm−1, gx=2.194, gy=2.160, gz=2.188. Polarized optical absorption spectra of Ni2+ were taken at room and liquid nitrogen temperature covering the range from 5500 to 42000 cm−1. The spectra are nearly independent of the polarization of light. Both EPR and optical spectra reveal that Ni2+ is ordered into M1. The local symmetry of the Ni(M1) complex is described by the point group D2h with the main symmetry axis along a. Heating experiments in air up to 1000° C and three days indicated no change in the Ni ordering in this synthetic sample.

Effect of solute-co-ordinating solvent interactions and temperature on the EPR and electronic spectra of bis(dithiophosphato)copper(II)

Due to the symmetry of its heterodimeric core, which is composed of the related PsaA and PsaB membrane polypeptides, photosystem I possesses two cofactor branches that traverse the membrane. There are two phylloquinones in PS I, at least one of which serves as a secondary electron acceptor called A1. Using a combination of site-directed mutagenesis and EPR spectroscopy, we have probed the phylloquinone sites and identified mutants that affect the properties of the A1 cofactor. The PsaA-W693F mutant causes loss of both the photo-accumulated A1- EPR signal and an alteration in the time-resolved P700+ A1- EPR spectrum. Mutation of the corresponding tryptophan in PsaB had no such effect. However, flash absorption spectroscopy of isolated PS I particles revealed that mutation of either tryptophan resulted in an acceleration of the back-reaction from the terminal iron-sulfur clusters. Examination of wild-type Chlamydomonas PS I by in vivo flash spectroscopy demonstrated that reoxidation of A1, as measured by monitoring decay of an absorption increase at 380-400 nm, occurs with biphasic kinetics (half-times of about 13 and 140 ns). The PsaA-W693F mutation specifically affected both the rate of the slower kinetic component (slowed 3-4 fold) and its decay-associated spectrum, while the PsaB-W673F mutation had similar effects on the faster phase. Thus it seems that, although the two phylloquinones of PS I are functionally different, both of them may be used as intermediates in forward and reverse electron transfer through PS I.

Reduction of Quinones by NADH Catalyzed by Organoiridium Complexes

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g approximately 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and approximately 2.0 (component I) and at 2.33, 2.24, and approximately 2.0 (component II). In the reduced state, after exposure to an H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers: center I (2.03, 1.89 and 1.86) detectable below 10 K, and center II (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 8942]. During the course of a redox titration at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The oxidation-reduction and spectroscopic properties of various forms of nitrous oxide reductase from Pseudomonas stutzeri were investigated. The high-activity form I of the enzyme (purple, 8 Cu, Mr 140,000) was reduced by a large variety of cationic, anionic and photochemically generated agents. The blue form III was the only product found in these experiments under anaerobic conditions. Reductive (dithionite) and oxidative (ferricyanide) titrations showed that the conversion of the purple form I to the blue species III was fully reversible in the absence of dioxygen. Two kinetically different phases of the reaction of form I with a stoichiometric amount of dithionite (1e- -equivalent/Cu) were detected: in the fast phase (seconds), the purple chromophore with lamba max at 540 nm disappeared almost completely, whereas in the slower phase (minutes) the blue species with lambda max around 650 nm was generated. Irrespective of the nature of the reductant the blue species did not react even at large excess of reductant. It was reoxidized by ferricyanide, hydrogen peroxide and nitric oxide. A new, catalytically inactive derivative of nitrous oxide reductase (form V, 2 Cu, Mr 140,000) was isolated from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The pink color of the mutant protein faded almost completely after addition of 0.5e- -equivalent/Cu. In this case no blue species was found, similar to earlier observations for the regenerated, catalytically inactive protein. Varying with the sample and the pH, 50-80% of the total copper of form I was in an electron-paramagnetic-resonance-(EPR)-silent state as compared to 47% in the mutant protein. The broad, featureless EPR signal recorded at 9.32 GHz for the blue, reduced form III of nitrous oxide reductase represented approximately 20% of the total copper. For the blue species no resolution enhancement was achieved at 34 GHz. At this frequency both forms I and V showed similar EPR signals with apparent g-values at 2.16 and 1.99. At 9.32 GHz, form V had an EPR signal with gII at 2.18, AII = 3.55 mT (4 or 5 lines, in contrast to form I) and gI at 2.03. Above 100 K the splitting of the gII region into seven equidistant lines in the EPR signal of the high-activity form I and the hyperfine structure of the perpendicular transition disappeared. Carbon monoxide and nitric oxide, but not nitrous oxide, had marked effects on the spectroscopic properties of the purple form I. Marked effects were also obtained for the exogenous ligands nitrite, azide, cyanate and thiocyanate.(ABSTRACT TRUNCATED AT 400 WORDS)