Improved understanding of longevity represents a significant welfare opportunity for the domestic dog, given its unparalleled morphological diversity. Epidemiological research using electronic patient records (EPRs) collected from primary veterinary practices overcomes many inherent limitations of referral clinic, owner questionnaire and pet insurance data. Clinical health data from 102,609 owned dogs attending first opinion veterinary practices (n=86) in central and southeast England were analysed, focusing on 5095 confirmed deaths. Of deceased dogs with information available, 3961 (77.9%) were purebred, 2386 (47.0%) were female, 2528 (49.8%) were neutered and 1105 (21.7%) were insured. The overall median longevity was 12.0 years (IQR 8.9-14.2). The longest-lived breeds were the Miniature poodle, Bearded collie, Border collie and Miniature dachshund, while the shortest-lived were the Dogue de Bordeaux and Great Dane. The most frequently attributed causes of death were neoplastic, musculoskeletal and neurological disorders. The results of multivariable modelling indicated that longevity in crossbred dogs exceeded purebred dogs by 1.2 years (95% confidence interval 0.9-1.4; P<0.001) and that increasing bodyweight was negatively correlated with longevity. The current findings highlight major breed differences for longevity and support the concept of hybrid vigour in dogs.

Studies of event-related brain potentials (ERPs) have shown that attributes of the ERP can be used as dependent variables in the study of human information processing. These variables can complement the information gained from the study of overt, skeletal responses. The manner in which the P300 component of the EPR can be used to study human information processing is illustrated in this report. Specifically, we show that through an analysis of the covariation of the latency of P300 component and reaction time, it is possible to examine the relation between the probability of a stimulus and the speed of response to that stimulus. Our data indicate that increased in the probability of a stimulus reduce reaction time by decreasing both stimulus-evaluation and response-production times. We also examine changes in reaction time and P300 latency induced by the match or mismatch between two stimuli presented consecutively, again as a function of probability. Models of the effects of stimulus matching on reaction time are evaluated.

MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.

Therapeutic nanoparticles (TNPs) have shown heterogeneous responses in human clinical trials, raising questions of whether imaging should be used to identify patients with a higher likelihood of NP accumulation and thus therapeutic response. Despite extensive debate about the enhanced permeability and retention (EPR) effect in tumors, it is increasingly clear that EPR is extremely variable; yet, little experimental data exist to predict the clinical utility of EPR and its influence on TNP efficacy. We hypothesized that a 30-nm magnetic NP (MNP) in clinical use could predict colocalization of TNPs by magnetic resonance imaging (MRI). To this end, we performed single-cell resolution imaging of fluorescently labeled MNPs and TNPs and studied their intratumoral distribution in mice. MNPs circulated in the tumor microvasculature and demonstrated sustained uptake into cells of the tumor microenvironment within minutes. MNPs could predictably demonstrate areas of colocalization for a model TNP, poly(d,l-lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG), within the tumor microenvironment with >85% accuracy and circulating within the microvasculature with >95% accuracy, despite their markedly different sizes and compositions. Computational analysis of NP transport enabled predictive modeling of TNP distribution based on imaging data and identified key parameters governing intratumoral NP accumulation and macrophage uptake. Finally, MRI accurately predicted initial treatment response and drug accumulation in a preclinical efficacy study using a paclitaxel-encapsulated NP in tumor-bearing mice. These approaches yield valuable insight into the in vivo kinetics of NP distribution and suggest that clinically relevant imaging modalities and agents can be used to select patients with high EPR for treatment with TNPs.

The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.

We have used chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) to probe the functional dynamics of phospholamban (PLB), which regulates the Ca-ATPase (SERCA) in cardiac sarcoplasmic reticulum. The transmembrane domain of PLB inhibits SERCA at low [Ca(2+)], but the cytoplasmic domain relieves this inhibition upon Ser16 phosphorylation. Monomeric PLB was synthesized with Ala11 replaced by the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label, which reports peptide backbone dynamics directly. PLB was reconstituted into membranes in the presence or absence of SERCA. TOAC-PLB showed normal inhibitory function, which was reversed by phosphorylation at Ser16 or by micromolar [Ca(2+)]. EPR showed that the PLB cytoplasmic domain exhibits two resolved conformations, a tense T state that is ordered and a relaxed R state that is dynamically disordered and extended. PLB phosphorylation shifts this equilibrium toward the R state and makes it more dynamic (hyperextended). Phosphorylation strongly perturbs the dynamics of SERCA-bound PLB without dissociating the complex, while micromolar [Ca(2+)] has no effect on PLB dynamics. A lipid anchor synthetically attached to the N terminus of PLB permits Ca-dependent SERCA inhibition but prevents the phosphorylation-induced disordering and reversal of inhibition. We conclude that the relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA. This mechanism is distinct from the one that relieves PLB-dependent SERCA inhibition upon the addition of micromolar [Ca(2+)].

Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 kDa and was composed of two presumably identical subunits. The isoelectric point was 5.1. Several spectroscopically distinct forms of the enzyme were identified. A 'pink' form of the enzyme was obtained when the purification was done aerobically. The specific activity of this species was around 30 nkat/mg protein as measured by the nitrous-oxide-dependent oxidation of photochemically reduced benzyl viologen. A 'purple' form of the enzyme, whose catalytic activity was 2-5-fold higher, was obtained when the purification was done anaerobically. The activity of both forms of the enzyme was substantially increased by dialyzing the protein against 2-(N-cyclohexylamino)ethanesulfonate buffer at pH approximately equal to 10. A maximal activity of 1000 nkat/mg protein has been obtained for the purple form using this procedure. A 'blue', enzymatically inactive form of the enzyme resulted when either the pink or the purple species was exposed to excess dithionite or ascorbate. Anaerobic, potentiometric titrations of both the purple and the pink form of the enzyme gave a Nernst factor, n540, of 0.95 and a midpoint potential, E'0,540 of +260 mV (vs SHE, 25 degrees C, Tris/HCl buffer, pH 7.5). Electron paramagnetic resonance (EPR) and optical spectra of N2O reductase suggested the presence of an unusual type 1 copper center. Type 2 copper was absent. The hyperfine splitting in the g parallel region consisted of a seven-line pattern. In the presence of excess of reductant, a broad EPR signal with g values at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple forms of the enzyme were similar; however, the spectrum of the purple form was better resolved with g parallel = 2.18 (A parallel = 3.83 mT) and g perpendicular = 2.03 (A perpendicular = 2.8 mT). Most of the copper in N2O reductase was removed by anaerobic dialysis against KCN. Reaction of the apoprotein with Cu(en)2SO4 partially regenerated the optical and EPR spectra of the holoprotein; the resulting protein was enzymatically inactive. Monospecific antibodies against the copper protein strongly inhibited the N2O reductase activity of purified samples and cell-free extracts.

The biodistribution of soluble macromolecules is governed extensively by their ability to penetrate endothelial layers. Many solid tumors possess vasculature that is hyperpermeable to macromolecules, not always correlating with the presence of interendothelial cell fenestrations. The exact physiological mechanisms responsible for this nonspecific leakiness are not yet fully understood. Together with enhanced vascular permeability, however, tumors usually lack effective lymphatic drainage; consequently, they selectively accumulate circulating macromolecules (up to 10% of an i.v. dose per gram in mice). This "enhanced permeability and retention effect" (EPR effect) has been studied extensively, and it is thought to constitute the mechanism of action of SMANCS (styrene-maleic/anhydride-neocarzinostatin), now in regular clinical use in Japan for the treatment of hepatoma. It seems likely that EPR also contributes to the anticancer activity of the N-(2-hydroxypropyl)methacrylamide copolymer-anthracycline conjugates which are shortly to undergo clinical evaluation in the U.K.

We have found that environmentally persistent free radicals (PFRs) are formed by adsorption of substituted aromatic molecular precursors on the surface of cupric oxide-containing particles at temperatures between 100 and 400 degrees C. This temperature range corresponds to the conditions in the postflame, cool zone of combustion, and thermal processes. Depending upon the nature of the precursor and the adsorption temperature, both substituted phenoxyl and semiquinone radicals are formed. The PFRs are formed through a mechanism of initial physisorption, followed by chemisorption via elimination of water or hydrogen chloride, and electron transfer resulting in the simultaneous reduction of Cu(II) to Cu(I) and formation of the PFR. The PFRs are still observable by electron paramagnetic resonance (EPR) after exposure to air for more than a day. Their lifetimes under vacuum appear to be infinite. Other redox-active transition metals such as iron are expected to also mediate or catalyze the formation of PFRs. The properties of the observed radicals are consistent with radicals previously observed on airborne and combustion-generated particulate matter. We propose a catalytic biochemical cycle for both the particle-associated semiquinone and phenoxyl PFRs that result in the formation of hydroxyl radical and other reactive oxygen species (ROS). This suggests that combustion-generated, particle-associated PFRs may be responsible for the oxidative stress resulting in cardiopulmonary disease and probably cancer that has been attributed to exposure to airborne fine particles.

Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ₁₋₄₀, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ₁₋₄₀ fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.

Paclitaxel is one of the most effective chemotherapeutic drugs ever developed and is active against a broad range of cancers, such as lung, ovarian, and breast cancers. Due to its low water solubility, paclitaxel is formulated in a mixture of Cremophor EL and dehydrated ethanol (50:50, v/v) a combination known as Taxol. However, Taxol has some severe side effects related to Cremophor EL and ethanol. Therefore, there is an urgent need for the development of alternative Taxol formulations. The encapsulation of paclitaxel in biodegradable and non-toxic nano-delivery systems can protect the drug from degradation during circulation and in-turn protect the body from toxic side effects of the drug thereby lowering its toxicity, increasing its circulation half-life, exhibiting improved pharmacokinetic profiles, and demonstrating better patient compliance. Also, nanoparticle-based delivery systems can take advantage of the enhanced permeability and retention (EPR) effect for passive tumor targeting, therefore, they are promising carriers to improve the therapeutic index and decrease the side effects of paclitaxel. To date, paclitaxel albumin-bound nanoparticles (Abraxane®) have been approved by the FDA for the treatment of metastatic breast cancer and non-small cell lung cancer (NSCLC). In addition, there are a number of novel paclitaxel nanoparticle formulations in clinical trials. In this comprehensive review, several types of developed paclitaxel nano-delivery systems will be covered and discussed, such as polymeric nanoparticles, lipid-based formulations, polymer conjugates, inorganic nanoparticles, carbon nanotubes, nanocrystals, and cyclodextrin nanoparticles.

Crystal structures of the molecular motor kinesin show conformational variability in a structural element called the neck linker. Conformational change in the neck linker, initiated by ATP exchange, is thought to drive the movement of kinesin along the microtubule track. We use site-specific EPR measurements to show that when microtubules are absent, the neck linker exists in equilibrium between two structural states (disordered and 'docked'). The active site nucleotide does not control the position taken by the neck linker. However, we find that sulfate can specifically bind near the nucleotide site and stabilize the docked neck linker conformation, which we confirmed by solving a new crystal structure. Comparing the crystal structures of our construct with the docked or undocked neck linker reveals how microtubule binding may activate the nucleotide-sensing mechanism of kinesin, allowing neck linker transitions to power motility.

Several novel antioxidant-iron chelators bearing 8-hydroxyoxyquinoline moiety were synthesized, and various properties related to their iron chelation, and neuroprotective action were investigated. All the chelators exhibited strong iron(III) chelating and high antioxidant properties. Chelator 9 (HLA20), having good permeability into K562 cells and moderate selective MAO-B inhibitory activity (IC50 110 microM), displayed the hightest protective effects against differentiated P19 cell death induced by 6-hydroxydopamine. EPR studies suggested that Chelator 9 also act as radical scavenger to directly scavenge hydroxyl radical.

The nitrophorins are NO-carrying heme proteins that are found in the saliva of two species of blood-sucking insects, the kissing bug (Rhodnius prolixus) and the bedbug (Cimex lectularius). In both insects the NO is bound to the ferric form of the protein, which gives rise to Kds in the micromolar to nanomolar range, and thus upon injection of the saliva into the tissues of the victim the NO can dissociate to cause vasodilation and inhibition of platelet aggregation. The structures of the proteins from each of these insects are unique, and each has a large component of beta-sheet structure, which is unusual for heme proteins. While the Rhodnius nitrophorins increase the effectiveness of their NO-heme proteins by also binding histamine, secreted by the victim in response to the bite, to the heme, the Cimex nitrophorin does not bind histamine but rather binds two molecules of NO reversibly, one to the heme and the other to the cysteine thiolate which serves as the heme ligand in the absence of NO. This requires homolytic cleavage of the Fe-S-Cys bond, which produces an EPR-active Fe(II)-NO complex having the {FeNO}7 electron configuration. For the Rhodnius nitrophorins, the heme of the {FeNO}6 stable NO complex could have the limiting electron configurations Fe(III)-NO+ or Fe(II)-NO+. While vibrational spectroscopy suggests the latter and Mossbauer spectroscopy cannot differentiate between a purely diamagnetic Fe(II) center and a strongly antiferromagnetically coupled Fe(III)-NO* center, the strong ruffling of the heme (with alternate meso-carbons shifted significantly above and below the mean plane of the porphyrin, and concomitant shifts of the beta-pyrrole carbons above and below the mean plane of the porphyrin ring, to produce a very nonplanar porphyrin macrocycle) may suggest at least an important contribution of the latter. The strong ruffling would help to stabilize the (dxz, dyz)4(dxy)1 electron configuration of low-spin Fe(III) (but not low-spin Fe(II)), and the dxy orbital does not have correct symmetry for overlap with the half-filled pi* orbital of NO. This Fe(III)-NO* electron configuration would facilitate reversible dissociation of NO.

Tumor oxygenation predicts cancer therapy response and malignant phenotype. This has spawned a number of oxymetries. Comparison of different oxymetries is crucial for the validation and understanding of these techniques. Electron paramagnetic resonance (EPR) imaging is a novel technique for providing quantitative high-resolution images of tumor and tissue oxygenation. This work compares sequences of tumor pO2 values from EPR oxygen images with sequences of oxygen measurements made along a track with an Oxylite oxygen probe. Four-dimensional (three spatial and one spectral) EPR oxygen images used spectroscopic imaging techniques to measure the width of a spectral line in each image voxel from a trityl spin probe (OX063, Amersham Health R&D) in the tissues and tumor of mice after spin probe injection. A simple calibration allows direct, quantitative translation of each line width to an oxygen concentration. These four-dimensional EPR images, obtained in 45 minutes from FSa fibrosarcomas grown in the legs of C3H mice, have a spatial resolution of approximately 1 mm and oxygen resolution of approximately 3 Torr. The position of the Oxylite track was measured within a 2-mm accuracy using a custom stereotactic positioning device. A total of nine images that involve 17 tracks were obtained. Of these, most showed good correlation between the Oxylite measured pO2 and a track located in the tumor within the uncertainties of the Oxylite localizability. The correlation was good both in terms of spatial distribution pattern and pO2 magnitude. The strong correlation of the two modalities corroborates EPR imaging as a useful tool for the study of tumor oxygenation.

Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

The beta-Amyloid peptide (A beta) is hypothesized to mediate the neurodegeneration seen in Alzheimer's disease. Recently, we proposed a new hypothesis to explain the toxicity of A beta based on the free-radical generating capacity of A beta. We have recently demonstrated using electron paramagnetic resonance (EPR) spectroscopy that A beta (1-40) generates free radicals in solution. It was therefore suggested that A beta radicals can attack cell membranes, initiate lipoperoxidation, damage membrane proteins, and compromise ion homeostasis resulting in neurodegeneration. To evaluate this hypothesis, the ability of A beta to induce neuronal oxidation, changes in calcium levels, enzyme inactivation, and neuronal death were compared with the ability of A beta to produce free-radicals. Using hippocampal neurons in culture, several methods for detection of oxidation were utilized such as the conversion of 2,7-dichlorofluorescin to 2,7-dichlorofluorescein, and a new fluorescence microscopic method for the detection of carbonyls. The ability of A beta to produce free-radicals was determined using EPR with the spin-trapping compound N-tert-butyl-alpha-phenylnitrone. Consistent with previous studies, we found that preincubation of A beta increased the toxicity of the peptide. There is a strong correlation between the intensity of radical generation by A beta and neurotoxicity. The highest neuronal oxidation and toxicity was seen at a time when A beta was capable of generating the most intense radical signal. Furthermore, little oxidation and toxicity was seen when cultures were treated with freshly dissolved A beta, which did not generate a detectable radical signal. These data are consistent with the hypothesis that free-radical-based oxidative damage induced by A beta contributes to the neurodegeneration of Alzheimer's disease.

Ferric uptake regulation protein (Fur) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. The function of Fur is not limited to iron homeostasis. A wide variety of genes involved in various mechanisms such as oxidative and acid stresses are under Fur control. Flavohemoglobin (Hmp) is an NO-detoxifying enzyme induced by NO and nitrosothiol compounds. Fur recently was found to regulate hmp in Salmonella typhimurium, and in Escherichia coli, the iron-chelating agent 2,2'-dipyridyl induces hmp expression. We now establish direct inhibition of E. coli Fur activity by NO. By using chromosomal Fur-regulated lacZ reporter fusion in E. coli, Fur activity is switched off by NO at micromolar concentration. In vitro Fur DNA-binding activity, as measured by protection of restriction site in aerobactin promoter, is directly sensitive to NO. NO reacts with Fe(II) in purified FeFur protein to form a S = 12 low-spin FeFur-NO complex with a g = 2.03 EPR signal. Appearance of the same EPR signal in NO-treated cells links nitrosylation of the iron with Fur inhibition. The nitrosylated Fur protein is still a dimer and is stable in anaerobiosis but slowly decays in air. This inhibition probably arises from a conformational switch, leading to an inactive dimeric protein. These data establish a link between control of iron metabolism and the response to NO effects.

Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy has emerged as a well-established method that can provide specific information on the location and environment of an individual residue within large and complex protein structures. The SDSL technique involves introducing a cysteine residue at the site of interest and then covalently labeling with a sulfhydryl-specific spin label containing a stable free radical, which is used as the EPR-detectable probe. SDSL directly probes the local environment, structure, and proximity of individual residues, and is often greatly advantageous over techniques that give global information on protein structure and changes. SDSL can detect and follow changes in local structure due to intramolecular conformational changes or dynamic interactions with other proteins, peptides, or substrates. In addition, this technique can detect changes in distances between two sites and provide information on the depth of spin labels located within a membrane bilayer. EPR is neither limited by the size of the protein or peptide nor limited by the optical properties of the sample and has the unique ability to address and answer structure and dynamics questions that are not solvable solely by genetic or crystal structure analysis, making it highly complementary to other structural methods. In this chapter, we introduce the basic methods for using SDSL EPR spectroscopy in the study of the structure and dynamics of proteins and peptides and illustrate the practical applications of this method through specific examples in the literature.

The oxidation of 2',7'-dichlorofluorescin (DCFH) and its diacetate form (DCFHDA) by the HRP/peroxynitrite system was investigated. Both DCFH and DCFHDA were oxidized to fluorescent products. A major anomaly, however, was the observation that fluorescence continued to build up long after peroxynitrite total decomposition and the initial HRP compound I reduction, suggesting the production of oxidants by the system. Indeed, preformed HRP compound I was instantly reduced by DCFH and DCFHDA to compound II with the obligate formation of DCF(-) semiquinone and DCFHDA-derived radicals. Catalase strongly inhibited fluorescence and EPR signals, suggesting the intermediate formation of H2O2. Taken together the data indicate that peroxynitrite rapidly oxidizes HRP to HRP compound I, which is reduced by DCFH and its diacetate form with the concomitant formation of DCF(-) semiquinone and DCFHDA-derived radicals. These are oxidized by O2, producing O2(-) (as demonstrated by EPR and oxygen consumption experiments), which dismutates to produce H2O2, which serves to fuel further DCFH/DCFHDA oxidation via HRP catalysis. Also DCFHDA was shown to be considerably more resistant to oxidation than its hydrolyzed product DCFH, presumably because of the absence of the easily oxidizable phenol moieties. DCFHDA/DCFH have been used to study free radical production in a variety of systems. Our findings demonstrate that this assay is subject to a serious artifact in that it produces what it is purported to measure; therefore, its use in biological systems should be approached with caution.

Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S](+) interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S](+) of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). (2)H and (13)C ENDOR spectroscopy was performed on [4Fe-4S](+)-PFL-AE (S = (1)/(2)) in the presence of AdoMet labeled at the methyl position with either (2)H or (13)C (denoted [1+/AdoMet]). The observation of a substantial (2)H coupling of approximately 1 MHz ( approximately 6-7 MHz for (1)H), as well as hyperfine-split signals from the (13)C, manifestly require that AdoMet lie close to the cluster. (2)H and (13)C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S](2+) state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe-4S](2+)/AdoMet complex to form the reduced state (denoted [2+/AdoMet](red)) trapped in the structure of the oxidized state. (2)H and (13)C ENDOR spectra for [2+/AdoMet](red) are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency (13)C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the (2)H and (13)C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be approximately 3.0-3.8 A, while the distance from the methyl carbon to the nearest iron is approximately 4-5 A. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S](2+/+) cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.

E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.

Stroke causes heterogeneous changes in tissue oxygenation, with a region of decreased blood flow, the penumbra, surrounding a severely damaged ischemic core. Treatment of acute ischemic stroke aims to save this penumbra before its irreversible damage by continued ischemia. However, effective treatment remains elusive due to incomplete understanding of processes leading to penumbral death. While oxygenation is central in ischemic neuronal death, it is unclear exactly what actual changes occur in interstitial oxygen tension (pO2) in ischemic regions during stroke, particularly the penumbra. Using the unique capability of in vivo electron paramagnetic resonance (EPR) oximetry to measure localized interstitial pO2, we measured both absolute values, and temporal changes of pO2 in ischemic penumbra and core during ischemia and reperfusion in a rat model. Ischemia rapidly decreased interstitial pO2 to 32% +/- 7.6% and 4% +/- 0.6% of pre-ischemic values in penumbra and core, respectively 1 hour after ischemia. Importantly, whilst reperfusion restored core pO2 close to its pre-ischemic value, penumbral pO2 only partially recovered. Hyperoxic treatment significantly increased penumbral pO2 during ischemia, but not in the core, and also increased penumbral pO2 during reperfusion. These divergent, important changes in pO2 in penumbra and core were explained by combined differences in cellular oxygen consumption rates and microcirculation conditions. We therefore demonstrate that interstitial pO2 in penumbra and core is differentially affected during ischemia and reperfusion, providing new insights to the pathophysiology of stroke. The results support normobaric hyperoxia as a potential early intervention to save penumbral tissue in acute ischemic stroke.

The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.