New, ultrasmall nanoparticles with sizes below 5 nm have been obtained. These small rigid platforms (SRP) are composed of a polysiloxane matrix with DOTAGA (1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid)-Gd(3+) chelates on their surface. They have been synthesised by an original top-down process: 1) formation of a gadolinium oxide Gd2O3 core, 2) encapsulation in a polysiloxane shell grafted with DOTAGA ligands, 3) dissolution of the gadolinium oxide core due to chelation of Gd(3+) by DOTAGA ligands and 4) polysiloxane fragmentation. These nanoparticles have been fully characterised using photon correlation spectroscopy (PCS), transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID) and electron paramagnetic resonance (EPR) to demonstrate the dissolution of the oxide core and by inductively coupled plasma mass spectrometry (ICP-MS), mass spectrometry, fluorescence spectroscopy, (29)Si solid-state NMR, (1)H NMR and diffusion ordered spectroscopy (DOSY) to determine the nanoparticle composition. Relaxivity measurements gave a longitudinal relaxivity r1 of 11.9 s(-1) mM(-1) per Gd at 60 MHz. Finally, potentiometric titrations showed that Gd(3+) is strongly chelated to DOTAGA (complexation constant logβ110 =24.78) and cellular tests confirmed the that nanoconstructs had a very low toxicity. Moreover, SRPs are excreted from the body by renal clearance. Their efficiency as contrast agents for MRI has been proved and they are promising candidates as sensitising agents for image-guided radiotherapy.

This review paper attempts to provide an overview of the principles and techniques that are often termed electron paramagnetic resonance (EPR) oximetry. The paper discusses the potential of such methods and illustrates they have been successfully applied to measure oxygen tension, an essential parameter of the tumor microenvironment. To help the reader understand the motivation for carrying out these measurements, the importance of tumor hypoxia is first discussed: the basic issues of why a tumor is hypoxic, why these hypoxic microenvironments promote processes driving malignant progression and why hypoxia dramatically influences the response of tumors to cytotoxic treatments will be explained. The different methods that have been used to estimate the oxygenation in tumors will be reviewed. To introduce the basics of EPR oximetry, the specificity of in vivo EPR will be discussed by comparing this technique with NMR and MRI. The different types of paramagnetic oxygen sensors will be presented, as well as the methods for recording the information (EPR spectroscopy, EPR imaging, dynamic nuclear polarization). Several applications of EPR for characterizing tumor oxygenation will be illustrated, with a special emphasis on pharmacological interventions that modulate the tumor microenvironment. Finally, the challenges for transposing the method into the clinic will also be discussed.

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30 mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30 mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.

The interface of bio-nano science and cancer medicine is an area experiencing much progress but also beset with controversy. Core concepts of the field-e.g., the enhanced permeability and retention (EPR) effect, tumor targeting and accumulation, and even the purpose of "nano" in cancer medicine-are hotly debated. In parallel, considerable advances in neighboring fields are occurring rapidly, including the recent progress of "immuno-oncology" and the fundamental impact it is having on our understanding and the clinical treatment of the group of diseases collectively known as cancer. Herein, we (i) revisit how cancer is commonly treated in the clinic and how this relates to nanomedicine; (ii) examine the ongoing debate on the relevance of the EPR effect and tumor targeting; (iii) highlight ways to improve the next-generation of nanomedicines; and (iv) discuss the emerging concept of working with (and not against) biology. While discussing these controversies, challenges, emerging concepts, and opportunities, we explore new directions for the field of cancer nanomedicine.

This paper argues that we should understand the process of IT design as the development of sociotechnical configurations. Drawing upon our experiences with an electronic patient record (EPR) on an Intensive Care Unit (ICU), we depict medical work practices as natural systems. Several considerations for design are developed. First, the EPR should not be overly structured with rationalistic and prefixed notions of the organization and content of medical work. Implementing structure is crucial, however, this should be derived from detailed, empirical knowledge of the practice involved. Second, it is crucial to ensure that the usage of the system will yield immediate benefits for primary users: the systems should support work, not generate it. Third, designing IT should include being aware of the socio political nature of seemingly 'neutral' tools as EPRs.

Despite the central importance of peripheral membrane proteins to cellular signaling and metabolic pathways, the structures of protein-membrane interfaces remain largely inaccessible to high-resolution structural methods. In recent years a number of laboratories have contributed to the development of an electron paramagnetic resonance (EPR) power saturation approach that utilizes site-directed spin labeling to determine the key geometric parameters of membrane-docked proteins, including their penetration depths and angular orientations relative to the membrane surface. Representative applications to Ca(2+)-activated, membrane-docking C2 domains are described.

Spectroscopic methods, density functional calculations, and ligand field analyses are combined to define the geometric models and electronic structure descriptions of the Cu(M) and Cu(H) sites in the oxidized form of the noncoupled binuclear copper protein peptidylglycine alpha-hydroxylating monooxygenase (PHM). The Cu(M) site has a square pyramidal geometry with a long axial Cu-methionine bond and two histidines, H(2)O, and OH(-) as equatorial ligands. The Cu(H) site has a slightly D(2)(d) distorted square planar geometry with three histidines and H(2)O ligands. The structurally inequivalent Cu(M) and Cu(H) sites do not exhibit measurable differences in optical and electron paramagnetic resonance (EPR) spectra, which result from their similar ligand field transition energies and ground-state Cu covalencies. The additional axial methionine ligand interaction and associated square pyramidal distortion of the Cu(M) site have the opposite effect of the strong equatorial OH(-) donor ligand on the Cu d orbital splitting pattern relative to the Cu(H) site leading to similar ligand field transition energies for both sites. The small molecule NO(2)(-) binds in different coordination modes to the Cu(M) and Cu(H) site because of differences in their exchangeable coordination positions resulting in these Cu(II) sites being spectroscopically distinguishable. Azide binding to PHM is used as a spectroscopic and electronic structure analogue to OOH(-) binding to provide a starting point for developing a geometric and electronic structural model for the putative Cu(II)(M)-OOH intermediate in the H-atom abstraction reaction of PHM. Possible electronic structure contributions of the Cu(II)(M)-OOH intermediate to reactivity are considered by correlation to the well-studied L3Cu(II)-OOH model complex (L3 = [HB[3-tBu-5-iPrpz](3)]). The Met-S ligand of the Cu(M) site is found to contribute to the stabilization of the Cu(II)(M)-oxyl species, which would be a product of Cu(II)(M)-OOH H-atom abstraction reaction. This Met-S contribution could have a significant effect on the energetics of a H-atom abstraction reaction by the Cu(II)(M)-OOH intermediate.

The laboratory synthesis of the oxygen-evolving complex (OEC) of photosystem II has been the objective of synthetic chemists since the early 1970s. However, the absence of structural information on the OEC has hampered these efforts. Crystallographic reports on photosystem II that have been appearing at ever-improving resolution over the past ten years have finally provided invaluable structural information on the OEC and show that it comprises a [Mn(3)CaO(4)] distorted cubane, to which is attached a fourth, external Mn atom, and the whole unit attached to polypeptides primarily by aspartate and glutamate carboxylate groups. Such a heterometallic Mn/Ca cubane with an additional metal attached to it has been unknown in the literature. This paper reports the laboratory synthesis of such an asymmetric cubane-containing compound with a bound external metal atom, [(1)]. All peripheral ligands are carboxylate or carboxylic acid groups. Variable-temperature magnetic susceptibility data have established 1 to possess an S = 9/2 ground state. EPR spectroscopy confirms this, and the Davies electron nuclear double resonance data reveal similar hyperfine couplings to those of other Mn(IV) species, including the OEC S(2) state. Comparison of the X-ray absorption data with those for the OEC reveal 1 to possess structural parameters that make it a close structural model of the asymmetric-cubane OEC unit. This geometric and electronic structural correspondence opens up a new front in the multidisciplinary study of the properties and function of this important biological unit.

Established pulse EPR approaches to the measurement of small dipole-dipole couplings between electron spins rely on constant-time echo experiments to separate relaxational contributions from dipolar time evolution. This requires a compromise between sensitivity and resolution to be made prior to the measurement, so that optimum data are only obtained if the magnitude of the dipole-dipole coupling is known beforehand to a good approximation. Moreover, the whole dipolar evolution function is measured with relatively low sensitivity. These problems are overcome by a variable-time experiment that achieves suppression of the relaxation contribution by reference deconvolution. Theoretical and experimental results show that this approach leads to significant sensitivity improvements for typical systems and experimental conditions. Further sensitivity improvements or, equivalently, an extension of the accessible distance range can be obtained by matrix deuteration or digital long-pass filtering of the time-domain data. Advantages and limitations of the new variable-time experiment are discussed by comparing it to the established analogous constant-time experiment for measurements of end-to-end distances of 5 and 7.5 nm on rod-like shape-persistent biradicals and for the measurement of a broadly distributed transmembrane distance in a doubly spin-labeled mutant of plant light harvesting complex II.

Mitochondrial complex I has previously been shown to release superoxide exclusively towards the mitochondrial matrix, whereas complex III releases superoxide to both the matrix and the cytosol. Superoxide produced at complex III has been shown to exit the mitochondria through voltage dependent anion channels (VDAC). To test whether complex I-derived, mitochondrial matrix-directed superoxide can be released to the cytosol, we measured superoxide generation in mitochondria isolated from wild type and from mice genetically altered to be deficient in MnSOD activity (TnIFastCreSod2(fl/fl)). Under experimental conditions that produce superoxide primarily by complex I (glutamate/malate plus rotenone, GM+R), MnSOD-deficient mitochondria release ∼4-fold more superoxide than mitochondria isolated from wild type mice. Exogenous CuZnSOD completely abolished the EPR-derived GM+R signal in mitochondria isolated from both genotypes, evidence that confirms mitochondrial superoxide release. Addition of the VDAC inhibitor DIDS significantly reduced mitochondrial superoxide release (∼75%) in mitochondria from either genotype respiring on GM+R. Conversely, inhibition of potential inner membrane sites of superoxide exit, including the matrix face of the mitochondrial permeability transition pore and the inner membrane anion channel did not reduce mitochondrial superoxide release in the presence of GM+R in mitochondria isolated from either genotype. These data support the concept that complex I-derived mitochondrial superoxide release does indeed occur and that the majority of this release occurs through VDACs.

Colloidal carriers have been shown to improve tumor therapy by increased drug delivery into tumor sites resulting directly from the enhanced permeability and retention effect (EPR). However, the clinical outcome of tumor therapy is often limited due to multidrug resistance. Several different types of resistance exist with expression of p-glycoprotein being the most commonly described. Paclitaxel entrapped in emulsifying wax nanoparticles (PX NPs) was shown to overcome drug resistance in a human colon adenocarcinoma cell line (HCT-15). In the present studies, the in-vivo efficacy of PX NPs in a HCT-15 mouse xenograft model was demonstrated. Significant inhibition in tumor growth was observed in mice receiving PX NPs treatment. Additionally, mice dosed with Taxol also demonstrated slower tumor growth; however, the efficacy of the Taxol treatment was shown in the in-vitro HUVEC model to be due to the antiangiogenic effect of paclitaxel. It was concluded that the enhanced efficacy of PX NPs over Taxol in the xenograft model was due to both overcoming paclitaxel resistance and an antiangiogenic effect.

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.

Beef heart aconitase, isolated under aerobic conditions, has been studied with Mössbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mössbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably of the [3Fe-3S] type, is in a paramagnetic state of interger electronic spin (S = 2); the Mössbauer spectra exhibit al the unique features reported for proteins with 3Fe clusters. On activation of aconitase with ferrous ion, the paramagnetic 3Fe cluster of dithionite-reduced enzyme is converted into a diamagnetic (S = 0) form. Activation studies with iron enriched in either 27 Fe or 56 Fe suggest that activation transforms the 3Fe cluster into a center that has a [4Fe-4S] core. This conclusion is supported by the observation that EPR signals characteristic of reduced [4Fe-4S] clusters can be elicited under appropriate conditions. It has frequently been assumed that the activation of aconitase with Fe2+ produces an active site containing a single ferrous ion. The data reported here suggest that a ferrous ion is used to rebuild a [4Fe-4S] cluster.

Eight new strains of deep-sea hyperthermophilic sulfur reducers were isolated from hydrothermal vent fields at 9 degrees 50'N East Pacific Rise (EPR) and at the Cleft and CoAxial segments along the Juan de Fuca Ridge (JdFR). 16S rRNA gene sequence analysis showed that each strain belongs to the genus Thermococcus. Restriction fragment length polymorphism patterns of the 16S/23S rRNA intergenic spacer region revealed that these isolates fell into three groups: those from the EPR, those from fluid and rock sources on the JdFR, and those isolated from Paralvinella spp. polychaete vent worms from the JdFR. The optimum-temperature specific growth rates and the temperature ranges for growth were significantly higher and broader for those strains isolated from worms relative to those isolated from low-temperature diffuse hydrothermal fluids. Furthermore, the worm-derived isolates generally produced a larger array of proteases and amylases based on zymogram analyses. The zymogram patterns also changed with growth temperature suggesting that these organisms alter their lytic protein suites in response to changes in temperature. This study suggests that there is significant phenotypic diversity in Thermococcus that is not apparent from their highly conserved 16S rRNA nucleotide sequences.

The crystal structure of the membrane-associated [NiFe] hydrogenase from Allochromatium vinosum has been determined to 2.1 Å resolution. Electron paramagnetic resonance (EPR) and Fourier transform infrared spectroscopy on dissolved crystals showed that it is present in the Ni-A state (>90%). The structure of the A. vinosum [NiFe] hydrogenase shows significant similarities with [NiFe] hydrogenase structures derived from Desulfovibrio species. The amino acid sequence identity is ∼ 50%. The bimetallic [NiFe] active site is located in the large subunit of the heterodimer and possesses three diatomic non-protein ligands coordinated to the Fe (two CN(-) , one CO). Ni is bound to the protein backbone via four cysteine thiolates; two of them also bridge the two metals. One of the bridging cysteines (Cys64) exhibits a modified thiolate in part of the sample. A mono-oxo bridging ligand was assigned between the metal ions of the catalytic center. This is in contrast to a proposal for Desulfovibrio sp. hydrogenases that show a di-oxo species in this position for the Ni-A state. The additional metal site located in the large subunit appears to be a Mg(2+) ion. Three iron-sulfur clusters were found in the small subunit that forms the electron transfer chain connecting the catalytic site with the molecular surface. The calculated anomalous Fourier map indicates a distorted proximal iron-sulfur cluster in part of the crystals. This altered proximal cluster is supposed to be paramagnetic and is exchange coupled to the Ni(3+) ion and the medial [Fe(3)S(4)](+) cluster that are both EPR active (S=1/2 species). This finding of a modified proximal cluster in the [NiFe] hydrogenase might explain the observation of split EPR signals that are occasionally detected in the oxidized state of membrane-bound [NiFe] hydrogenases as from A. vinosum.

The ferritins are a family of proteins produced in a variety of amounts and types depending on the state of development of an animal, or the state of differentiation of a particular cell type, or the environment. Iron storage is the main function of the ferritins when iron is needed for intracellular use (housekeeping) for iron proteins such as ribonucleotide reductase, cytochromes, oxidases, nitrogenases, or photosynthetic reaction centers or for extracellular use by other cells (specialized). Under abnormal conditions, such as the breach of transferrin-receptor-controlled incorporation of iron, ferritin can also serve to detoxify excess intracellular iron. The structure of ferritin is very complex, consisting of a protein coat of 24 polypeptide subunits, approximately 20 kDa, which surrounds an inorganic phase of hydrous ferric oxide. The polypeptide subunits, bundles of four alpha helices, display remarkable conservation of sequence among plants and animals, which is probably related to the necessity of forming the hollow sphere pierced by 14 channels through which iron may pass. In spite of the conserved regions of sequence, there are multiple genes for ferritin polypeptide subunits within an organism; at the moment three distinct subunit types, H H'(or M), and L, have been identified which are expressed in a cell-specific fashion. How many different subunit types exist, the influence on function, and the number of genes required to encode them are currently being actively investigated. Not only does the protein coat of ferritin display variations, the inorganic phase of ferritin can vary as well. For instance, differences can occur in the number of Fe atoms (up to 4500), as well as in the phosphorus content and in the degree of hydration and order. Such observations have depended on the use of a variety of physical techniques such as X-ray diffraction, EXAFS, and Mössbauer spectroscopy. The same approaches, as well as EPR spectroscopy, have been used to monitor the path taken by Fe as it passes from mononuclear Fe(II) outside the protein coat to polynuclear Fe(III) inside the protein coat. Both mononuclear Fe(II) and Fe(III) have been observed, as well as dimeric Fe(II)-O-Fe(III), and Fe(III)-oxo bridged clusters attached to the protein. A possible protein site for the Fe(III) cluster is a groove on the inner surface of the dimeric interface, suggested by the structure and from the affect of natural cross-links between subunit pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct structural insights on the fundamental mechanisms of permeation, selectivity, and gating remain unavailable for the Na(+) and Ca(2+) channel families. Here, we report the spectroscopic structural characterization of the isolated Voltage-Sensor Domain (VSD) of the prokaryotic Na(+) channel NaChBac in a lipid bilayer. Site-directed spin-labeling and EPR spectroscopy were carried out for 118 mutants covering all of the VSD. EPR environmental data were used to unambiguously assign the secondary structure elements, define membrane insertion limits, and evaluate the activated conformation of the isolated-VSD in the membrane using restrain-driven molecular dynamics simulations. The overall three-dimensional fold of the NaChBac-VSD closely mirrors those seen in KvAP, Kv1.2, Kv1.2-2.1 chimera, and MlotiK1. However, in comparison to the membrane-embedded KvAP-VSD, the structural dynamics of the NaChBac-VSD reveals a much tighter helix packing, with subtle differences in the local environment of the gating charges and their interaction with the rest of the protein. Using cell complementation assays we show that the NaChBac-VSD can provide a conduit to the transport of ions in the resting or "down" conformation, a feature consistent with our EPR water accessibility measurements in the activated or "up" conformation. These results suggest that the overall architecture of VSD's is remarkably conserved among K(+) and Na(+) channels and that pathways for gating-pore currents may be intrinsic to most voltage-sensors. Cell complementation assays also provide information about the putative location of the gating charges in the "down/resting" state and hence a glimpse of the extent of conformational changes during activation.

The amyloid beta peptide (Abeta) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer's disease. However, the nature of the toxic Abeta species and its precise mechanism of action remain to be determined. It has been reported that the methionine residue at position 35 has a pivotal role to play in the toxicity of Abeta. We examined the effect of mutating the methionine to valine in Abeta42 (AbetaM35V). The neurotoxic activity of AbetaM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Abeta42. AbetaM35V binds Cu2+ and produces similar amounts of H2O2 as Abeta42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AbetaM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Abeta and copper in a lipid environment as shown by EPR analysis, which indicated that the valine substitution made the peptide less rigid in the bilayer region with a resulting higher affinity for the bilayer. Circular dichroism spectroscopy showed that both Abeta42 and AbetaM35V displayed a mixture of alpha-helical and beta-sheet conformations. These findings provide further evidence that the toxicity of Abeta is regulated by binding to neuronal cells.

Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O(2)(*)(-)) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O(2)(*)(-) and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague-Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 micromol/kg bw, orally), and TNF+ Tempol (Temp; 300 micromol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O(2)(*)(-) production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.

The lac permease of E. coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer. The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane. Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover. Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E. coli is proposed. Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII). In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles. Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa. Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa. In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex. In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325. The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane. Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism. On the other hand, widespread, cooperative conformational changes appear to be involved in turnover. As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover. This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e. helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism. (ABSTRACT TRUNCATED)

Superoxide reductases (SORs) belong to a new class of metalloenzymes that degrade superoxide by reducing it to hydrogen peroxide. These enzymes contain a catalytic iron site that cycles between the Fe(II) and Fe(III) states during catalysis. A key step in the reduction of superoxide has been suggested to involve HO(2) binding to Fe(II), followed by innersphere electron transfer to afford an Fe(III)-OO(H) intermediate. In this paper, the mechanism of the superoxide-induced oxidation of a synthetic ferrous SOR model ([Fe(II)(S(Me2)N(4)(tren))](+) (1)) to afford [Fe(III)(S(Me2)N(4)(tren)(solv))](2+) (2-solv) is reported. The XANES spectrum shows that 1 remains five-coordinate in methanolic solution. Upon reaction of 1 with KO(2) in MeOH at -90 degrees C, an intermediate (3) is formed, which is characterized by a LMCT band centered at 452(2780) nm, and a low-spin state (S = 1/2), based on its axial EPR spectrum (g(perpendicular) = 2.14; g(parallel) = 1.97). Hydrogen peroxide is detected in this reaction, using both (1)H NMR spectroscopy and a catalase assay. Intermediate 3 is photolabile, so, in lieu of a Raman spectrum, IR was used to obtain vibrational data for 3. At low temperatures, a nu(O-O) Fermi doublet is observed in the IR at 788(2) and 781(2) cm(-)(1), which collapses into a single peak at 784 cm(-1) upon the addition of D(2)O. This vibrational peak diminishes in intensity over time and essentially disappears after 140 s. When 3 is generated using an (18)O-labeled isotopic mixture of K(18)O(2)/K(16)O(2) (23.28%), the vibration centered at 784 cm(-1) shifts to 753 cm(-1). This new vibrational peak is close to that predicted (740 cm(-1)) for a diatomic (18)O-(18)O stretch. In addition, a nu(O-O) vibrational peak assigned to free hydrogen peroxide is also observed (nu(O-O) = 854 cm(-1)) throughout the course of the reaction between Fe(II)-1 and superoxide and is strongest after 100 s. XAS studies indicate that 3 possesses one sulfur scatterer at 2.33(2) A and four nitrogen scatterers at 2.01(1) A. Addition of two Fe-O shells, each containing one oxygen, one at 1.86(3) A and one at 2.78(3) A, improved the EXAFS fits, suggesting that 3 is an end-on peroxo or hydroperoxo complex, [Fe(III)(S(Me2)N(4)(tren))(OO(H))](+). Upon warming above -50 degrees C, 3 is converted to 2-MeOH. In methanol and methanol:THF (THF = tetrahydrofuran) solvent mixtures, 2-MeOH is characterized by a LMCT band at lambda(max) = 511(1765) nm, an intermediate spin-state (S = 3/2), and, on the basis of EXAFS, a relatively short Fe-O bond (assigned to a coordinated methanol or methoxide) at 1.94(10) A. Kinetic measurements in 9:1 THF:MeOH at 25 degrees C indicate that 3 is formed near the diffusion limit upon addition of HO(2) to 1 and converts to 2-MeOH at a rate of 65(1) s(-1), which is consistent with kinetic studies involving superoxide oxidation of the SOR iron site.

A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3-N=NH by the alpha-195Gln variant and during reduction of H2N-NH2 by the alpha-70Ala/alpha-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of NN reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of NN reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.