The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H(2)O(2) and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H(2)O(2)- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the "untargeted" carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.

It has previously been reported that exposure of purified mitochondrial or cytoplasmic aconitase to superoxide (O(2)(-)(*) or hydrogen peroxide (H(2)O(2)) leads to release of the Fe-alpha from the enzyme's [4Fe-4S](2+) cluster and to inactivation. Nevertheless, little is known regarding the response of aconitase to pro-oxidants within intact mitochondria. In the present study, we provide evidence that aconitase is rapidly inactivated and subsequently reactivated when isolated cardiac mitochondria are treated with H(2)O(2). Reactivation of the enzyme is dependent on the presence of the enzyme's substrate, citrate. EPR spectroscopic analysis indicates that enzyme inactivation precedes release of the labile Fe-alpha from the enzyme's [4Fe-4S](2+) cluster. In addition, as judged by isoelectric focusing gel electrophoresis, the relative level of Fe-alpha release and cluster disassembly does not reflect the magnitude of enzyme inactivation. These observations suggest that some form of posttranslational modification of aconitase other than release of iron is responsible for enzyme inactivation. In support of this conclusion, H(2)O(2) does not exert its inhibitory effects by acting directly on the enzyme, rather inactivation appears to result from interaction(s) between aconitase and a mitochondrial membrane component responsive to H(2)O(2). Nevertheless, prolonged exposure of mitochondria to steady-state levels of H(2)O(2) or O(2)(-)(*) results in disassembly of the [4Fe-4S](2+) cluster, carbonylation, and protein degradation. Thus, depending on the pro-oxidant species, the level and duration of the oxidative stress, and the metabolic state of the mitochondria, aconitase may undergo reversible modulation in activity or progress to [4Fe-4S](2+) cluster disassembly and proteolytic degradation.

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

This review discusses the development and therapeutic potential of prototype macromolecular drugs for use in cancer chemotherapy, in particular the development and use of SMANCS, a conjugate of neocarzinostatin and poly(styrene-comaleic acid). The various topics covered include a brief description of the chemistry and polymer conjugation, the binding of the conjugate to albumin and the biological behaviour in vitro and in vivo after arterial injection in animals, including plasma half-life, and the lipid solubility of SMANCS in medium chain triglycerides and Lipiodol, a lipid contrast medium suitable for use in X-ray-computed tomography. The biological response-modifying effects and the tumor-targeting mechanism of SMANCS and other macromolecular drugs are also discussed. The latter mechanism is accounted for in terms of a tumor 'enhanced permeability and retention' (or EPR) effect. A principal advantage in the use of SMANCS or other macromolecular drugs is the potential for a reduction or elimination of toxicity. Macromolecular drugs such as a pyran copolymer-NCS conjugate show a marked reduction in bone marrow toxicity normally associated with the use of NCS. This is believed to be due to a hypothetical blood-bone marrow 'barrier' which, relative to NCS, restricts or limits access of the macromolecular drug to the bone marrow. In addition, the clinical possibilities for SMANCS are discussed, including the suggestion that angiotensin II-induced hypertension has clinical potential in improving the selective delivery of macromolecular drugs (i.e. SMANCS) to tumors. Aqueous SMANCS formulations have been tested in pilot studies in patients with solid tumors of the ovary, esophagus, lung, stomach, adrenal gland and in the brain. Formulations based on SMANCS/Lipiodol have been shown to be effective both as a diagnostic tool and for therapeutic use in solid tumors where the formulations are given arterially via a catheter. In a pilot study in primary unresectable hepatoma, an objective reduction in tumor size was observed for about 90% of cases when an adequate amount of the macromolecular drug was administered. A patient receiving such treatment with no active liver cirrhosis and tumor nodules/lesion confined within one liver segment might expect to have a 90% chance of survival after treatment for at least 5 years.

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a 'storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.

Human islet amyloid polypeptide (hIAPP) misfolding is thought to play an important role in the pathogenesis of type II diabetes mellitus. It has recently been shown that membranes can catalyze the misfolding of hIAPP via an alpha-helical intermediate of unknown structure. To better understand the mechanism of membrane-mediated misfolding, we used site-directed spin labeling and EPR spectroscopy to generate a three-dimensional structural model of this membrane-bound form. We find that hIAPP forms a single alpha-helix encompassing residues 9-22. The helix is flanked by N- and C-terminal regions that do not take up a clearly detectable secondary structure and are less ordered. Residues 21 and 22 are located in a transitional region between the alpha-helical structure and C terminus and exhibit significant mobility. The alpha-helical structure presented here has important implications for membrane-mediated aggregation. Anchoring hIAPP to the membrane not only increases the local concentration but also reduces the encounter between peptides to essentially a two-dimensional process. It is significant to note that the alpha-helical membrane-bound form leaves much of an important amyloidogenic region of hIAPP (residues 20-29) exposed for misfolding. Misfolding of this and other regions is likely further aided by the low dielectric environment near the membrane that is known to promote secondary structure formation. Based upon these considerations, a structural model for membrane-mediated aggregation is discussed.

The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 Å-80 Å) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.

Transcription factors AP-1 and NF-kappaB have been implicated in the inducible expression of a variety of genes in response to oxidative stress. Recently, based on the observation that butylated hydroxyanisole (BHA) and pyrrolidine dithiocarbamate (PDTC) induce AP-1 binding activity and AP-1-dependent gene expression and assuming that these compounds exert an antioxidant effect, it was claimed that AP-1 is an antioxidant-responsive factor. To determine whether AP-1 can be responsive to both oxidant and antioxidant, we examined the nature of BHA and PDTC inducing activity. Using EPR spectroscopy to detect semiquinone radicals, we demonstrate the autoxidation of BHA metabolite tert-butylhydroquinone (TBHQ) to tert-butylquinone. The kinetics of TBHQ-mediated generation of .OH radicals were monitored in intact hepatoma HepG2 cells by EPR spin trapping technique. Exogenous catalase inhibited the rate and amount of .OH radical formation and the induction of AP-1-mediated glutathione S-transferase (GST) Ya gene expression by BHA and TBHQ, thus indicating the intermediate formation of H2O2 in the metabolism of these chemicals. Furthermore, we show that the induction of AP-1 and NF-kappaB activities and GST Ya gene expression by BHA and TBHQ is due to a pro-oxidant activity, since this induction was inhibited by thiol compounds N-acetyl cysteine and GSH. Similarly, induction of AP-1 and GST Ya gene expression by PDTC was inhibited by N-acetyl cysteine and GSH. The present findings do not support the notion that the induction of AP-1 by BHA, TBHQ, or PDTC is an antioxidant response and demonstrate that both AP-1 and NF-kappaB activities are induced by oxygen radicals.

Doxorubicin (DXR) commonly used in cancer therapy produces undesirable side effects such as cardiotoxicity. To minimize these, attempts have been made to couple the drug with dextran (DEX) and then to encapsulate this drug conjugate in hydrogel nanoparticles. By encapsulation of the drug conjugate in biodegradable, biocompatible long circulating hydrogel nanoparticles, we further improved the therapeutic efficacy of the conjugate. The size of these nanoparticles as determined by quasi-elastic light scattering, was found to be 100+/-10 nm diameter, which favors the enhanced permeability and retention effect (EPR) as observed in most solid tumors. The antitumor effect of these DEX-DXR nanoparticles, was evaluated in J774A.1 macrophage tumor cells implanted in Balb/c mice. The in vivo efficacy of these nanoparticles as antitumor drug carriers, was determined by tumor regression and increased survival time as compared to drug conjugate and free drug. These results suggest that encapsulation of the conjugate in nanoparticles not only reduces the side effects, but also improves its therapeutic efficacy in the treatment of solid tumors.

Early detection is a crucial element for the timely diagnosis and successful treatment of all human cancers but is limited by the sensitivity of current imaging methodologies. We have synthesized and studied bioresorbable calcium phosphate nanoparticles (CPNPs) in which molecules of the near-infrared (NIR) emitting fluorophore, indocyanine green (ICG), are embedded. The ICG-CPNPs demonstrate exceptional colloidal and optical characteristics. Suspensions consisting of 16 nm average diameter particles are colloidally stable in physiological solutions (phosphate buffered 0.15 M saline (PBS), pH 7.4) with carboxylate or polyethylene glycol (PEG) surface functionality. ICG-doped CPNPs exhibit significantly greater intensity at the maximum emission wavelength relative to the free constituent fluorophore, consistent with the multiple molecules encapsulated per particle. The quantum efficiency per molecule of the ICG-CPNPs is 200% greater at 0.049 +/- 0.003 over the free fluorophore in PBS. Photostability based on fluorescence half-life of encapsulated ICG in PBS is 500% longer under typical clinical imaging conditions relative to the free dye. PEGylated ICG-CPNPs accumulate in solid, 5 mm diameter xenograft breast adenocarcinoma tumors via enhanced retention and permeability (EPR) within 24 h after systemic tail vein injection in a nude mouse model. Ex situ tissue imaging further verifies the facility of the ICG-CPNPs for deep-tissue imaging with NIR signals detectable from depths up to 3 cm in porcine muscle tissue. Our ex vivo and in vivo experiments verify the promise of the NIR CPNPs for diagnostic imaging in the early detection of solid tumors.

Nanoparticles for cancer therapy and imaging are designed to accumulate in the diseased tissue by exploiting the Enhanced Permeability and Retention (EPR) effect. This limits their size to about 100nm. Here, using intravital microscopy and elemental analysis, we compare the in vivo localization of particles with different geometries and demonstrate that plateloid particles preferentially accumulate within the tumor vasculature at unprecedented levels, independent of the EPR effect. In melanoma-bearing mice, 1000×400nm plateloid particles adhered to the tumor vasculature at about 5% and 10% of the injected dose per gram organ (ID/g) for untargeted and RGD-targeted particles respectively, and exhibited the highest tumor-to-liver accumulation ratios (0.22 and 0.35). Smaller and larger plateloid particles, as well as cylindroid particles, were more extensively sequestered by the liver, spleen, and lungs. Plateloid particles appeared well-suited for taking advantage of hydrodynamic forces and interfacial interactions required for efficient tumoritropic accumulation, even without using specific targeting ligands.

Three new intermediates of the catalytic cycle of the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been detected using transient kinetic techniques. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains an oxygen-bridged [Fe(III).Fe(III)] cluster that catalyzes O2 activation and insertion chemistry. At 4 degrees C, rapid mixing of O2 with diferrous MMOH ([Fe(II).Fe(II)]) in the presence of a 2-fold excess of MMOB resulted in loss of the g = 16 EPR signal characteristic of the diferrous cluster at an apparent first order rate of 22 +/- 5 s-1 (O2 approximately 700 microM). Subsequently, an EPR silent, chromophoric (lambda max = 330 and 430 nm, epsilon approximately 7500 M-1 cm-1 at each wavelength) intermediate (compound Q) formed with an average first order rate constant of 1 +/- 0.1 s-1 and then decayed at 0.05 +/- 0.01 s-1. Since compound Q formed much more slowly than diferrous MMOH disappeared, at least one other undetected intermediate (compound P) must have formed before compound Q. MMO substrates had little or no effect on the formation rate of compound Q, but they caused the decay rate to increase linearly with the concentration added. The substrates methane, furan, and nitrobenzene caused compound Q decay to occur with second order rate constants of 19,000 M-1 s-1, 9000 M-1 s-1, and 200 M-1 s-1 (+/- 5%), respectively. When nitrobenzene was used as a substrate, a second chromophoric intermediate (compound T, lambda max = 325 nm, with a shoulder at 395 nm, epsilon 395 approximately 6000 M-1 cm-1) formed at the same rate as compound Q decay. Chemical quench studies showed that compound T is an enzyme-product complex that decays with a rate constant of 0.02 +/- 0.005 s-1. This rate is approximately the same as kcat for nitrobenzene turnover at 4 degrees C catalyzed by the reconstituted MMO system, suggesting that product release is the rate-limiting step in catalysis. The characteristics of compound Q suggest that it may be the activated form of the enzyme that directly catalyzes substrate oxidation.

A new computational approach has been developed to determine the spatial arrangement of proteins in membranes by minimizing their transfer energies from water to the lipid bilayer. The membrane hydrocarbon core was approximated as a planar slab of adjustable thickness with decadiene-like interior and interfacial polarity profiles derived from published EPR studies. Applicability and accuracy of the method was verified for a set of 24 transmembrane proteins whose orientations in membranes have been studied by spin-labeling, chemical modification, fluorescence, ATR FTIR, NMR, cryo-microscopy, and neutron diffraction. Subsequently, the optimal rotational and translational positions were calculated for 109 transmembrane, five integral monotopic and 27 peripheral protein complexes with known 3D structures. This method can reliably distinguish transmembrane and integral monotopic proteins from water-soluble proteins based on their transfer energies and membrane penetration depths. The accuracies of calculated hydrophobic thicknesses and tilt angles were approximately 1 A and 2 degrees, respectively, judging from their deviations in different crystal forms of the same proteins. The hydrophobic thicknesses of transmembrane proteins ranged from 21.1 to 43.8 A depending on the type of biological membrane, while their tilt angles with respect to the bilayer normal varied from zero in symmetric complexes to 26 degrees in asymmetric structures. Calculated hydrophobic boundaries of proteins are located approximately 5 A lower than lipid phosphates and correspond to the zero membrane depth parameter of spin-labeled residues. Coordinates of all studied proteins with their membrane boundaries can be found in the Orientations of Proteins in Membranes (OPM) database:http://opm.phar.umich.edu/.

The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, three distinct coordination modes are identified. Each mode is fully characterized to reveal a series of copper-dependent octarepeat domain structures. Multiple His coordination is clearly identified at low copper stoichiometry. In addition, EPR detected copper-copper interactions at full occupancy suggest that the octarepeat domain partially collapses, perhaps stabilizing this specific binding mode and facilitating cooperative copper uptake. This work provides the first complete characterization of all dominant copper coordination modes at pH 7.4.

The initiation of lipid peroxidation by Fe2+ and H2O2 (Fenton's reagent) is often proposed to be mediated by the highly reactive hydroxyl radical. Using Fe2+, H2O2, and phospholipid liposomes as a model system, we have found that lipid peroxidation, as assessed by malondialdehyde formation, is not initiated by the hydroxyl radical, but rather requires Fe3+ and Fe2+. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide and the bleaching of para-nitrosodimethylaniline confirmed the generation of the hydroxyl radical in this system. Accordingly, catalase and the hydroxyl radical scavengers mannitol and benzoate efficiently inhibited the generation and the detection of hydroxyl radical. However, catalase, mannitol, and benzoate could either stimulate or inhibit lipid peroxidation. These unusual effects were found to be consistent with their ability to modulate the extent of Fe2+ oxidation by H2O2 and demonstrated that lipid peroxidation depends on the Fe3+:Fe2+ ratio, maximal initial rates occurring at 1:1. These studies suggest that the initiation of liposomal peroxidation by Fe2+ and H2O2 is mediated by an oxidant which requires both Fe3+ and Fe2+ and that the rate of the reaction is determined by the absolute Fe3+:Fe2+ ratio.

Elemental analyses, Mössbauer, and EPR data are reported to show that endonuclease III of Escherichia coli is an iron-sulfur protein. Mössbauer spectra of protein freshly prepared from E. coli grown on 57Fe-enriched medium demonstrate that the native enzyme contains a single 4Fe-4S cluster in the 2+ oxidation state, with a net spin of zero. Upon treatment with ferricyanide, a fraction (less than 25%) of the clusters is oxidized into a state which yields an EPR spectrum near g = 2.01 typical of a 3Fe-4S cluster. The magnetic field dependence of the linear electric field effect verifies this assignment. Electron spin echo modulation on the g = 2.01 form of the protein in deuterated solvent indicates the presence of exchangeable protons in the vicinity of the 3Fe-4S cluster. The data obtained show that the [4Fe-4S]2+ cluster of the native enzyme is resistant to either oxidation or reduction, although photoreduction elicited a g = 1.94 type EPR signal characteristic of a [4Fe-4S]1+ cluster. These studies show that endonuclease III is unique in being both a DNA repair enzyme and an iron-sulfur protein. The function of the 4Fe-4S cluster remains to be established.

In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.

Cu,Zn superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) is known to be inhibited slowly by H2O2. Using EPR and the spin traps 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN), we have shown that Cu,Zn-SOD catalyzes the formation of "free" .OH radicals from H2O2 in pH 7.6 bicarbonate buffer. Supporting evidence includes the following: (i) H2O2 and active Cu,Zn-SOD are required to yield significant signals from spin-trap-OH adducts. (ii) With O2-., Cu,Zn-SOD causes the appearance of intense resonance signals due to DMPO-OH adducts. These signals were inhibited strongly by catalase. (iii) With H2O2, Cu,Zn-SOD, and DMPO, radical scavengers formate and azide, but not ethanol, decrease DMPO-OH signals while causing new intense signals due to their corresponding DMPO-radical adducts. Failure of ethanol to quench DMPO-OH signals is discussed in light of the positively charged active channel of the enzyme. (iv) With PBN as a spin trap, ethanol quenches .OH radical signals and yields PBN-trapped hydroxyethyl radical signals. (v) Mn-SOD does not catalyze "free" .OH radical formation and it also exerts no effect on the signals of DMPO-OH adducts when added together with the Cu,Zn-SOD. The capacity of Cu,Zn-SOD to generate "free" .OH radicals from H2O2 may in part explain the biological damage associated with elevated intracellular SOD activity.

In addition to the reaction-center chlorophyll, at least two other organic cofactors are involved in the photosynthetic oxygen-evolution process. One of these cofactors, called "Z," transfers electrons from the site of water oxidation to the reaction center of photosystem II. The other species, "D," has an uncertain function but gives rise to the stable EPR signal known as signal II. Z+. and D+. have identical EPR spectra and are generally assumed to arise from species with the same chemical structure. Results from a variety of experiments have suggested that Z and D are plastoquinones or plastoquinone derivatives. In general, however, the evidence to support this assignment is indirect. To address this situation, we have developed more direct methods to assign the structure of the Z+./D+. radicals. By selective in vivo deuteration of the methyl groups of plastoquinone in cyanobacteria, we show that hyperfine couplings from the methyl protons cannot be responsible for the partially resolved structure seen in the D+. EPR spectrum. That is, we verify by extraction and mass spectrometry that quinones are labeled in algae fed deuterated methionine, but no change is observed in the line shape of signal II. Considering the spectral properties of the D+. radical, a tyrosine origin is a reasonable alternative. In a second series of experiments, we have found that deuteration of tyrosine does indeed narrow the D+. signal. Extraction and mass spectral analysis of the quinones in these cultures show that they are not labeled by tyrosine. These results eliminate a plastoquinone origin for D+.; we conclude instead that D+., and most likely Z+., are tyrosine radicals.

Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins M1 and M2. Protein M2 contains a tyrosyl free radical, essential for activity, which can be quantified directly in frozen, packed cells by EPR spectroscopy. A 3-7-fold increase in the concentration of tyrosyl radical-containing M2 subunit was observed when mouse mammary tumor TA 3 cells passed from the G1 to the S phase of the cell cycle. Similar results were obtained with cells synchronized by isoleucine starvation or separated by centrifugal elutriation. Addition of deuterated tyrosine to cells give rise to a different EPR signal in newly synthesized protein M2. Pulse-chase experiments with deuterated tyrosine showed unequivocally that the S phase-correlated increase in radical-containing M2 subunit was due to de novo protein synthesis. Labeled M2 molecules disappeared with a half-life of 3 h, and therefore new molecules must be synthesized at a high rate during the S phase. In contrast, after hydroxyurea inactivation, cells rapidly regenerated the tyrosyl radical in already existing protein M2 molecules. This enzyme activation mechanism is clearly different from the one responsible for regulating protein M2 activity during the cell cycle.

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

Apolipoprotein A-I (apoA-I) plays a central role in the reverse cholesterol transport pathway; however, the structural basis for its antiatherogenic effects remains poorly understood. Here we employ EPR spectroscopy and fluorescence resonance energy transfer to elucidate the conformation and relative alignment of apoA-I monomers on discoidal (9.4 nm) reconstituted high density lipoprotein (rHDL). EPR spectroscopy provided evidence for an extended helical secondary structure. Position 139 since it was the only residue examined to display a dynamic motional character consistent with a flexible loop structure. The EPR spectra of nitroxide probes at positions 133 and 146 exhibit spin coupling, indicating that these positions are proximal to an apoA-I paired counterpart on the perimeter of rHDL. fluorescence resonance energy transfer studies employing engineered apoA-I variants possessing a single tryptophan (energy donor) and/or a single cysteine (whose thiol moiety was covalently labeled with an extrinsic energy acceptor) provided evidence that paired apoA-I molecules around the perimeter of rHDL align in an extended antiparallel conformation. Taken together with the observation that the EPR spectra of nitroxide probes positioned at intervening sequence positions (134-145) do not exhibit spin coupling, this has led us to propose a "looped belt" model, wherein residues 133-146 comprise a flexible loop segment that confers to apoA-I an intrinsic ability to adapt its structure to accommodate changing particle lipid content. Specifically, in the looped belt model, with the exception of amino acids 134-145, apoA-I aligns with its counterpart in a helix 5-helix 5 registry, centered at position 139.

OBJECTIVE

The involvement of transition metals in atherosclerosis is controversial. Some epidemiological studies have reported a relationship between iron (Fe) and cardiovascular disease, whereas others have not. Experimental studies have reported elevated levels of iron and copper (Cu) in diseased human arteries but have often used methods that release metal ions from proteins.

RESULTS

In this study, we have used the minimally invasive technique of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICPMS) to quantify iron and copper in ex vivo healthy human arteries and carotid lesions. The EPR spectra detected are characteristic of nonheme Fe(III) complexes. Statistically elevated levels of iron were detected in the intima of lesions compared with healthy controls (0.370 versus 0.022 nmol/mg tissue for EPR, 0.525 versus 0.168 nmol/mg tissue by ICPMS, P<0.05 in each cases). Elevated levels of copper were also detected (7.51 versus 2.01 pmol/mg tissue, lesion versus healthy control, respectively, P<0.05). Iron levels did not correlate with the gender or age of the donor, or tissue protein or calcium levels, but cholesterol levels correlated positively with iron accumulation, as measured by EPR.

CONCLUSIONS

These data support the hypothesis that iron accumulates in human lesions and may contribute to disease progression.