Iron-sulfur (Fe-S) clusters are versatile biological cofactors that require biosynthetic systems in vivo to be assembled. In Escherichia coli, the Isc (iscRSUA-hscBA-fdx-iscX) and Suf (sufABCDSE) pathways fulfill this function. Despite extensive biochemical and genetic analysis of these two pathways, the physiological function of the A-type proteins of each pathway (IscA and SufA) is still unclear. Studies conducted in vitro suggest two possible functions for A-type proteins, as Fe-S scaffold/transfer proteins or as iron donors during cluster assembly. To resolve this issue, SufA was coexpressed in vivo with its cognate partner proteins from the suf operon, SufBCDSE. Native SufA purified anaerobically using this approach was unambiguously demonstrated to be a [2Fe-2S] protein by biochemical analysis and UV-vis, Mossbauer, resonance Raman, and EPR spectroscopy. Furthermore, native [2Fe-2S] SufA can transfer its Fe-S cluster to both [2Fe-2S] and [4Fe-4S] apoproteins. These results clearly show that A-type proteins form Fe-S clusters in vivo and are competent to function as Fe-S transfer proteins as purified. This study resolves the contradictory results from previous in vitro studies and demonstrates the critical importance of providing in vivo partner proteins during protein overexpression to allow correct biochemical maturation of metalloproteins.

A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation. The resulting increased levels of "free iron" catalyze deleterious oxidative reactions in the cell. In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in whole cells of the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific chelator, to promote oxidation of all of the "free iron" to the Fe(III) state wherein it is EPR-detectable. Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1 delta), Mn-SOD (sod2 delta), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1 delta mutant), by pmr1, a genetic suppressor of the sod delta mutant phenotype (in the sod1 delta sod2 delta double knockout strain), and by anaerobic growth. In wild-type cells, an increase in the EPR-detectable iron pool could be induced by treatment with paraquat, a redox-cycling drug that generates superoxide. Cells that were not pretreated with desferrioxamine had Fe(III) EPR signals that were equally as strong as those from treated cells, indicating that "free iron" accumulated in the ferric form in our strains in vivo. Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.

The mechanism of cobalt(II) porphyrin-catalyzed benzylic C-H bond amination of ethylbenzene, toluene, and 1,2,3,4-tetrahydronaphthalene (tetralin) using a series of different organic azides [N(3)C(O)OMe, N(3)SO(2)Ph, N(3)C(O)Ph, and N(3)P(O)(OMe)(2)] as nitrene sources was studied by means of density functional theory (DFT) calculations and electron paramagnetic resonance (EPR) spectroscopy. The DFT computational study revealed a stepwise radical process involving coordination of the azide to the metal center followed by elimination of dinitrogen to produce unusual "nitrene radical" intermediates (por)Co(III)-N(•)Y (4) [Y = -C(O)OMe, -SO(2)Ph, -C(O)Ph, -P(O)(OMe)(2)]. Formation of these nitrene radical ligand complexes is exothermic, predicting that the nitrene radical ligand complexes should be detectable species in the absence of other reacting substrates. In good agreement with the DFT calculations, isotropic solution EPR signals with g values characteristic of ligand-based radicals were detected experimentally from (por)Co complexes in the presence of excess organic azide in benzene. They are best described as nitrene radical anion ligand complexes (por)Co(III)-N(•)Y, which have their unpaired spin density located almost entirely on the nitrogen atom of the nitrene moiety. These key cobalt(III)-nitrene radical intermediates readily abstract a hydrogen atom from a benzylic position of the organic substrate to form the intermediate species 5, which are close-contact pairs of the thus-formed organic radicals R'(•) and the cobalt(III)-amido complexes (por)Co(III)-NHY ({R'(•)···(por)Co(III)-NHY}). These close-contact pairs readily collapse in a virtually barrierless fashion (via transition state TS3) to produce the cobalt(II)-amine complexes (por)Co(II)-NHYR', which dissociate to afford the desired amine products NHYR' (6) with regeneration of the (por)Co catalyst. Alternatively, the close-contact pairs {R'(•)···(por)Co(III)-NHY} 5 may undergo β-hydrogen-atom abstraction from the benzylic radical R'(•) by (por)Co(III)-NHY (via TS4) to form the corresponding olefin and (por)Co(III)-NH(2)Y, which dissociates to give Y-NH(2). This process for the formation of olefin and Y-NH(2) byproducts is also essentially barrierless and should compete with the collapse of 5 via TS3 to form the desired amine product. Alternative processes leading to the formation of side products and the influence of different porphyrin ligands with varying electronic properties on the catalytic activity of the cobalt(II) complexes have also been investigated.

Nanoparticles are frequently suggested as diagnostic agents. However, except for iron oxide nanoparticles, diagnostic nanoparticles have been barely incorporated into clinical use so far. This is predominantly due to difficulties in achieving acceptable pharmacokinetic properties and reproducible particle uniformity as well as to concerns about toxicity, biodegradation, and elimination. Reasonable indications for the clinical utilization of nanoparticles should consider their biologic behavior. For example, many nanoparticles are taken up by macrophages and accumulate in macrophage-rich tissues. Thus, they can be used to provide contrast in liver, spleen, lymph nodes, and inflammatory lesions (eg, atherosclerotic plaques). Furthermore, cells can be efficiently labeled with nanoparticles, enabling the localization of implanted (stem) cells and tissue-engineered grafts as well as in vivo migration studies of cells. The potential of using nanoparticles for molecular imaging is compromised because their pharmacokinetic properties are difficult to control. Ideal targets for nanoparticles are localized on the endothelial luminal surface, whereas targeted nanoparticle delivery to extravascular structures is often limited and difficult to separate from an underlying enhanced permeability and retention (EPR) effect. The majority of clinically used nanoparticle-based drug delivery systems are based on the EPR effect, and, for their more personalized use, imaging markers can be incorporated to monitor biodistribution, target site accumulation, drug release, and treatment efficacy. In conclusion, although nanoparticles are not always the right choice for molecular imaging (because smaller or larger molecules might provide more specific information), there are other diagnostic and theranostic applications for which nanoparticles hold substantial clinical potential.

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.

Ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, the latter of which contains an iron-tyrosyl free radical center essential for activity. We have studied the in vivo effects on the R2 protein of the potent iron chelators parabactin and desferrioxamine using R2-overproducing mouse cells with a tyrosyl free radical signal easily quantifiable by EPR spectroscopy. Both chelators inhibited cell growth, and the inhibition was reversible by iron. Furthermore, both chelators, which penetrate cells and chelate the intracellular iron pool, caused a disappearance of the R2 tyrosyl free radical. In parallel, there was an accumulation of apo-R2 protein in the inhibited cells. In vitro studies using pure, 59Fe-labeled recombinant mouse R2 protein unexpectedly showed that its iron center is labile at physiological temperatures and that iron is spontaneously lost from the protein even in the absence of chelators in a temperature-dependent process. Our conclusion is that parabactin or desferrioxamine inhibits ribonucleotide reduction and cell growth not by directly attacking the iron-radical center of the R2 protein, but instead by chelating the intracellular iron pool. This prevents the regeneration of the iron-radical center both in newly synthesized apo-R2 protein and in apo-R2 protein continuously formed from active R2 protein by the loss of iron.

Sulfate-reducing organisms use sulfate as an electron acceptor in an anaerobic respiratory process. Despite their ubiquitous occurrence, sulfate respiration is still poorly characterized. Genome analysis of sulfate-reducing organisms sequenced to date permitted the identification of only two strictly conserved membrane complexes. We report here the purification and characterization of one of these complexes, DsrMKJOP, from Desulfovibrio desulfuricans ATCC 27774. The complex has hemes of the c and b types and several iron-sulfur centers. The corresponding genes in the genome of Desulfovibrio vulgaris were analyzed. dsrM encodes an integral membrane cytochrome b; dsrK encodes a protein homologous to the HdrD subunit of heterodisulfide reductase; dsrJ encodes a triheme periplasmic cytochrome c; dsrO encodes a periplasmic FeS protein; and dsrM encodes another integral membrane protein. Sequence analysis and EPR studies indicate that DsrJ belongs to a novel family of multiheme cytochromes c and that its three hemes have different types of coordination, one bis-His, one His/Met, and the third a very unusual His/Cys coordination. The His/Cys-coordinated heme is only partially reduced by dithionite. About 40% of the hemes are reduced by menadiol, but no reduction is observed upon treatment with H2 and hydrogenase, irrespective of the presence of cytochrome c3. The aerobically isolated Dsr complex displays an EPR signal with similar characteristics to the catalytic [4Fe-4S]3+ species observed in heterodisulfide reductases. Further five different [4Fe-4S](2+/1+) centers are observed during a redox titration followed by EPR. The role of the DsrMKJOP complex in the sulfate respiratory chain of Desulfovibrio spp. is discussed.

Mammalian cysteine dioxygenase (CDO) is a non-heme iron metalloenzyme that catalyzes the first committed step in oxidative cysteine catabolism. The active site coordination of CDO comprises a mononuclear iron ligated by the Nepsilon atoms of three protein-derived histidines, thus representing a new variant on the 2-histidine-1-carboxylate (2H1C) facial triad motif. Nitric oxide was used as a spectroscopic probe in investigating the order of substrate-O2 binding by EPR spectroscopy. In these experiments, CDO exhibits an ordered binding of l-cysteine prior to NO (and presumably O2) similar to that observed for the 2H1C class of non-heme iron enzymes. Moreover, the CDO active site is essentially unreactive toward NO in the absence of substrate, suggesting an obligate ordered binding of l-cysteine prior to NO. Typically, addition of NO to a mononuclear non-heme iron center results in the formation of an {FeNO}7 (S = 3/2) species characterized by an axial EPR spectrum with gx, gy, and gz values of approximately 4, approximately 4, and approximately 2, respectively. However, upon addition of NO to CDO in the presence of substrate l-cysteine, a low-spin {FeNO}7 (S = 1/2) signal that accounts for approximately 85% of the iron within the enzyme develops. Similar {FeNO}7 (S = 1/2) EPR signals have been observed for a variety of octahedral mononuclear iron-nitrosyl synthetic complexes; however, this type of iron-nitrosyl species is not commonly observed for non-heme iron enzymes. Substitution of l-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol did not produce any analogous {FeNO}7 signals (S = 1/2 or 3/2), thus reflecting the high substrate specificity of the enzyme observed by a number of researchers. The unusual {FeNO}7 (S = 1/2) electronic configuration adopted by the substrate-bound iron-nitrosyl CDO (termed {ES-NO}7) is a result of the bidentate thiol/amine coordination of l-cysteine in the NO-bound CDO active site. DFT computations were performed to further characterize this species. The DFT-predicted geometric parameters for {ES-NO}7 are in good agreement with the crystallographically determined substrate-bound active site configuration of CDO and are consistent with known iron-nitrosyl model complexes. Moreover, the computed EPR parameters (g and A values) are in excellent agreement with experimental results for this CDO species and those obtained from comparable synthetic {FeNO}7 (S = 1/2) iron-nitrosyl complexes.

Glutamyl-prolyl tRNA synthetase (EPRS) is a component of the heterotetrameric gamma-interferon-activated inhibitor of translation (GAIT) complex that binds 3'UTR GAIT elements in multiple interferon-gamma (IFN-gamma)-inducible mRNAs and suppresses their translation. Here, we elucidate the specific EPRS phosphorylation events that regulate GAIT-mediated gene silencing. IFN-gamma induces sequential phosphorylation of Ser(886) and Ser(999) in the noncatalytic linker connecting the synthetase cores. Phosphorylation of both sites is essential for EPRS release from the parent tRNA multisynthetase complex. Ser(886) phosphorylation is required for the interaction of NSAP1, which blocks EPRS binding to target mRNAs. The same phosphorylation event induces subsequent binding of ribosomal protein L13a and GAPDH and restores mRNA binding. Finally, Ser(999) phosphorylation directs the formation of a functional GAIT complex that binds initiation factor eIF4G and represses translation. Thus, two-site phosphorylation provides structural and functional pliability to EPRS and choreographs the repertoire of activities that regulates inflammatory gene expression.

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.

Melanin synthesis of follicular melanocytes is strictly coupled to the growth stage of the hair cycle (anagen), ceases during follicle regression (catagen), and is absent throughout the resting stage (telogen). Having previously characterized the expression and activity of melanogenesis-related proteins during the telogen-anagen transition of the murine hair cycle (JID 96:172, 1991), we here report a biophysical and biochemical analysis of follicular melanogenesis during the anagen-catagen-telogen transformation of the C57 BL-6 mouse hair cycle. Tyrosinase activity and concentration as well as dopachrome tautomerase activity were compared with melanin synthesis, as measured by electron paramagnetic resonance spectroscopy (EPR). The visible changes in skin color and the histologically appreciable switch-off of melanin formation during the anagen-catagen transformation were accompanied by a steep decline in 1) the melanin-associated EPR signal of full-thickness mouse skin, 2) tyrosinase and dopachrome tautomerase activities, and 3) the skin concentration of 80-85-kD melanogenesis related protein and 66-68-kD tyrosinase protein. Telogen skin displayed a minimum of the EPR amplitude as well as of tyrosinase and dopachrome tautomerase activity detected. By EPR, only eumelanin was identified during all hair cycle stages. The gradual switch-off of melanogenesis during anagen VI started with an unexpectedly early decline of the EPR melanin signal, followed by dopachrome tautomerase activity and the concentration of 80-85-kD melanogenesis related protein. The initiation of catagen was characterized by a significant and rapid decrease in activity and concentration of tyrosinase, and was accompanied by a second drop in dopachrome tautomerase activity. Together, these biochemical and biophysical parameters of follicular melanogenesis serve as novel and differential markers for the imminent termination of anagen and the development of catagen. They also show that the switch-off of melanogenesis during the anagen-catagen-telogen transition is a stochastic process commencing already in mid anagen VI.

Gender is a profound determinant of aging and lifespan, but little is known about gender differences in free radical homeostasis. Free radicals are proposed as key elements in the multifactorial process of aging and it is predicted that the longer-lived gender should have lower levels of oxidative stress. While the majority of studies on aging have included a single gender, recent studies in rats compared genders and found that females, the longer-lived sex, had lower oxidative stress and mitochondrial dysfunction than males. We explored the association between oxidative stress and gender-specific aging in C57BL6 mice, in which females are the shorter-lived gender. Reactive oxygen species (ROS) were measured in young and old mice by confocal imaging of dihydroethidium (DHE) oxidation in the brain, and by electron paramagnetic resonance (EPR) spectrometry of isolated brain mitochondria. Both genders exhibited significant age-dependent increases in ROS. However, females had a greater increase with age than males in DHE oxidation but not mitochondrial EPR. Superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (GPx1) protein levels were lower in old females. To determine whether enhancing antioxidant defenses would eliminate gender differences in lifespan, mice were treated chronically with a superoxide dismutase mimetic. Treatment blocked the age-dependent increase in ROS, with a greater effect in females on DHE oxidation, but not mitochondrial EPR. Treatment also increased lifespan to a greater degree in females. Our results indicate that differences in ROS homeostasis contribute to gender divergence in survival, but also suggest that mitochondrial superoxide production may not be primarily responsible for gender differences in lifespan.

Molecular dynamics in spin-labeled muscle proteins was studied with a recently developed electron paramagnetic resonance (EPR) technique, saturation transfer spectroscopy, which is uniquely sensitive to rotational motion in the range of 10(-7)-10(-3) sec. Rotational correlation time (tau2) were determined for a spin label analog of iodoacetamide bound to the subfragment-1 (S-1) region of myosin under a variety of conditions likely to shed light on the molecular mechanism of muscle contraction. Results show that (a) the spin labels are rigidly bound to the isolated S-1 (tau2 = 2 x 10(-7) sec) and can be used to estimate values of tau2 for the S-1 region of the myosin molecule; (b) in solutions of intact myosin, S-1 has considerable mobility relative to the rest of the myosin molecule, the value of tau2 for the S-1 segment of myosin being less than twice that for isolated S-1, while the molecular weights differ by a factor of 4 to 5; (c) in myosin filaments, tau2 increases by a factor of only about 10, suggesting motion of the S-1 regions independent of the backbone of the myosin filament, but slower than that in a single molecule; (d) addition of F-actin to solutions of myosin or S-1 increases tau2 by a factor of nearly 10(3), indicating strong immobilization of S-1 upon binding to actin. Saturation transfer spectroscopy promises to provide an extremely useful tool for the study of the motions of the crossbridges and thin filaments in reconstituted systems and in glycerinated muscle fibers.

The development and use of in vivo techniques for strictly experimental applications in animals has been very successful, and these results now have made possible some very attractive potential clinical applications. The area with the most obvious immediate, effective and widespread clinical use is oximetry, where EPR almost uniquely can make repeated and accurate measurements of pO2 in tissues. Such measurements can provide clinicians with information that can impact directly on diagnosis and therapy, especially for oncology, peripheral vascular disease and wound healing. The other area of immediate and timely importance is the unique ability of in vivo EPR to measure clinically significant exposures to ionizing radiation 'after-the-fact', such as may occur due to accidents, terrorism or nuclear war. There are a number of other capabilities of in vivo EPR that also potentially could become extensively used in human subjects. In pharmacology the unique capabilities of in vivo EPR to detect and characterize free radicals could be applied to measure free radical intermediates from drugs and oxidative process. A closely related area of potential widespread applications is the use of EPR to measure nitric oxide. These often unique capabilities, combined with the sensitivity of EPR spectra to the immediate environment (e.g. pH, molecular motion, charge) have already resulted in some very productive applications in animals and these are likely to expand substantially in the near future. They should provide a continually developing base for extending clinical uses of in vivo EPR. The challenges for achieving full implementation include adapting the spectrometer for safe and comfortable measurements in human subjects, achieving sufficient sensitivity for measurements at the sites of the pathophysiological processes that are being measured, and establishing a consensus on the clinical value of the measurements.

Dose regimens of anticancer agents are usually designed on the basis of the maximum tolerable drug doses, and toxicity prevents drug usage at higher doses, even though the drugs may be more effective at the higher doses. We previously studied macromolecular anticancer drugs, i.e. those larger than 40 kDa, and observed their accelerated accumulation in tumors. Their concentration in tumors was more than 5-100-fold their blood concentration because of the enhanced permeability and retention (EPR) effect. Here, we report that the EPR effect was enhanced by applying nitroglycerin (NG) ointment on the skin of tumor-bearing animals. Tumors studied included breast cancer, which was induced in Sprague-Dawley rats by the chemical carcinogen 7,12-dimethylbenz[a]anthracene, and three different transplanted tumor models in mice. NG was applied on tumor or nontumorous normal skin as well. Two to three times more putative macromolecular drug (an Evans blue/albumin complex) was delivered to solid tumors with NG than without NG. We also demonstrated that NG enhanced tumor delivery with another macromolecular drug candidate, PZP, i.e. polyethylene glycol-conjugated zinc protoporphyrin IX, which inhibits heme oxygenase-1. In addition, we investigated the therapeutic effect of NG using a combination with low molecular weight anthracycline or high molecular weight PZP in mouse tumor models. NG had no apparent toxicity at the doses used, and showed significantly increased therapeutic effects in both cases. Regardless of its site of application, NG thus enhanced the delivery of the drug to tumors, and enhanced therapeutic effects.

A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of the Synechocystis sp. PCC 6803 gene slr2034 and Arabidopsis thaliana HCF136, and the psbEFLJ operon, was cloned from the cyanobacterium Synechococcus sp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type photosystem (PS) II levels. Inactivation of the rubA gene of Synechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was approximately 80% of the wild-type level. RubA contains a domain of approximately 50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane alpha-helix at its C terminus. Overproduction of the water-soluble rubredoxin domain in Escherichia coli led to a product with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type ( approximately 40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced, and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis of PS I.

By application of microsecond light flashes the oxygen-evolving complex (OEC) was driven through its functional cycle, the S-state cycle. The S-state population distribution obtained by the application of n flashes (n = 0. 6) was determined by analysis of EPR spectra; Mn K-edge X-ray absorption spectra were collected. Taking into consideration the likely statistical error in the data and the variability stemming from the use of three different approaches for the determination of edge positions, we obtained an upshift of the edge position by 0.8-1.5, 0.5-0.9, and 0.6-1.3 eV for the S0-S1, S1-S2, and S2-S3 transitions, respectively, and a downshift by 2.3-3.1 eV for the S3-S0 transition. These results are highly suggestive of Mn oxidation state changes for all four S-state transitions. In the S0-state spectrum, a clearly resolved shoulder in the X-ray spectrum around 6555 eV points toward the presence of Mn(II). We propose that photosynthetic oxygen evolution involves cycling of the photosystem II manganese complex through four distinct oxidation states of this tetranuclear complex: Mn(II)-Mn(III)-Mn(IV)2 in the S0-state, Mn(III)2-Mn(IV)2 in the S1-state, Mn(III)1-Mn(IV)3 in the S2-state, and Mn(IV)4 in the S3-state.

Originally thought of as simply damaging or toxic "accidents" of in vivo chemistry, free radicals are becoming increasingly recognized as redox signaling molecules implicit in cellular homeostasis. Indeed, at the vascular level, it is plausible that oxidative stress plays a regulatory role in normal vascular function. Using electron paramagnetic resonance (EPR) spectroscopy, we sought to document the ability of an oral antioxidant cocktail (vitamins C, E, and alpha-lipoic acid) to reduce circulating free radicals, and we employed Doppler ultrasound to examine the consequence of an antioxidant-mediated reduction in oxidative stress on exercise-induced vasodilation. A total of 25 young (18-31 yr) healthy male subjects partook in these studies. EPR spectroscopy revealed a reduction in circulating free radicals following antioxidant administration at rest ( approximately 98%) and as a consequence of exercise ( approximately 85%). Plasma total antioxidant capacity and vitamin C both increased following the ingestion of the antioxidant cocktail, whereas vitamin E levels were not influenced by the ingestion of the antioxidants. Brachial artery vasodilation during submaximal forearm handgrip exercise was greater with the placebo (7.4 +/- 1.8%) than with the antioxidant cocktail (2.3 +/- 0.7%). These data document the efficacy of an oral antioxidant cocktail in reducing free radicals and suggest that, in a healthy state, the aggressive disruption of the delicate balance between pro- and antioxidant forces can negatively impact vascular function. These findings implicate an exercise-induced reliance upon pro-oxidant-stimulated vasodilation, thereby revealing an important and positive vascular role for free radicals.

The major DNA photoproduct of dormant, UV-irradiated Bacillus subtilis spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)]. During spore germination, SP is reversed to two intact thymines in situ by the DNA repair enzyme SP lyase, an S-adenosylmethionine (S-AdoMet)-dependent iron-sulfur ([Fe-S]) protein encoded by the splB gene. In the present work, cross-linking, SDS/PAGE, and size exclusion chromatography revealed that SplB protein dimerized when incubated with iron and sulfide under anaerobic reducing conditions. SplB isolated under aerobic conditions generated an EPR spectrum consistent with that of a partially degraded [3Fe-4S] center, and reduction of SplB with dithionite shifted the spectrum to that of a [4Fe-4S] center. Addition of S-AdoMet to SplB converted some of the [4Fe-4S] centers to an EPR-silent form consistent with electron donation to S-AdoMet. HPLC and electrospray ionization MS analyses showed that SP lyase cleaved S-AdoMet to generate 5'-deoxyadenosine. The results indicate that (i) SP lyase is a homodimer of SplB; (ii) dimer formation is coordinated by a [4Fe-4S] center; and (iii) the reduced [4Fe-4S] center is capable of donating electrons to S-AdoMet to generate a 5'-adenosyl radical that is then used for the in situ reversal of SP. Thus, SP lyase belongs to the "radical SAM" superfamily of enzymes that use [Fe-S] centers and S-AdoMet to generate adenosyl radicals to effect catalysis. SP lyase is unique in being the first and only DNA repair enzyme known to function via this novel enzymatic mechanism.

We propose a molecular mechanism of force generation in muscle, based primarily on site-specific spectroscopic probe studies of myosin heads in contracting muscle fibers and myofibrils. Electron paramagnetic resonance (EPR) and time-resolved phosphorescence anisotropy (TPA) of probes attached to SH1 (Cys 707, in the catalytic domain of the head) have consistently shown that most myosin heads in contracting muscle are dynamically disordered, undergoing large-amplitude rotations in the microsecond time range. Some of these disordered heads are bound to actin, especially in the early (weak-binding, preforce) phase of the ATPase cycle. The small ordered population (10-20%) is rigidly oriented precisely as in rigor, with no other distinct angle observed in contraction or in the presence of intermediate states trapped by nucleotide analogs. These results are not consistent with the classical model in which the entire head undergoes a 45 degree transition between two distinct orientations. Therefore, it has been proposed that the catalytic domain of the myosin head has only one stereospecific (rigor-like) actin-binding angle, and that the head's internal structure changes during force generation, causing the distal light-chain-binding domain to rotate. To test this model, we have performed EPR and TPA studies of probes attached to regulatory light chains (RLCs) in rabbit and scallop myofibrils and fibers. The RLC results confirm the predominance of dynamic (microsecond) rotational disorder in both relaxation and contraction, and show that the different mechanisms of calcium regulation in the two muscles produce different rotational dynamics. In rabbit myofibrils, RLC probes are more dynamically disordered than SH1 probes, especially in rigor and contraction,indicating that the light-chain-binding domain undergoes rotational motions relative to the catalytic domain when myosin heads interact with actin. An SH1-bound spin label, which is sensitive to myosin's internal dynamics, resolves three distinct conformations during contraction, and time-resolved EPR shows that these transitions are coupled to specific steps in the ATPase cycle. We propose that force is generated during contraction by a disorder-to-order transition, in which myosin heads first attach weakly to actin in a nonstereospecific mode characterized by large-scale dynamic disorder, then undergo at least two conformational transitions involving large-scale structural (rotational) changes within the head, culminating in a highly ordered strong-binding state that bears force.

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.

Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducer Desulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at approximately 2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 +/- 15 mV for the quinone/semiquinone couple and at -130 +/- 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is -350 +/- 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.

Microwave driven dynamic nuclear polarization (DNP) is a process in which the large polarization present in an electron spin reservoir is transferred to nuclei, thereby enhancing NMR signal intensities. In solid dielectrics there are three mechanisms that mediate this transfer--the solid effect (SE), the cross effect (CE), and thermal mixing (TM). Historically these mechanisms have been discussed theoretically using thermodynamic parameters and average spin interactions. However, the SE and the CE can also be modeled quantum mechanically with a system consisting of a small number of spins and the results provide a foundation for the calculations involving TM. In the case of the SE, a single electron-nuclear spin pair is sufficient to explain the polarization mechanism, while the CE requires participation of two electrons and a nuclear spin, and can be used to understand the improved DNP enhancements observed using biradical polarizing agents. Calculations establish the relations among the electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) frequencies and the microwave irradiation frequency that must be satisfied for polarization transfer via the SE or the CE. In particular, if δ, Δ < ω(0I), where δ and Δ are the homogeneous linewidth and inhomogeneous breadth of the EPR spectrum, respectively, we verify that the SE occurs when ω(M) = ω(0S) ± ω(0I), where ω(M), ω(0S) and ω(0I) are, respectively, the microwave, and the EPR and NMR frequencies. Alternatively, when Δ>> ω(0I)>> δ, the CE dominates the polarization transfer. This two-electron process is optimized when ω(0S(1))-ω(0S(2)) = ω(0I) and ω(M)~ω(0S(1)) or ω(0S(2)), where ω(0S(1)) and ω(0S(2)) are the EPR Larmor frequencies of the two electrons. Using these matching conditions, we calculate the evolution of the density operator from electron Zeeman order to nuclear Zeeman order for both the SE and the CE. The results provide insights into the influence of the microwave irradiation field, the external magnetic field, and the electron-electron and electron-nuclear interactions on DNP enhancements.

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo.