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Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Ru61a and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the [alpha]/[beta]-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1-H-3-hydroxy-4-oxoquinaldine substrate, its N-acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical [alpha]/[beta]-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The 'oxyanion hole' of the [alpha]/[beta]-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions. oxygenase | oxygen chemistry | structural enzymology www.pn as.org/cgi/doi/10.1073/pnas.0909033107

In enteric bacteria, the cellular response to oxidative stress is activated by oxidation of the iron-sulfur clusters in SoxR, which then induces transcription of soxS, turning on a battery of defense genes. Here we demonstrate both in vitro and in cells that activation of SoxR can occur in a DNA-mediated reaction with guanine radicals, an early genomic signal of oxidative stress, serving as the oxidant. SoxR in its reduced form is found to inhibit guanine damage by repairing guanine radicals. Moreover, cells treated with a DNA-binding photooxidant, which generates guanine radicals, promotes the expression of soxS. In vitro, this photooxidant, tethered to DNA 80 bp from the soxS promoter, induces transcription by activating SoxR upon irradiation. Thus, transcription can be activated from a distance through DNA-mediated charge transport. This chemistry offers a general strategy for DNA-mediated signaling of oxidative stress. DNA charge transport | guanine radicals | oxidative stress

Typical 2-Cys peroxiredoxins (Prxs) are ubiquitous peroxidases that are involved in peroxide scavenging and/or the regulation of peroxide signaling in eukaryotes. Despite their prevalence, very few Prxs have been reliably characterized in terms of their substrate specificity profile and redox potential even though these values are important for gaining insight into physiological function. Here, we present such studies focusing on Salmonella typhimurium alkyl hydroperoxide reductase C component (StAhpC), an enzyme that has proven to be an excellent prototype of this largest and most widespread class of Prxs that includes mammalian Prx I-Prx IV. The catalytic efficiencies of StAhpC ([k.sub.cat]/[K.sub.m]) are >[10.sup.7] [M.sup.-1] x [s.sup.-1] for inorganic and primary hydroperoxide substrates and [approximately equal to] 100-fold less for tertiary hydroperoxides, with the difference being exclusively caused by changes in [K.sub.m]. The oxidative inactivation of AhpC through reaction with a second molecule of peroxide shows parallel substrate specificity. The midpoint reduction potential of StAhpC is determined to be -178 [+ or -] 0.4 mV, a value much higher than most other thiol-based redox proteins. The relevance of these results for our understanding of Prx and the physiological role of StAhpC is discussed. antioxidants | peroxidases | redox regulation | redox-active disulfide

Yeast cytokinesis entails a rejuvenation process by which the aged mother cell generates daughter cells enjoying full replicative potential. Here we show that this process includes a precipitous reduction in the levels of reactive oxygen species in the progeny immediately after completion of cytokinesis. The reduction in hydrogen peroxide is the result of a Sir2p and actin cytoskeleton-dependent segregation of the cytosolic catalase Ctt1p such that the daughter cell receives a higher load of undamaged and active Ctt1p than the progenitor cell. Such spatial quality control provides the daughter cells with a superior capacity to combat external oxidative stress and delays self-inflicted oxidative damage to their cellular proteins. aging | catalase | damage segregation | rejuvenation

Helicobacter pylori elicits an oxidative stress during host colonization. This oxidative stress is known to cause lesions in the host DNA. Here we addressed the question as to whether the pathogen DNA is subject to lethal or mutational damage by the host-generated oxidative response. H. pylori Hpnth mutants unable to repair oxidized pyrimidines from the bacterial DNA were generated. H. pylori strains lacking a functional endonuclease III (HpNth) showed elevated spontaneous and induced mutation rates and were more sensitive than the parental strain to killing by exposure to oxidative agents or activated macrophages. Although under laboratory conditions the Hpnth mutant strain grows as well as the wild-type strain, in a mouse infection the stomach bacterial load gradually decreases while the population in the wild-type strain remains stable, showing that endonuclease III deficiency reduces the colonization capacity of the pathogen. In coinfection experiments with a wild-type strain, Hpnth cells are eradicated 15 days postinfection (p.i.) even when inoculated in a 1:9 wild-type:mutant strain ratio, revealing mutagenic lesions that are counterselected under competition conditions. These results show that the host effectively induces lethal and premutagenic oxidative DNA adducts on the H. pylori genome. The possible consequences of these DNA lesions on the adaptability of H. pylori strains to new hosts are discussed.

Mismatch repair (MMR) proteins contribute to genome integrity by correcting replication errors. In higher eukaryotes, MMR proteins also regulate the cellular response to DNA lesions such as oxidized, alkylated, or crosslinked bases. Previous studies have linked MMR proteins to the activation of apoptosis through p53-dependent and p53-independent mechanisms. MMR-deficient cells exhibit variable defects in the induction of p53 and its related p73, which are activators of apoptosis. However, the specific role of each MMR protein in the regulation of apoptosis has not been determined. Here, we describe an interaction between PMS2, an MMR protein, and p73. This interaction causes the stabilization of p73 and the redistribution of PMS2 to the nuclear compartment. Exposure to cisplatin enhances the association between PMS2 and p73. Moreover, stimulation of the p73 proapoptotic function by cisplatin requires PMS2. These results suggest that PMS2 contributes to genome integrity not only through DNA repair but also by enhancing DNA damage-induced apoptosis.

Recent work in our laboratory showed that products formed by the antibody-catalyzed water-oxidation pathway can kill bacteria. Dihydrogen peroxide, the end product of this pathway, was found to be necessary, but not sufficient, for the observed efficiency of bacterial killing. The search for further bactericidal agents that might be formed along the pathway led to the recognition of an oxidant that, in its interaction with chemical probes, showed the chemical signature of ozone. Here we report that the antibody-catalyzed water-oxidation process is capable of regioselectively converting antibody-bound benzoic acid into para-hydroxy benzoic acid as well as regioselectively hydroxylating the 4-position of the phenyl ring of a single tryptophan residue located in the antibody molecule. We view the occurrence of these highly selective chemical reactions as evidence for the formation of a short-lived hydroxylating radical species within the antibody molecule. In line with our previously presented hypothesis according to which the singlet-oxygen ([sup.1][O.sup.*.sub.2]) induced antibody-catalyzed wateroxidation pathways proceeds via the formation of dihydrogen trioxide ([H.sub.2][O.sub.3]), we now consider the possibility that the hydroxylating species might be the hydrotrioxy radical H[O.sup.*.sub.3], and we point to the remarkable potential of this either [H.sub.2][O.sub.3]- or [O.sub.3]-derivable species to act as a masked hydroxyl radical (H[O.sup.*]) in a biological environment.

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and L-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo. Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDL[R.sup.-/-]) mice showed increased activities of redox-transcription factors (ELK-1, p-Jun, and p-CREB) and decreased expression of eNOS. Intervention with antioxidants and L-arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo. These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and L-arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations. eNOS | ELK-1 | p-CREB | p-Jun | vascular disease

Photosystem II (PSII) catalyzes the light-driven oxidation of water and the reduction of plastoquinone; the oxidation of water occurs at a cluster of four manganese. The PSII CP43 subunit functions in light harvesting, and mutations in the fifth luminal loop (E) of CP43 have established its importance in PSII structure and/or assembly [Kuhn, M. G. & Vermaas, V. F. J. (1993) Plant Mol. Biol. 23, 123-133]. The sequence [A.sup.350]PWLEPL[R.sup.357] in luminal loop E is conserved in CP43 genes from 50 organisms. To map important posttranslational modifications in this sequence, tandem mass spectrometry (MS/ MS) was used. These data show that the indole side chain of Trp-352 is posttranslationally modified to give mass shifts of +4, +16, and +18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (+4), a keto-/ amino-/hydroxy- (+16) derivative, and a dihydro-hydroxy- (+18) derivative of the indole side chain. Peptide synthesis and MS/MS confirmed the kynurenine assignment. The +16 and +18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The site-directed mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics. mass spectrometry | collision-induced dissociation | tryptophan | kynurenine | photoinhibition

The cancer chemopreventive properties of selenium compounds are well documented, yet little is known of the mechanism(s) by which these agents inhibit carcinogenesis. We show that selenium in the form of selenomethionine (SeMet) can activate the p53 tumor suppressor protein by a redox mechanism that requires the redox factor Ref1. Assays to measure direct reduction/oxidation of p53 showed a SeMet-dependent response that was blocked by a dominant-negative Ref 1. By using a peptide containing only p53 cysteine residues 275 and 277, we demonstrate the importance of these residues in the SeMet-induced response. SeMet induced sequence-specific DNA binding and transactivation by p53. Finally, cellular responses to SeMet were determined in mouse embryo fibroblasts wild-type or null for p53 genes. The evidence suggests that the DNA repair branch of the p53 pathway was activated. The central relevance of DNA repair to cancer prevention is discussed. cancer chemoprevention | selenium | APE/Ref 1 | p53 tumor suppressor gene | DNA repair

C75, a known inhibitor of fatty acid synthase is postulated to cause significant weight loss through decreased hypothalamic neuropeptide Y (NPY) production. Peripherally, C75, an [alpha]-methylene-[gamma]-butyrolactone, reduces adipose tissue and fatty liver, despite high levels of malonyl-CoA. To investigate this paradox, we studied the effect of C75 on fatty acid oxidation and energy production in diet-induced obese (DIO) mice and cellular models. Whole-animal calorimetry showed that C75-treated DIO mice had a 50% greater weight loss, and a 32.9% increased production of energy because of fatty acid oxidation, compared with paired-fed controls. Etomoxir, an inhibitor of carnitine O-palmitoyltransferase-1 (CPT-1), reversed the increased energy expenditure in DIO mice by inhibiting fatty acid oxidation. C75 treatment of rodent adipocytes and hepatocytes and human breast cancer cells increased fatty acid oxidation and ATP levels by increasing CPT-1 activity, even in the presence of elevated concentrations of malonyl-CoA. Studies in human cancer cells showed that C75 competed with malonyl-CoA, as measured by CPT-1 activity assays. Thus, C75 acts both centrally to reduce food intake and peripherally to increase fatty acid oxidation, leading to rapid and profound weight loss, loss of adipose mass, and resolution of fatty liver. The pharmacological stimulation of CPT-1 activity is a novel finding. The dual action of the C75 class of compounds as fatty acid synthase inhibitors and CPT-1 agonists has therapeutic implications in the treatment of obesity and type II diabetes.

Larvae of Tyria jacobaeae feed solely upon the pyrrolizidine alkaloid-containing plant Senecio jacobaea. Ingested pyrrolizidine alkaloids (PAs), which are toxic to unspecialized insects and vertebrates, are efficiently N-oxidized in the hemolymph of T. jacobaeae by senecionine N-oxygenase (SNO), a flavin-dependent monooxygenase (FMO) with a high substrate specificity for PAs. Peptide microsequences obtained from purified T. jacobaeae SNO were used to clone the corresponding cDNA, which was expressed in active form in Escherichia coli. T. jacobaeae SNO possesses a signal peptide characteristic of extracellular proteins, and it belongs to a large family of mainly FMO-like sequences of mostly unknown function, including two predicted Drosophila melanogaster gene products. The data indicate that the gene for T. jacobaeae SNO, highly specific for toxic pyrrolizidine alkaloids, was recruited from a preexisting insect-specific FMO gene family of hitherto unknown function. The enzyme allows the larvae to feed on PA-containing plants and to accumulate predation-deterrent PAs in the hemolymph. N-oxidation | alkaloid sequestering insects | insect adaptation | chemical defense | gene recruitment

N-acetylcysteine (NAC), a strong antioxidant, has antigenotoxic and anticarcinogenic properties currently being investigated in clinical trials. NAC detoxifies free radicals (e.g., the hydroxyl radical, *OH) that cause DNA changes implicated in disease (e.g., cancer). The *OH reacts with purines to form mutagenic 8-hydroxypurine (8-OH) and putatively nonmutagenic formamidopyrimidine (Fapy) lesions. Fapy lesions inhibit DNA synthesis likely modulating the mutagenic potential of the 8-OH lesions, which would suggest that the ratio of these oxidized bases is biologically important. However, little is known about how NAC modifies oxidized DNA structure or how such modifications may affect cellular processes, such as replication and transcription. By using gas chromatography-mass spectrometry and Fourier transform-infrared spectroscopy, we found that dietary NAC (5% in the diet for 14 days) affected *OH-induced structural changes in DNA of the hind leg of the BALB/c mouse. For example, mutagenic 8-hydroxyguanine (8-OH-Gua) was reduced [approximately equal to] 50% (P = 0.02) in mice fed NAC compared with controls. NAC reduced the [log.sub.10] (8-OH-Gua/FapyGua) ratio from 0.58 [+ or -] 0.15 to essentially zero, a virtually neutral redox status. DNA from control mice had a remarkably high variance compared with mice fed NAC. Moreover, the DNA from treated and control mice was distinct with respect to base structure and vertical base-stacking interactions. The findings showing that NAC lowered the concentration of 8-OH-Gua, the log ratio, and the variance (previously associated with neoplastic changes) suggest that NAC reduces the mutagenic potential of oxidized DNA. These benefits could be offset by the other structural changes found after NAC exposure, which may affect the fidelity of DNA synthesis. DNA damage | free radicals | N-acetylcysteine | oxidation | redox

Nitric oxide (N[O.sup.*]) is a short-lived physiological messenger. Its various biological activities can be preserved in a more stable form of S-nitrosothiols (RS-NO). Here we demonstrate that at physiological N[O.sup.*] concentrations, plasma albumin becomes saturated with N[O.sup.*] and accelerates formation of low-molecular-weight (LMW) RS-NO in vitro and in vivo. The mechanism involves micellar catalysis of N[O.sup.*] oxidation in the albumin hydrophobic core and specific transfer of N[O.sup.+] to LMW thiols. Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating LMW thiols. Results suggest that the hydrophobic phase formed by albumin serves as a major reservoir of N[O.sub.*] and its reactive oxides and controls the dynamics of N[O.sup.*]-dependant processes in the vasculature.

Cumulative oxidative damages to cell constituents are considered to contribute to aging and age-related diseases. The enzyme peptide methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized methionine in proteins by reducing methionine sulfoxide back to methionine. However, whether MSRA plays a role in the aging process is poorly understood. Here we report that overexpression of the msrA gene predominantly in the nervous system markedly extends the lifespan of the fruit fly Drosophila. The MSRA transgenic animals are more resistant to paraquat-induced oxidative stress, and the onset of senescence-induced decline in the general activity level and reproductive capacity is delayed markedly. The results suggest that oxidative damage is an important determinant of lifespan, and MSRA may be important in increasing the lifespan in other organisms including humans.

We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-L-carnitine (ALCAR) and/or the mitochondrial antioxidant precursor R-[alpha]-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction.

Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2[e.sup.-]. Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [[sup.14]C]ADP to [[sup.14]C]ATP, which was [approximately equal to] 15 % of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR ([alpha][beta][gamma][delta]-composition) autophosphorylated its [gamma]-subunit when incubated with [[[gamma]-.sup.32]P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionitereduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.

Vestimentiferan tubeworms, symbiotic with sulfur-oxidizing chemoautotrophic bacteria, dominate many cold-seep sites in the Gulf of Mexico. The most abundant vestimentiferan species at these sites, Lamellibrachia cf. luymesi, grows quite slowly to lengths exceeding 2 meters and lives in excess of 170-250 years. L. cf. luymesi can grow a posterior extension of its tube and tissue, termed a 'root,' down into sulfidic sediments below its point of original attachment. This extension can be longer than the anterior portion of the animal. Here we show, using methods optimized for detection of hydrogen sulfide down to 0.1 [micro]M in seawater, that hydrogen sulfide was never detected around the plumes of large cold-seep vestimentiferans and rarely detectable only around the bases of mature aggregations. Respiration experiments, which exposed the root portions of L. cf. luymesi to sulfide concentrations between 51-561 [micro]M, demonstrate that L. cf. luymesi use their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 [micro]mol*[g.sup.-1]*[h.sup.-1]. Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior (root) portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan L. cf. luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake.

Oxidized low density lipoprotein (LDL) is a key factor in the pathogenesis of atherosclerosis and its thrombotic complications, such as stroke and myocardial infarction. It activates endothelial cells and platelets through mechanisms that are largely unknown. Here, we show that lysophosphatidic acid (LPA) was formed during mild oxidation of LDL and was the active compound in mildly oxidized LDL and minimally modified LDL, initiating platelet activation and stimulating endothelial cell stress-fiber and gap formation. Antagonists of the LPA receptor prevented platelet and endothelial cell activation by mildly oxidized LDL. We also found that LPA accumulated in and was the primary platelet-activating lipid of atherosclerotic plaques. Notably, the amount of LPA within the human carotid atherosclerotic lesion was highest in the lipid-rich core, the region most thrombogenic and most prone to rupture. Given the potent biological activity of LPA on platelets and on cells of the vessel wall, our study identifies LPA as an atherothrombogenic molecule and suggests a possible strategy to prevent and treat atherosclerosis and cardiocerebrovascular diseases.

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide [Mathematical Expression Omitted] to [O.sub.2] and [H.sub.2][O.sub.2]. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of [H.sub.2][O.sub.], leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of L-[Alpha]-lecithin liposomes was enhanced greatly during the SOD/[H.sub.2][O.sub.2] reaction in the presence of nitrite anion (N[O.sub.[2.sup.-]]) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of N[O.sub.[2.sup.-]] also greatly enhanced [Alpha]-tocophenol ([Alpha]-TH) oxidation by SOD/[H.sub.2][O.sub.2] in saturated 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was [Alpha]-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing [Gamma]-to-copherol ([Gamma]-TH) were incubated with SOD/[H.sub.2][O.sub.2]/N[O.sub.[2.sup.-]], the major product identified was 5-N[O.sub.2]-[Gamma]-TH. Nitrone spin traps significantly inhibited the formation of [Alpha]-tocopheryl quinone and 5-N[O.sub.2]-[Gamma]-TH. N[O.sub.[2.sup.-]] inhibited [H.sub.2][O.sub.2]-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of N[O.sub.[2.sup.-]] by an SOD-bound oxidant to the nitrogen dioxide radical [Mathematical Expression Omitted]. In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that N[O.sub.[2.sup.-]] is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.

Oxidants arising from mitochondria lead to many age-related oxidative lesions. Inherent proton leakage rate across the inner mitochondrial membrane, reduced membrane fluidity and reduced cardiolipin levels and function lead to the failure of many mitochondrial functions with age. Acetyl-L-carnitine, a high-energy mitochondrial substrate, corrects many of the cellular functional defects due to age, by increasing cellular ATP production. Oxidative damage-induced, age-related mitochondrial defects may mediate cellular, tissue, and organismal aging.

Fast photoinduced long range electron transfer occurs between metal complexes that are intercalators of DNA. A comparison of donors and acceptors that bind to DNA through intercalative or nonintercalative modes using noncovalently bound species is done. Base stacking mechanics regulate electronic coupling through the double helix, which helps in intramolecular electron transfer.

Studies on plant protoplasts reveal that high oxidizing sulfhydryl/disulfide redox potentials may limit the cotranslational and/or early post translational steps leading to low total accumulation of cystein-rich proteins of bacterial origin. The addition of dithiothereitol increases the amount of cystein-rich protein and enhances the activity of neomycin phosphotransferase II but not of phosphinothricin acetyltransferase.

The blood plasma from healthy, fasting donors were analyzed for the presence and distribution oflipid hyperperoxides (LOOHs). The results showed that low density lipoproteins (LDLs) are relatively peroxide-free. In contrast, a preferential accumulation of LOOHs were observed in high density lipoproteins (HDLs). However, the antioxidant ubiquinol-10 was found preferentially in the LDLs. These results demonstrate that the HDLs are the principle carriers of LOOHs, partly because of the antioxidant action of ubiquinol-10 in LDLs. Therefore, HDLs may have a beneficial function in the hepatic clearance of circulating oxidized lipids.

Calorie restriction (CR) and protein restriction (PR) in the diet is associated with increased lifespan and decreased incidence of carcinogenesis. Since the accumulation of oxidative damage products has also been linked with aging, an investigation was conducted to determine the effects of CR and PR on the accumulation of oxidatively damaged DNA and proteins. The results showed that irradiated rats fed on both calorie and protein restricted diets had significantly lower levels of oxidatively damaged proteins than control groups. Both aging and irradiation increased induced the accumulation of oxidatively damaged proteins.

Arachidonate 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) are required in the synthesis of leukotrienes (LTs), which mediate certain inflammatory and immunologic reactions. The genes encoding these proteins were expressed in human B and T lymphocytes. Investigations on the action of diamide and 1-chloro-2, 4-dinitrobenzene showed that these drugs inhibited the synthesis of LTB4. The mode of inhibitory action appeared to involve the activation of 5-lipoxygenase by altering the redox status of a cellular factor. However, such activation did not involve intracellular glutatione levels.

CuB site has a possible potential role in the proton pumping activity of cytochrome c oxidase. A model identical to redox-linked exchange mechanism for CuA site can be applied to CuB center as indicated by qualitative analysis. However, the small distance between CuB and cytochrome a3renders the uncoupling electron transfer pathways as facile. This argument coupled with the CuB location in the membrane contradicts the redox linkage proposal for CuB site.

Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.

The correct formation of disulfide bonds in the periplasm of Escherichia coli involves Dsb proteins, including two related periplasmic disulfide-bond isomerases, DsbC and DsbG. DsbD is a membrane protein required to maintain the functional oxidation state of DsbC and DsbG. In this work, purified proteins were used to investigate the interaction between DsbD and DsbC. A 131-residue N-terminal fragment of DsbD (DsbD[Alpha]) was expressed and purified and shown to form a functional folded domain. Gel filtration results indicate that DsbD[Alpha] is monomeric. DsbD[Alpha] was shown to interact directly with and to reduce the DsbC dimer, thus increasing the isomerase activity of DsbC. The DsbC-DsbDa complex was characterized, and formation of the complex was shown to require the N-terminal dimerization domain of DsbC. These results demonstrate that DsbD interacts directly with full-length DsbC and imply that no other periplasmic components are required to maintain DsbC in the functional reduced state.

Studies of initial activities of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum show that CODH is mostly inactive at redox potentials higher than -300 mV. Initial activities measured at a wide range of redox potentials (0-500 mV) fit a function corresponding to the Nernst equation with a midpoint potential of -316 mV. Previously, extensive EPR studies of CODH have suggested that CODH has three distinct redox states: (i) a spin-coupled state at -60 to -300 mV that gives rise to an EPR signal termed [C.sub.red1]; (ii) uncoupled states at [is less than] -320 mV in the absence of [CO.sub.2] referred to as [C.sub.unc]; and (iii) another spin-coupled state at [is less than] -320 mV in the presence of [CO.sub.2] that gives rise to an EPR signal termed [C.sub.red2B]. Because there is no initial CODH activity at potentials that give rise to [C.sub.red1], the state ([C.sub.red1]) is not involved in the catalytic mechanism of this enzyme. At potentials more positive than -380 mV, CODH recovers its full activity over time when incubated with CO. This reductant-dependent conversion of CODH from an inactive to an active form is referred to hereafter as 'autocatalysis.' Analyses of the autocatalytic activation process of CODH suggest that the autocatalysis is initiated by a small fraction of activated CODH; the small fraction of active CODH catalyzes CO oxidation and consequently lowers the redox potential of the assay system. This process is accelerated with time because of accumulation of the active enzyme.

All photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, [Q.sub.B]. This process involves the transfer of two protons from the aqueous solution to the protein-bound [Q.sub.B] molecule. The second proton, [H.sup.+](2), is supplied to [Q.sub.B] by Glu-L212, an internal residue protonated in response to formation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] In this work, the pathway for [H.sup.+](2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], studied by using pH-sensitive dyes. The other was the electron transfer [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. At pH 8.5, binding of [Zn.sup.2+], [Cd.sup.2+], or [Ni.sup.2+] reduced the rates of proton uptake upon [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] formation as well as [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] by [approximately equals] an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] is formed [10.sup.5]-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212 [right arrow] Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon [Zn.sup.2+] binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton [H.sup.+](2) was thus identified to be the same as for the first proton [H.sup.+](1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Proteins of the ERV1/ALR family are encoded by all eukaryotes and cytoplasmic DNA viruses for which substantial sequence information is available. Nevertheless, the roles of these proteins are imprecisely known. Multiple alignments of ERV1/ALR proteins indicated an invariant C-X-X-C motif, but no similarity to the thioredoxin fold was revealed by secondary structure predictions. We chose a virus model to investigate the role of these proteins as thiol oxidoreductases. When cells were infected with a mutant vaccinia virus in which the E10R gene encoding an ERV1/ALR family protein was repressed, the disulfide bonds of three other viral proteins--namely, the L1R and F9L proteins and the G4L glutaredoxin--were completely reduced. The same outcome occurred when Cys-43 or Cys-46, the putative redox cysteines of the E10R protein, was mutated to serine. These two cysteines were disulfide bonded during a normal virus infection but not if the synthesis of other viral late proteins was inhibited or the E10R protein was expressed by itself in uninfected cells, suggesting a requirement for an upstream viral thiol oxidoreductase. Remarkably, the cysteine-containing domains of the E10R and L1R viral membrane proteins and the glutaredoxin are in the cytoplasm, in which assembly of vaccinia virions occurs, rather than in the oxidizing environment of the endoplasmic reticulum. These data indicated a viral pathway of disulfide bond formation in which the E10R protein has a central role. By extension, the ERV1/ALR family may represent a ubiquitous class of cellular thiol oxidoreductases that interact with glutaredoxins or thioredoxins. poxvirus | vaccinia virus | thiol oxidoreductase | glutaredoxin

Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence [-Cys.sup.59]-Val-Asn-Val-Gly-[Cys.sup.64], identical to the active site of GR; the other was a selenenylsulfide formed from [Cys.sup.497]-Se[Cys.sup.498] and confirmed by mass spectrometry. In the NADPH reduced enzyme, these centers were present as a dithiol and a selenolthiol, respectively. Based on the structure of GR, we propose that in TrxR, the C-terminal [Cys.sup.497]-Se[Cys.sup.498] residues of one monomer are adjacent to the [Cys.sup.59] and [Cys.sup.64] residues of the second monomer. The reductive half-reaction of TrxR is similar to that of GR followed by exchange from the nascent [Cys.sup.59] and [Cys.sup.64] dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. Characterization of recombinant mutant rat TrxR with Se[Cys.sup.498] replaced by Cys having a 100-fold lower [k.sub.cat] for Trx reduction revealed the C-terminal redox center was present as a dithiol when the [Cys.sup.59]-[Cys.sup.64] was a disulfide, demonstrating that the selenium atom with its larger radius is critical for formation of the unique selenenylsulfide. Spectroscopic redox titrations with dithionite or NADPH were consistent with the structure model. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been postulated and are compatible with known enzyme activities and the effects of inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.

Cytokines and reactive oxygen intermediates (ROI) are frequent companions at sites of acute inflammation. We have shown previously that in human monocytes, bacterial lipopolysaccharide, IL-1, and tumor necrosis factor-[varies] induce a rapid down-regulation of the monocyte chemotactic protein-1 receptor CCR2 (CC chemokine receptor-2). These stimuli also induce production of ROI. In this paper, we investigate the influence of antioxidants and/or ROI on chemokine-receptor expression. In human monocytes, the antioxidant pyrrolidine dithiocarbamate (PDTC) rapidly inhibited CCR2 (95-100% of inhibition) and CCR5 (77-100% of inhibition) mRNA expression by strongly decreasing transcript stability. CCR2 half-life was decreased from 1.5 h to 45 min; CCR5 half-life was decreased from 2 h to 70 min. This inhibitory activity also included CXCR4 (CXC chemokine receptor-4) but not CXCR2 receptor and, although to a lesser extent, was shared by the antioxidants N-acetyl-L-cysteine and 2-mercaptoethanol. In contrast, the ROI-generating system xanthine/xanthine oxidase increased CCR5 and CXCR4 mRNA expression and counteracted the inhibitory effect of PDTC. Accordingly, [H.sub.2][O.sub.2] and the glutathione-depleting drug buthionine sulfoximine increased to different extents CCR2, CCR5, and CXCR4 mRNA expression. The PDTC-mediated inhibition of CCR5 and CXCR4 mRNA expression was associated with decreased chemotactic responsiveness ([is greater than] 90% inhibition) and with a marked inhibition of surface-receptor expression. In contrast, xanthine/ xanthine oxidase opposed the bacterial lipopolysaccharide- and tumor necrosis factor-[varies]-mediated inhibition of CCR5 and CXCR4 mRNA expression and increased both the CCR5 surface expression and the cell migration (3-fold) in response to macrophage inflammatory protein-1 [Beta]. These results suggest that the redox status of cells is a crucial determinant in the regulation of the chemokine system.