Self-assembling amphiphilic siderophores from marine bacteria.
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Most aerobic bacteria secrete siderophores to facilitate iron acquisition. Two families of siderophores were isolated from strains belonging to two different genera of marine bacteria. The aquachelins, from Halomonas aquamarina strain DS40M3, and the marinobactins, from Marinobacter sp. strains DS40M6 and DS40M8, each contain a unique peptidic head group that coordinates iron(III) and an appendage of one of a series of fatty acid moieties. These siderophores have low critical micelle concentrations (CMCs). In the absence of iron, the marinobactins are present as micelles at concentrations exceeding their CMC; upon addition of iron(III), the micelles undergo a spontaneous phase change to form vesicles. These observations suggest that unique iron acquisition mechanisms may have evolved in marine bacteria. (+info)
Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1.
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Heme oxygenase converts heme to biliverdin, iron, and CO in a reaction with two established intermediates, alpha-meso-hydroxyheme and verdoheme. Transient kinetic studies show that the conversion of Fe(3+)-heme to Fe(3+)-verdoheme is biphasic. Electron transfer to the heme (0.11 s(-1) at 4 degrees C and 0.49 s(-1) at 25 degrees C) followed by rapid O(2) binding yields the ferrous dioxy complex. Transfer of an electron (0.056 s(-1) at 4 degrees C and 0.21 s(-1) at 25 degrees C) to this complex triggers the formation of alpha-meso-hydroxyheme and its subsequent O(2)-dependent fragmentation to Fe(3+)-verdoheme. The conversion of Fe(3+)-verdoheme to Fe(3+)-biliverdin is also biphasic. Thus, reduction of Fe(3+) to Fe(2+)-verdoheme (0.15 s(-1) at 4 degrees C and 0.55 s(-1) at 25 degrees C) followed by O(2) binding and an electron transfer produces Fe(3+)-biliverdin (0.025 s(-1) at 4 degrees C and 0.10 s(-1) at 25 degrees C). The conversion of Fe(3+)-biliverdin to free biliverdin is triphasic. Reduction of Fe(3+)-biliverdin (0.035 s(-1) at 4 degrees C and 0.15 s(-1) at 25 degrees C), followed by rapid release of Fe(2+) (0.19 s(-1) at 4 degrees C and 0.39 s(-1) at 25 degrees C), yields the biliverdin-enzyme complex from which biliverdin slowly dissociates (0.007 s(-1) at 4 degrees C and 0.03 s(-1) at 25 degrees C). The rate of Fe(2+) release agrees with the rate of Fe(3+)-biliverdin reduction. Fe(2+) release clearly precedes biliverdin dissociation. In the absence of biliverdin reductase, biliverdin release is the rate-limiting step, but in its presence biliverdin release is accelerated and the overall rate of heme degradation is limited by the conversion of Fe(2+)-verdoheme to the Fe(3+)-biliverdin. (+info)
Nitric oxide modulates the catalytic activity of myeloperoxidase.
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Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin six-coordinate complexes, MPO-Fe(III).NO and MPO-Fe(II).NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reduced versus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo. (+info)
Protease activity of CND41, a chloroplast nucleoid DNA-binding protein, isolated from cultured tobacco cells.
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CND41 is a 41 kDa DNA-binding protein isolated from chloroplast nucleoids of cultured tobacco cells. The presence of the active domain of aspartic protease in the deduced amino acid sequence of CND41 suggests that it has proteolytic activity. To confirm this, CND41 was highly purified from cultured tobacco cells and its proteolytic activity was characterized with fluorescein isothiocyanate-labeled hemoglobin as the substrate. The purified CND41 had strong proteolytic activity at an acidic pH (pH 2-4). This activity was inhibited by various chemicals, including the nucleoside triphosphates, NADPH, Fe(3+) and sodium dodecyl sulfate. (+info)
Radiation synovectomy using 165Dy ferric-hydroxide and oxidative DNA damage in patients with different types of arthritis.
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Radiation synovectomy is an effective treatment for chronic synovitis refractory to pharmacological treatment in patients with rheumatoid or seronegative arthritis. Concerns persist about possible radiation-induced cytogenetic damage after radiation synovectomy leading to recommendations to use this technique only in the elderly. Micronucleus (MN) frequency in lymphocytes and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8OHdG) as an indicator of cellular oxidative DNA base damage are biomarkers of radiation-induced cytogenetic damage. The course of both biomarkers was studied in patients with different types of chronic synovitis undergoing radiation synovectomy with very short-lived 165Dy-ferric-hydroxide (DFH). METHODS: Radiation synovectomy of the knee was performed in 13 men and 12 women (mean age, 44+/-15 y) using a mean activity of 9.48+/-1.65 GBq 165Dy-DFH in 27 consecutive treatments. MN frequency in lymphocytes and urinary excretion of 8OHdG, measured by high-performance liquid chromatography, were assessed before and 4 (MN only) and 20 h after radiation synovectomy. RESULTS: Urinary excretion of 8OHdG in patients (in micromol/mol creatinine; pretreatment mean, 3.1+/-3.4; median, 2.27) was not significantly different from that in healthy volunteers (mean, 2.0+/-1.2; median, 1.87) and not altered by radiation synovectomy (post-treatment mean, 2.5+/-1.5; median, 2.04, NS). An increase in 8OHdG levels after radiation synovectomy of more than 1 SD was found in only 1 patient, who experienced leakage to the lymph nodes but who already had elevated urinary 8OHdG levels before treatment. The frequency of MN/500 binucleated cells (BNCs) was slightly lower in patients (pretreatment mean, 4.3+/-2.6; median, 4.25) than in healthy volunteers (mean, 5.4+/-2.3; median, 5.3) and did not significantly change after therapy, either (4-h post-treatment mean, 3.9+/-2.1, median, 3.8; 20-h post-treatment mean, 4.1+/-2, median 3.8 MN/500 BNC). In 22 of 27 treatments, no leakage to nontarget organs could be monitored, whereas leakage to the local lymph nodes and the liver was detected after 5 treatments. CONCLUSION: Radiation synovectomy using 165Dy-DFH causes no significant radiation burden to most patients as indicated by the absence of adverse changes in levels of biomarkers of cytogenetic damage and a low incidence of leakage. These data suggest that the risk of malignancy may not be elevated. (+info)
Porphyrin and phthalocyanine antiscrapie compounds.
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The transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative diseases for which no effective treatments are available. The likelihood that a bovine form of TSE has crossed species barriers and infected humans underscores the urgent need to identify anti-TSE drugs. Certain cyclic tetrapyrroles (porphyrins and phthalocyanines) have recently been shown to inhibit the in vitro formation of PrP-res, a protease-resistant protein critical for TSE pathogenesis. We now report that treatment of TSE-infected animals with three such compounds increased survival time from 50 to 300%. The significant inhibition of TSE disease by structurally dissimilar tetrapyrroles identifies these compounds as anti-TSE drugs. (+info)
Effects of interferon-gamma and lipopolysaccharide on macrophage iron metabolism are mediated by nitric oxide-induced degradation of iron regulatory protein 2.
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Iron regulatory proteins (IRP-1 and IRP-2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements, which are located in the 3'-untranslated region and the 5'-untranslated region of their respective mRNAs. Cellular iron levels affect binding of IRPs to iron-responsive elements and consequently expression of TfR and ferritin. Moreover, NO(*), a redox species of nitric oxide that interacts primarily with iron, can activate IRP-1 RNA binding activity resulting in an increase in TfR mRNA levels. Recently we found that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA binding of IRP-2 followed by IRP-2 degradation, and these changes were associated with a decrease in TfR mRNA levels (Kim, S., and Ponka, P. (1999) J. Biol. Chem. 274, 33035-33042). In this study, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP-1 binding activity, whereas RNA binding of IRP-2 decreased and was followed by a degradation of this protein. Moreover, the decrease of IRP-2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. Furthermore, LPS/IFN-gamma-stimulated RAW 264.7 cells showed increased rates of ferritin synthesis. These results suggest that NO(+)-mediated degradation of IRP-2 plays a major role in iron metabolism during inflammation. (+info)
The catalytic pathway of cytochrome p450cam at atomic resolution.
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Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction. (+info)