Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Dithionite”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources
PubMed2004

Abiotic reductive dechlorination of chlorinated ethylenes by soil.

Abiotic reductive dechlorination of chlorinated ethylenes by soil in anaerobic environments was characterized to improve knowledge of the behavior of chlorinated ethylenes in natural systems, including systems modified to promote attenuation of contaminants. Target organics in the soil suspension reached sorption equilibrium in 2 days and the sorption isotherm of target organics was properly described by the linear sorption model. A modified Langmuir-Hinshelwood model was developed to describe the kinetics of reductive dechlorination of target organics by soil. The rate constants for the reductive dechlorination of chlorinated ethylenes at the reactive surfaces of reduced soils were found in the range between 0.055 (+/- 8.9%) and 2.60 (+/- 3.2%) day(-1). The main transformation products in reduced soil suspensions were C2 hydrocarbons. No chlorinated intermediates were observed at concentrations above detection limits. Five cycles of reduction of the soil followed by oxidation of the soil with trichloroethylene (TCE) did not affect the removal of TCE. The removal was affected by the reductants used and increased in the order: Fe(II) < dithionite < Fe(II) + dithionite.

Anaerobiosis↗
PubMed1991

Characterisation of 'fast' and 'slow' forms of bovine heart cytochrome-c oxidase.

We have prepared cytochrome-c oxidase from bovine heart (using a modification of the method of Kuboyama et al. (1972) J. Biol. Chem. 247, 6375-6383) which binds cyanide rapidly, shows no kinetic distinction between the two haems on reduction by dithionite, has a Soret absorption maximum above 424 nm, and has a negligible 'g' = 12' EPR signal. On incubation at pH 6.5 this 'fast' oxidase reverts to the 'slow' ('resting') form characterised by slow cyanide binding, slow reduction of haem a3 by dithionite, a blue-shifted Soret maximum and a large 'g' = 12' signal. Incubation of 'fast' oxidase with formate produces a form of the enzyme with properties almost identical to those of 'slow' oxidase. The kinetics of formate binding to 'fast' oxidase are found to be biphasic, revealing the presence of at least two 'fast' subpopulations in our preparations. Evidence is presented that there is an equilibrium mixture of high-spin and low-spin forms of haem a3 in both 'fast' subpopulations at room temperature. Incubation of 'fast' oxidase with chloride or bromide at pH 6.5 produces forms of oxidase with much lower rates of cyanide binding. Our working hypothesis is that formate mimics a binuclear centre ligand which is present in the 'slow' form of cytochrome oxidase. Although we show that chloride and bromide can also be ligands of the binuclear centre, possibly onto CuB, we can rule out either of these being the ligand present in the 'slow' enzyme. We will argue that the 'fast' and 'slow' forms of oxidase are equivalent to the 'pulsed' and 'resting' forms of oxidase, respectively.

Animals↗
PubMed1999

The integrity of the disulfide bond in a cyclic somatostatin analog during 99mTc complexation reactions.

Recent development of a variety of thiol-free chelating agents has facilitated the design of 99mTc-labeled somatostatin analogs suitable for receptor imaging of somatostatin-positive tumors. However, it remains ambiguous whether the disulfide bonds in cyclic peptides are stable during 99mTc complexation reactions, and contradictory results have been reported regarding the integrity of disulfide bonds in cyclic somatostatin analogs. To estimate the stability of the disulfide bond in a synthetic somatostatin analog at low peptide concentrations, [125I]I-RC-160, in which radioiodine was incorporated into the 3-Tyr residue, was synthesized and the integrity of the disulfide bond of the peptide was investigated in the presence of reducing agents such as ascorbic acid, dithionite, and stannous ions. The disulfide bond in [125I]I-RC-160 remained stable in the presence of ascorbic acid in boiling water. The disulfide bond was also stable when treated with stannous ions at concentrations sufficient to reduce 99mTc for complexation with a thiol-free chelating agent, bis(hydroxamamide) analog when the 99mTc complexation reaction was performed at room temperature. However, the disulfide bond of [125I]I-RC-160 was slightly cleaved in the presence of a small amount of stannous ions when the reaction was performed in boiling water. Treatment of [125I]I-RC-160 with dithionite in boiling water markedly reduced the disulfide bond of the parental peptide. These findings indicated that synthetic somatostatin analogs may be labeled with 99mTc with stannous ions as the reducing agent without impairing their structure after conjugation of thiol-free chelating agents that provide 99mTc chelates under mild reaction conditions.

Ascorbic Acid↗
PubMed1994

Biotin synthase: purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product.

We report here the first purification of the protein encoded by the Escherichia coli bioB gene. One species of this protein runs on native gels with an electrophoretic mobility typical of a protein with m = 82 kDa, suggesting the protein is a dimer (gene sequence predicts m = 38.7 kDa). There are two iron- and two acid-labile sulfur atoms per protein monomer. Solutions containing the protein are red and have an absorbance spectrum characteristic of proteins with [2Fe-2S] clusters. In its oxidized native state, the protein is EPR-silent. Upon addition of dithionite, the protein's UV-visible absorbance spectrum is very slowly bleached, and an EPR active species is produced that displays a signal at gavg = 1.95. All these results are consistent with this protein containing one [2Fe-2S] cluster per monomer. The EPR spin quantitation is only 5-10% of expected. Since this protein loses iron upon reduction with dithionite, the low-spin quantitation is probably due to cluster instability in the reduced state. Another species of the bioB gene products has also been purified which runs on native gels with an electrophoretic mobility typical of a protein with m = 104 kDa. This species appears to be a dimer with one [2Fe-2S] cluster per dimer. The 104-kDa protein can be converted to the 82-kDa protein upon incubation with Fe3+ and S2-. The bioB gene product we have isolated is active in the conversion of dethiobiotin to biotin in vitro in the presence of NADPH, AdoMet, Fe3+ or Fe2+, and additional unidentified factors from the crude extracts of E. coli. The Km for dethiobiotin in this reaction has been found to be 2 microM.

Amino Acid Sequence↗
PubMed1994

Cyanide binding at the non-heme Fe2+ of the iron-quinone complex of photosystem II: at high concentrations, cyanide converts the Fe2+ from high (S = 2) to low (S = 0) spin.

The primary electron acceptor complex of photosystem II, QAFe2+, can bind a number of small molecules at the iron site, including cyanide [Koulougliotis, D., Kostopoulos, T., Petrouleas, V., & Diner, B. A. (1993) Biochim. Biophys. Acta 1141, 275-282)]. In the presence of NaCN (30-300 mM) at pH 6.5, the reduced state, QA-Fe2+, produced either by illumination at < or = 200 K or by reduction in the dark with sodium dithionite, is characterized by a g = 1.98 EPR signal. The light- or dithionite-induced g = 1.98 signal decays with increasing pH above 6.5 and is almost totally absent at pH 8.1 and NaCN concentrations above 300 mM. However, at high pH (8.1), the g = 1.98 signal still forms transiently before it decays with a t1/2 of approximately 30 min in spinach BBY preparations treated with 100 mM NaCN. Complementary to the disappearance of the g = 1.98 signal with increasing pH or incubation time, a new EPR signal develops at g = 2.0045. This signal has the characteristics of the semiquinone, QA-, uncoupled from its magnetic interaction with the iron. Prolonged incubation of a high pH, high cyanide treated sample in a cyanide-free medium at pH 6 restores the ability of the sample to develop the cyanide-induced g = 1.98 signal at pH 6.5. This indicates that the iron is not physically dissociated during the high pH cyanide treatment. The high pH, high cyanide effects are accompanied by the conversion of the characteristic Fe2+ (S = 2) Mössbauer doublet [isomer shift (Fe) = 1.19 mm/s, quadrupole splitting = 2.95 mm/s] to a new one with parameters (isomer shift = 0.26 mm/s, quadrupole splitting = 0.36 mm/s) characteristic of an Fe2+(S = 0) state.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗
PubMed2000

Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein.

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.

Acyl Carrier Protein↗
PubMed2001

Single turnover EPR studies of benzoyl-CoA reductase.

Benzoyl-CoA reductase (BCR) catalyzes the ATP-driven transport of two electrons from a reduced 2[4Fe-4S] ferredoxin to the aromatic ring of benzoyl-CoA. A mechanism involving radical species and very low potential electrons similar to the Birch reduction of aromatics has been suggested for this reaction. The redox centers of BCR have previously been identified, by EPR- and Mössbauer spectroscopy, to be three cysteine-ligated [4Fe-4S] clusters [Boll et al. (2000) J. Biol. Chem. 275, 31857-31868] with redox potentials more negative than -500 mV. In this work, the catalytic cycle of BCR was studied by freeze-quench experiments; the dithionite reduced enzyme was rapidly mixed with equimolar amounts of benzoyl-CoA and excess MgATP plus dithionite, and subjected to EPR spectroscopic analysis. The turnover period of the enzyme under the conditions used was 3 s. The total S = (1)/(2) spin concentration increased 3-fold very rapidly (within approximately 25 ms). In the course of a single turnover the extent of enzyme reduction decreased again, finally reaching the starting value. An increased magnetic interaction of [4Fe-4S] clusters and the rise of an S = (7)/(2) high-spin EPR signal occurred as second simultaneous and transient events (at approximately 200 ms). Previous work showed that binding of the nucleotide affects the magnetic interaction of [4Fe-4S] clusters, whereas hydrolysis of MgATP is required for the switch to high-spin EPR signals. Finally, two novel transient EPR signals with an isotropic line-shape developed maximally in the late phase of the catalytic cycle ( approximately 1-2 s). These signals differed from those of typical free radicals by shifted g values at g = 2.015 and g = 2.033 and by an unusually fast relaxation rate, suggesting an interaction of these paramagnetic species with [4Fe-4S](+1) clusters. On the basis of these results, we present a proposal for a catalytic cycle involving radical species.

Acyl Coenzyme A↗
PubMed1984

Structural analysis of myeloperoxidase by resonance Raman spectroscopy.

Soret excitation of canine myeloperoxidase (MPO) gives rise to a complex resonance Raman (RR) spectrum characterized by multiple bands in the core size and oxidation state marker regions. Relative intensities of the bands obtained by 406- and 454-nm laser excitation were nearly identical and were temperature independent from 77 to 273 K. Spectra of dithionite-reduced and cyanide-coordinated derivatives are also reported. In the native and dithionite-reduced enzyme, there are no detectable bands between 1620 and 1700 cm-1, indicating that the hemes do not contain formyl substituents in conjugation with the macrocyclic ring. Excitation of the visible absorption band at 568 nm gave rise to only very weakly resonance-enhanced spectra. The RR spectra are interpreted within the context of other physical measurements to indicate that MPO contains two equivalent or nearly equivalent chlorin prosthetic groups. Possible mechanistic consequences of these structural features are discussed.

Animals↗
PubMed1985

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase.

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.

Aerobiosis↗
PubMed1987

Characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins of Azotobacter vinelandii using magnetic circular dichroism spectroscopy.

Low-temperature magnetic circular dichroism (MCD) spectroscopy has been used to investigate the metal clusters in the conventional nitrogenase MoFe protein and alternative VFe protein from Azotobacter vinelandii. In the dithionite-reduced state, the MCD spectrum of the MoFe protein is extremely similar to that previously observed for the S = 3/2 spin state of the M clusters in the MoFe protein of Klebsiella pneumoniae. A paramagnetic cluster with an S = 3/2 ground state is also responsible for the temperature-dependent MCD transitions of dithionite-reduced VFe protein. However, the electronic and magnetic properties of this cluster are quite distinct from those of M centers in conventional nitrogenase. When these proteins are oxidized with thionine, the MoFe protein exhibits MCD spectra and magnetization characteristics identical with those observed for the P clusters in K. pneumoniae, while those of the VFe protein are only similar. However, the paramagnetism in the thionine-oxidized VFe protein, like the conventional enzyme, probably arises from an S = 5/2 spin system with near-axial symmetry and a negative zero-field splitting. Novel clusters with electronic, magnetic, and redox properties similar to those of conventional P clusters are, therefore, present in the VFe protein.

Azotobacter↗
PubMed1981

Stabilization of the red semiquinone form of pig kidney general acyl-CoA dehydrogenase by acyl coenzyme A derivatives.

Pig kidney general acyl-CoA dehydrogenases forms the blue neutral radical on dithionite or photochemical reduction (Thorpe, C., Matthews, R. G., & Williams, C. H. (1979) Biochemistry 18, 331-337] in accord with its classification as a flavoprotein dehydrogenase. However, dithionite reduction of the enzyme in the presence of crotonyl coenzyme A (crotonyl-CoA) or octenoyl-CoA generates the red radical anion as the predominant species at pH 7.6. Crotonyl-CoA binds preferentially to the red radical form, depressing the apparent pK by at least 2.5 pH units to a value of 7.3. Butyryl-, octanoyl-, and palmitoyl-CoA induce very similar spectral changes to those induced by enoyl-CoA derivatives when added anaerobically to the blue semiquinone enzyme. In contrast, the competitive inhibitors acetoacetyl-CoA and heptadecyl-SCoA do not markedly perturb the spectrum of the neutral flavosemiquinone species. The stability of the enzyme radical complexes with either crotonyl- or octanoyl-CoA suggests that there is not effective intraflavin transfer of reducing equivalents between subunits. Perturbation of the spectrum of the one-electron-reduced enzyme by ligands may complicate interpretation of the reaction enzyme by ligands may complicate interpretation of the reaction between substrate complexes of the general acyl-CoA dehydrogenase and electron-transferring flavoprotein.

Acyl Coenzyme A↗
PubMed1978

Kinetic studies on electron transfer and interaction between nitrogenase components from Azotobacter vinelandii.

Kinetic properties of electron transfer by nitrogenase of Azotobacter vinelandii are dependent on the concentration of the two components of nitrogenase. An excess of the MoFe protein inhibits electron transfer in a distinctive manner, and the inhibition is reversed by increasing levels of reductant. The saturation curve for Fe protein is hyperbolic, indicating that only one Fe protein molecule per MoFe protein is required for full activity in ATP hydrolysis and electron transfer. These results can be interpreted on the basis of a complex between the Fe protein and the MoFe protein that dissociates rapidly during turnover. Both 2:1 and 1:1 complexes (Fe-MoFe) are active. Dithionite appears to be a relatively poor reductant for nitrogenase from Azotobacter vinelandii, whereas azotobacter flavodoxin is much better. In the presence of the flavodoxin it is possible to increase the specific activity of the Fe protein more than 50% relative to its activity with dithionite alone as a reductant; specific activities greater than 3000 nmol of C2H4 formed min(-1) (mg of Fe protein)(-1) have been observed.

Adenosine Triphosphate↗
PubMed1996

Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3.

The flavoprotein domain of P450BM-3 (BMR), which is functionally analogous to eukaryotic NADPH-P450 oxidoreductases, contains both FAD and FMN. When BMR is titrated with NADPH or sodium dithionite under anaerobic conditions, addition of 2 electron equivalents per mole of BMR results in the reduction of the high potential flavin (FMN) without the accumulation of semiquinone intermediates. Additional sodium dithionite first produces some neutral, blue flavin semiquinone radical and, finally, fully reduced FADH2. During reduction with NADPH, an absorbance increase characteristic of the formation of a flavin-pyridine nucleotide charge-transfer complex was observed only during the addition of the second mole of NADPH per mole of BMR. On the basis of these results, we conclude that the midpoint reduction potential for the FMN semiquinone/FMNH2 couple is more positive than that for FMN/FMN semiquinone. The kinetics of reduction of BMR with NADPH were studied by stopped-flow spectrophotometry. With a 1:1 ratio of NADPH to BMR, the absorbance changes can be fit to five consecutive first order reactions with rate constants of 350 s-1, 130 s-1, 27 s-1, 2.3 s-1, and 0.05 s-1. These reactions are most probably the following: (a) complex formation between BMR and NADPH; (b) reduction of FAD with formation of the NADP(+)-FADH- charge-transfer complex; (c) transfer of the first electron from FADH- to FMN to form an anionic, red FMN semiquinone leaving the FAD as the neutral, blue semiquinone. Precise identification of intermediates beyond this point is difficult. In the presence of a 10-fold molar excess of NADPH, the absorbance changes and rate constants are somewhat different due to the formation of several additional reduced species of BMR. The rate of the first step increases, confirming that this is the formation of the NADPH-BMR complex. Our results indicate that the kinetic and thermodynamic control of the flavins in BMR is significantly different from that in microsomal P450 reductase. The low potential of the anionic FMN semiquinone can be utilized to reduce the P450 heme. When the anionic semiquinone becomes protonated, its potential becomes more positive and it is readily reduced to FMNH2, which is not capable of reducing P450.

Anaerobiosis↗
PubMed1997

Spectroscopic and enzymatic characterization of the active site dinuclear metal center of calcineurin: implications for a mechanistic role.

The active site of bovine brain calcineurin contains an Fe3+-Zn2+ dinuclear metal center. Replacement of Zn2+ with Fe2+ yields a mixed valence Fe3+-Fe2+ center that exhibits a characteristic EPR signal that can be used as a convenient spectroscopic probe of the active site. Addition of product phosphate to both the Fe3+-Fe2+ and Fe3+-Zn2+ forms of calcineurin led to perturbations of the respective EPR signals, indicating that phosphate affects the environment of the paramagnetic centers. Anaerobic titrations of the iron-substituted Fe3+-Fe2+ enzyme with dithionite resulted in a gradual loss of activity toward pNPP that paralleled the loss of intensity of the EPR signal of the mixed valence diiron center. During dithionite reduction, an EPR resonance with g approximately 12 appeared. The intensity of this resonance increased when the spectrum was recorded in a parallel mode cavity and was therefore attributed to a paramagnetic center with integer spin. Oxidation of the Fe3+-Fe2+ cluster to the diferric state by hydrogen peroxide also led to a loss of activity. These results indicate that the mixed valence oxidation state represents the catalytically competent form of the cluster. The dependence of the enzyme activity on the redox state of the cluster has implications for a mechanistic role.

Aniline Compounds↗
PubMed1999

The heme component of the neutrophil NADPH oxidase complex is a target for aryliodonium compounds.

The redox core of the neutrophil NADPH oxidase complex is a membrane-bound flavocytochrome b in which FAD and heme b are the two prosthetic redox groups. Both FAD and heme b are able to react with diphenylene iodonium (DPI) and iodonium biphenyl (IBP), two inhibitors of NADPH oxidase activity. In this study, we show that the iodonium modification of heme b contributes predominantly to the inhibition of NADPH oxidase. This conclusion is based on the finding that both iodonium compounds decreased the absorbance of the Soret peak of flavocytochrome b in neutrophil membranes incubated with NADPH, and that this decrease was strictly correlated with the loss of oxidase activity. Furthermore, the heme component of purified flavocytochrome b reduced to no more than 95% by a limited amount of sodium dithionite could be oxidized by DPI or IBP. Butylisocyanide which binds to heme iron precludes heme b oxidation. In activated neutrophil membranes, competitive inhibition of O2 uptake by DPI or IBP occurred transiently and was followed by a noncompetitive inhibition. These results, together with those of EPR spectroscopy experiments, lead us to postulate that DPI or IBP first captures an electron from the reduced heme iron of flavocytochrome b to generate a free radical. Then, the binding of this radical to the proximate environment of the heme iron, most probably on the porphyrin ring, results in inhibition of oxidase activity. In the presence of an excess of sodium dithionite, DPI and IBP produced a biphasic decrease of the Soret band of flavocytochrome b, with a break in the dose effect curve occurring at 50% of the absorbance loss. This was consistent with the presence of two hemes in flavocytochrome b that differ by their sensitivity to DPI or IBP.

Animals↗
PubMed1999

Spectroscopic characterization of carbon monoxide complexes generated for copper/topa quinone-containing amine oxidases.

Carbon monoxide complexes have been generated for copper/topa quinone (TPQ)-containing amine oxidases from Arthrobactor globiformis (AGAO) and Aspergillus niger (AO-I) and characterized by various spectroscopic measurements. Addition of CO to AGAO anaerobically reduced with its substrate 2-phenylethylamine led to a slight increase of absorption bands at 440 and 470 nm derived from the semiquinone form (TPQ(sq)) of the TPQ cofactor, concomitantly giving rise to new CO-related absorption bands at 334 and 434 nm. The intensity of the TPQ(sq) radical EPR signal at g = 2.004 also increased in the presence of CO, while its hyperfine coupling structure was affected insignificantly. FT-IR measurements revealed C-O stretching bands (nu(CO)) at 2063 and 2079 cm(-1) for the CO complex of the substrate-reduced AGAO (at 2085 cm(-1) for AO-I), which shifted nearly 100 cm(-1) to lower frequencies upon using (13)C(18)O. Collectively, these results suggest that CO is bound to the Cu(I) ion in the Cu(I)/TPQ(sq) species formed in the reductive half-reaction of amine oxidation, thereby shifting the Cu(II)/aminoresorcinol right arrow over left arrow Cu(I)/semiquinone equilibrium toward the latter. When AGAO was reduced with dithionite, an intermediary form of the enzyme with Cu(II) reduced to Cu(I) but TPQ still in the oxidized state (TPQ(ox)) was produced. Dithionite reduction of AGAO in the presence of CO resulted in the immediate formation of FT-IR bands at 2064 and 2083 cm(-1), which were assigned to the nu(CO) bands of the CO bound to the TPQ(ox) enzyme. The intense 2083 cm(-1) band was then displaced by a new band at 2077 cm(-1), corresponding to the formation of the fully reduced topa. Significant variation of these nu(CO) frequencies indicates that vibrational properties of CO bound to copper amine oxidases are sensitively influenced by the coordination structure of the Cu(I) ion, which may be modulated by the chemical and redox states of the TPQ cofactor.

Amine Oxidase (Copper-Containing)↗
PubMedDate not supplied

Covalent interaction of 5-nitroimidazoles with DNA and protein in vitro: mechanism of reductive activation.

Human hepatic microsomal enzymes catalyzed the NADPH-dependent anaerobic reductive activation of [1-14C]metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and [4,5-14C]ronidazole [(1-methyl-5-nitroimidazole-2-yl)methyl carbamate] to species that became covalently bound to proteins. Due to the low efficiency of the enzyme-catalyzed covalent binding of metronidazole, the stoichiometry of anaerobic reductive activation was studied with dithionite as the reductant. Two moles of dithionite was consumed per mole of [1-14C]metronidazole for maximal covalent binding to either DNA or immobilized sulfhydryl groups, demonstrating that four electrons are required for the reductive activation of metronidazole. These data implicate the involvement of a hydroxylamine in covalent binding. Maximal covalent binding of [4,5-14C]ronidazole to DNA also required four-electron reduction, consistent with previous studies of the covalent binding of this agent to immobilized sulfhydryl groups [Kedderis et al. (1988) Arch. Biochem. Biophys. 262, 40-48]. Studies of the covalent binding of variously radiolabeled ronidazole molecules to DNA suggested that the imidazole ring was intact while greater than 80% of the 2-carbamoyl group and the C4 proton were not present in the DNA adduct. Studies of both the chemical and human hepatic microsomal reduction of [4-3H]metronidazole demonstrated that covalent binding occurred with the stoichiometric loss of this label, implicating binding at the C4 position. These results suggest that the reductive activation of 5-nitroimidazoles generally proceeds via four-electron reduction to form hydroxylamines followed by nucleophilic attack at C4.

Adult↗
PubMed2003

Binding of novel inhibitors of electron transfer in photosystem 2, derivatives of perfluoroisopropyldinitrobenzene, with polypeptide D2 of the reaction center.

A binding site for novel inhibitors of K15 type (derivatives of perfluoroisopropyldinitrobenzene) with the components of reaction center (RC) of photosystem 2 (PS-2) of higher plants has been investigated. It has been shown that multiple washing the PS-2 submembrane chloroplast fragments (BBY-particles) treated with the K15 inhibitor, including multiple dilution in buffer in the presence of high concentrations of mono- and divalent ions, prolonged (up to 2-5 h) incubation, centrifugation, and subsequent resuspension in buffer deprived of the inhibitor, does not lead to restoration of functional activity of the PS-2. After addition of dithionite, inducing reduction and consequent decomposition of the inhibitor, and subsequent removal of dithionite by washing, the functional activity of PS-2 was completely restored. Incubation in the presence of sodium dodecyl sulfate (SDS), leading to solubilization of the sample to the level of protein components, induced the appearance of a fraction of free K15 retaining the initial inhibitory efficiency. To create a covalent binding of the inhibitor with protein, retained under the conditions of denaturing SDS polyacrylamide gel electrophoresis, the azido-containing analog of K15 (K15-N(3)) was used. The need for radioactive label for identification of K15 was avoided by the revealed ability of K15-type inhibitors to emit fluorescence, which retained its features under the experimental conditions. With the technique of photoaffinity binding and denaturing SDS-PAGE in the presence of 6 M urea of submembrane chloroplast fragments enriched in PS-2 the D2-polypeptide, an integral component of the reaction center of PS-2, has been shown to be a binding site for K15-type inhibitors. This conclusion is in agreement with a suggestion (put forward in our earlier publications) that K15-type inhibitors are bound to PS-2 reaction center, replacing Q(A) in its binding site. Hence, an agent specifically binding to polypeptide D2 has been found for the first time. The data are compared with information about inhibitory action and binding sites of the known inhibitors of electron transfer in PS-2.

Azides↗