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Accurate detection of labile analytes through activity based fluorogenic sensing is meaningful but remains a challenge because of nonrapid reaction kinetic. Herein, we present a signaling reporter engineering strategy to accelerate azoreduction reaction by positively charged fluorophore promoted unstable anion recognition for rapidly sensing sodium dithionite (Na 2 S 2 O 4 ), a kind of widespread used but harmful inorganic reducing agent. Its quick decomposition often impedes application reliability of traditional fluorogenic probes in real samples because of their slow responses. In this work, four azo-based probes with different charged fluorophores (positive, zwitterionic, neutral, and negative) were synthesized and compared. Among of them, with sequestration effect of positively charged anthocyanin fluorophore for dithionite anion via electrostatic attraction, the cationic probe Azo-Pos displayed ultrafast fluorogenic response (∼2 s) with the fastest response kinetic ( k pos ' = 0.373 s -1 ) that is better than other charged ones ( k zwi ' = 0.031 s -1 , k neu ' = 0.013 s -1 , k neg ' = 0.003 s -1 ). Azo-Pos was demonstrated to be capable to directly detect labile Na 2 S 2 O 4 in food samples and visualize the presence of Na 2 S 2 O 4 in living systems in a timely fashion. This new probe has potential as a robust tool to fluorescently monitor excessive food additives and biological invasion of harmful Na 2 S 2 O 4 . Moreover, our proposed accelerating strategy would be versatile to develop more activity-based sensing probes for quickly detecting other unstable analytes of interest.

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H 2 ). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S] H ) covalently attached to a unique [2Fe] subcluster ([2Fe] H ), where both sites are redox active. Heterolytic splitting and formation of H 2 takes place at [2Fe] H , while [4Fe-4S] H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state H ox , named H ox H, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. H ox H was previously suggested to have a protonated [4Fe-4S] H . Here, we show that H ox H forms by simple addition of sodium sulfite (Na 2 SO 3 , the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO 2 ) is the species involved. Spectroscopy supports binding at or near [4Fe-4S] H , causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that H ox H and its one-electron reduced counterpart H red 'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states H ox -DT i and H red -DT i . The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

DNA-encoded chemical libraries have emerged as a cost-effective alternative to high-throughput screening (HTS) for hit identification in drug discovery. A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines. The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The reaction is performed in aqueous solution and does not compromise DNA structural integrity. The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.

Colloids may facilitate the transport of trace elements and nutrients like phosphate in soil. In this study, we characterized soil colloids (<0.45 μm), extracted from four agricultural soils by Na-bicarbonate and Na-pyrophosphate, by two complementary analytical techniques; asymmetric flow field-flow fractionation (AF4) and X-ray absorption spectroscopy (XAS). The combined results from AF4 and XAS show that colloidal Fe is present as (i) free Fe-(hydr)oxide nanoparticles, (ii) Fe-(hydr)oxides associated with clay minerals, and (iii) Fe in clay minerals. Free Fe-(hydr)oxide nanoparticles, which can be as small as 2-5 nm, are extracted with Na-pyrophosphate but not with Na-bicarbonate, except for one soil. In contrast, Fe-(hydr)oxides associated with clay minerals are dispersed by both extractants. XAS results show that the speciation of Fe in the colloidal fractions closely resembles the speciation of Fe in the bulk soil, indicating that dispersion of colloidal Fe from the studied soils was rather unselective. In one Fe-rich soil, colloidal Fe was dominantly dispersed in the form of free Fe-(hydr)oxide nanoparticles. In the other three soils, dispersed Fe-(hydr)oxides were dominantly associated with clay minerals, suggesting that their dispersion as free nanoparticles was inhibited by strong attachment. However, in these soils, Fe-(hydr)oxides can be dispersed as oxide-clay associations and may as such facilitate the transport of trace elements.

Arsenic-contaminated rice grain may threaten human health globally. Since H₃AsO₃⁰ is the predominant As species found in paddy pore-waters, and H₄SiO₄⁰ and H₃AsO₃⁰ share an uptake pathway, silica amendments have been proposed to decrease As uptake and consequent As concentrations in grains. Here, we evaluated the impact of two silicate mineral additions differing in solubility (+Si(L), diatomaceous earth, 0.29 mM Si; +Si(H), Si-gel, 1.1 mM Si) to soils differing in mineralogy on arsenic concentration in rice. The +Si(L) addition either did not change or decreased As concentration in pore-water but did not change or increased grain-As levels relative to the (+As--Si) control. The +Si(H) addition increased As in pore-water, but it significantly decreased grain-As relative to the (+As--Si) control. Only the +Si(H) addition resulted in significant increases in straw- and husk-Si. Total grain- and straw-As was negatively correlated with pore-water Si, and the relationship differed between two soils exhibiting different mineralogy. These differing results are a consequence of competition between H₄SiO₄⁰ and H₃AsO₃⁰ for adsorption sites on soil solids and subsequent plant-uptake, and illustrate the importance of Si mineralogy on arsenic uptake.

Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT(0), TMT(6), and iTRAQ(8) reagents and discuss limitations of the strategy.

Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents., (© 2012 American Chemical Society)

A low-spin, macrocyclic iron(II) complex in an aqueous solution responds to the addition of a chemical reactant (dithionite) by transformation into a high-spin complex, detectable by measurement of the longitudinal relaxation time (T(1)) of surrounding water hydrogen nuclear spins. The initial compound does not modify T(1) of pure water at concentrations as high as 4 mM. The response is pH-dependent, and the complex is robust at a variety of conditions.

Nanoscale zero-valent iron (NZVI) represents a promising approach for source zone control, but concerns over its reactive lifetime might limit application. Here, we demonstrate that dithionite (S₂O₄²⁻), a reducing agent for in situ redox manipulation, can restore the reducing capacity of passivated NZVI. Slurries of NZVI were aged in the presence (3 days) and absence (60 days) of dissolved oxygen over a range of pH values (6-8). Upon loss of reactivity toward model pollutants{1,1,1,2-tetrachloroethane, hexavalent chromium [Cr(VI)], nitrobenzene}, aged suspensions were reacted with dithionite, and the composition and reactivity of the dithionite-treated materials were determined. NZVI aging products generally depended on pH and the presence of oxygen, whereas the amount of dithionite influenced the nature and reducing capacity of products generated from reaction with aged NZVI suspensions. Notably, air oxidation at pH ≥ 8 quickly exhausted NZVI reactivity despite preservation of significant Fe(0) in the particle core. Under these conditions, formation of a passive surface layer hindered the complete transformation of NZVI particles into iron(III) oxides, which occurred at lower pH. Reduction of this passive layer by low dithionite concentrations( 1 g/g of NZVI) restored suspension reactivity to levels equal to, and occasionally greater than, that of unaged NZVI. Multiple dithionite additions further improved pollutant removal, allowing at least a 15-fold increase in Cr(VI) removal [∼300 mg of Cr(VI)/g of NZVI] relative to that of as-received NZVI [∼20 mg of Cr(VI)/g of NZVI].

Mo-nitrogenase catalyzes the reduction of dinitrogen to ammonia at the cofactor (i.e., FeMoco) site of its MoFe protein component. Biosynthesis of FeMoco involves NifEN, a scaffold protein that hosts the maturation of a precursor to a mature FeMoco before it is delivered to the target location in the MoFe protein. Previously, we have shown that the NifEN-bound precursor could be converted in vitro to a fully complemented "FeMoco" in the presence of 2 mM dithionite. However, such a conversion was incomplete, and Mo was only loosely associated with the NifEN-bound "FeMoco". Here we report the optimized maturation of the NifEN-associated precursor in 20 mM dithionite. Activity analyses show that upon the optimal conversion of precursor to "FeMoco", NifEN is capable of activating a FeMoco-deficient form of MoFe protein to the same extent as the isolated FeMoco. Furthermore, EPR and XAS/EXAFS analyses reveal the presence of a tightly organized Mo site in NifEN-bound "FeMoco", which allows the observation of a FeMoco-like S = 3/2 EPR signal and the modeling of a NifEN-bound "FeMoco" that adopts a conformation very similar to that of the MoFe protein-associated FeMoco. The sensitivity of FeMoco maturation to dithionite concentration suggests an essential role of redox chemistry in this process, and the optimal potential of dithionite solution could serve as a guideline for future identification of in vivo electron donors for FeMoco maturation.

Hydroxytyrosol, a naturally occurred o-phenolic compound exhibiting antioxidant properties, was synthesized by a three-step high-yielding procedure from natural and low-cost compounds such as tyrosol or homovanillyl alcohol. First, the efficient chemoselective protection of the alcoholic group of these compounds was performed by using dimethyl carbonate (DMC) as reagent/solvent; second, the oxidation with 2-iodoxybenzoic acid (IBX) or Dess-Martin periodinane reagent (DMP) and in situ reduction with sodium dithionite (Na2S2O4) allowed the preparation of carboxymethylated hydroxytyrosol; finally, by a mild hydrolytic step, hydroxytyrosol was obtained in high yield and purity, as confirmed by NMR spectra and HPLC profile. By using a similar methodology, lipophilic hydroxytyrosol derivatives, utilized as additives in pharmaceutical, food, and cosmetic preparations, were prepared. In fact, at first the chemoselective protection of the alcoholic group of tyrosol and homovanillyl alcohol was performed by using acyl chlorides without any catalyst to obtain the corresponding lipophilic derivatives, and then these compounds were converted in good yield and high purity into the hydroxytyrosol derivatives by oxidative/reductive pathway with IBX or DMP and Na2S2O4.

A new clay-liposome complex was developed for reducing leaching of herbicides and contamination of groundwater. The liposomes were composed of the neutral and Environmental Protection Agency approved phospholipid phosphatidylcholine (PC). Adsorption of PC liposomes on the clay mineral montmorillonite could exceed the cation exchange capacity of the clay, and was well simulated by the Langmuir equation. X-ray diffraction results for 6 mM PC and 1.6 g/L clay (3 day incubation) yielded a basal spacing of 7.49 nm, which was interpreted as the formation of a supported planar bilayer on montmorillonite platelets. Fluorescence methods demonstrated structural changes which reflected adsorption of PC followed by loss of vesicle integrity as measured by the penetration of dithionite into the internal monolayer of fluorescently labeled liposomes, resulting in a decrease in fluorescence intensity to 18% of initial after 4 h. Energy transfer was demonstrated after 1 h from labeled liposomes to montmorillonite labeled by an acceptor. The neutral herbicide alachlor adsorbed on the liposome-clay complex, yielding a formulation of up to 40% active ingredient, and 1.6-fold reduction in herbicide release in comparison to the commercial formulation. Hence, the PC-montmorillonite complex can form a basis for environmentally friendly formulations of herbicides, which would yield reduced leaching.

The reaction of deoxyhemoglobin with nitrite was characterized in the presence of dithionite using hemoglobin in solution or bound to the cytoplasmic domain of band 3 (CDB3). Deoxyhemoglobin was generated by predeoxygenation (nitrogen flushing followed by addition of dithionite), or transiently, by rapidly mixing oxyhemoglobin with nitrite and dithionite simultaneously. Wavelength-dependent kinetic studies confirmed the formation of nitrosyl hemoglobin. Furthermore, the rate of reaction was independent of dithionite concentration, indicating that dithionite does not reduce nitrite to nitric oxide directly. Model simulation studies showed that superoxide anion generated by dithionite reduction of molecular oxygen was not a factor in the reaction kinetics. CDB3-bound hemoglobin reacted faster with nitrite than did hemoglobin in solution. This difference was most pronounced for predeoxygenated hemoglobin and least pronounced for rapidly deoxygenated hemoglobin. The smaller difference observed in the rapid deoxygenation experiment was associated with much faster kinetics compared to the predeoxygenation experiment. Model simulation studies showed, and literature evidence indicates, that faster kinetics in the rapid deoxygenation experiment were related to the initial presence of R-state Hb(II)O 2 alphabeta dimers, both in dilute solution and when bound to CDB3. Thus, rapidly deoxygenated CDB3-bound hemoglobin alphabeta dimers react 5-fold faster with nitrite than predeoxygenated tetrameric hemoglobin in solution. Faster nitrite reductase kinetics for CDB3-bound hemoglobin suggests the possibility of preferential nitric oxide generation at the inner surface of the erythrocyte membrane, thus coupling the release of oxygen from hemoglobin to the production and successful release of nitric oxide from the erythrocyte, and the regulation of blood flow.

We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The Mn (IV) site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host's immune system [Högbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gräslund, A., and Nordlund, P. (2004) Science 305, 245-248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (Mn (II)/Fe (II), Mn (III)/Fe (III), and Mn (IV)/Fe (III)) toward hydrogen peroxide. Not only is the activity of the Mn (IV)/Fe (III)-R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H 2O 2, but both the fully reduced (Mn (II)/Fe (II)) and one-electron-reduced (Mn (III)/Fe (III)) forms of the protein are also efficiently activated by H 2O 2. The Mn (III)/Fe (III)-R2 species reacts with a second-order rate constant of 8 +/- 1 M (-1) s (-1) to yield the Mn (IV)/Fe (IV)-R2 intermediate previously observed in the reaction of Mn (II)/Fe (II)-R2 with O 2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709-8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active Mn (IV)/Fe (III)-R2 complex. The reaction of the Mn (II)/Fe (II)-R2 species with H 2O 2 proceeds in three resolved steps: sequential oxidation to Mn (III)/Fe (III)-R2 ( k = 1.7 +/- 0.3 mM (-1) s (-1)) and Mn (IV)/Fe (IV)-R2, followed by decay of the intermediate to the active Mn (IV)/Fe (III)-R2 product. The efficient reaction of both reduced forms with H 2O 2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe 2 (II/II)) to the "met" (Fe 2 (III/III)) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780-786] but is then only very inefficiently converted from the met to the active (Fe 2 (III/III)-Y (*)) form [Sahlin, M., Sjöberg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813-818].

A homemade instrument for the measurement of oxygen concentration in aqueous solutions measures the decay rate of the phosphorescence of a Pd-porphyrin complex (phosphor) dissolved in the solution, which is flashed every 0.1 s with 630 nm light. The concentration of O2 is a linear function of the decay rate. The instrument is used to study the reaction of dithionite (S2O42-) with O2 at 25 degrees C and 37 degrees C. It is found that the ratio of dithionite to oxygen consumed in the reaction is 1.2 +/- 0.2 at 25 degrees C and 1.7 +/- 0.1 at 37 degrees C, suggesting a temperature-dependent stoichiometry. At both temperatures, the initial rate of O2 consumption, -d[O2]/dt, is found to be 1/2 order in S2O42- and first order in O2. This finding is consistent with a previously proposed mechanism: S2O42- <--> 2SO2- comes to a rapid equilibrium, and SO2- reacts with O2 in the rate-determining step.

The FAD-dependent alpha-glycerophosphate oxidase (GlpO) from Enterococcus casseliflavus and Streptococcus sp. was originally studied as a soluble flavoprotein oxidase; surprisingly, the GlpO sequence is 30-43% identical to those of the alpha-glycerophosphate dehydrogenases (GlpDs) from mitochondrial and bacterial sources. The structure of a deletion mutant of Streptococcus sp. GlpO (GlpODelta, lacking a 50-residue insert that includes a flexible surface region) has been determined using multiwavelength anomalous dispersion data and refined at 2.3 A resolution. Using the GlpODelta structure as a search model, we have also determined the intact GlpO structure, as refined at 2.4 A resolution. The first two domains of the GlpO fold are most closely related to those of the flavoprotein glycine oxidase, where they function in FAD binding and substrate binding, respectively; the GlpO C-terminal domain consists of two helix bundles and is not closely related to any known structure. The flexible surface region in intact GlpO corresponds to a segment of missing electron density that links the substrate-binding domain to a betabetaalpha element of the FAD-binding domain. In accordance with earlier biochemical studies (stabilizations of the covalent FAD-N5-sulfite adduct and p-quinonoid form of 8-mercapto-FAD), Ile430-N, Thr431-N, and Thr431-OG are hydrogen bonded to FAD-O2alpha in GlpODelta, stabilizing the negative charge in these two modified flavins and facilitating transfer of a hydride to FAD-N5 (from Glp) as well. Active-site overlays with the glycine oxidase-N-acetylglycine and d-amino acid oxidase-d-alanine complexes demonstrate that Arg346 of GlpODelta is structurally equivalent to Arg302 and Arg285, respectively; in both cases, these residues interact directly with the amino acid substrate or inhibitor carboxylate. The structural and functional divergence between GlpO and the bacterial and mitochondrial GlpDs is also discussed.

Nonviral gene carriers must associate with and become internalized by cells in order to mediate efficient transfection. Methods to quantitatively measure and distinguish between cell association and internalization of delivery vectors are necessary to characterize the trafficking of vector formulations. Here, we demonstrate the utility of nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled oligonucleotides for discrimination between bound and internalized gene carriers associated with cells. Dithionite quenches the fluorescence of extracellular NBD-labeled material, but is unable to penetrate the cell membrane and quench internalized material. We have verified that dithionite-mediated quenching of extracellular materials occurs in both polymer- and lipid-based gene delivery systems incorporating NBD-labeled oligonucleotides. By exploiting this property, the efficiencies of cellular binding and internalization of lipid- and polymer-based vectors were studied and correlated to their transfection efficiencies. Additionally, spatiotemporal information regarding binding and internalization of NBD-labeled gene carriers can be obtained using conventional wide-field fluorescence microscopy, since dithionite-mediated quenching of extracellular materials reveals the intracellular distribution of gene carriers without the need for optical sectioning. Hence, incorporation of environmentally sensitive NBD-oligos into gene carriers allows for facile assessment of binding and internalization efficiencies of vectors in live cells.

Self-assembled thin films of 3,4,9,10-perylenediimides (PDIs) containing up to 50 PDI layers were grown on quartz slides using the zirconium phosphonate technique. When the films were immersed in aqueous solutions of the sodium dithionite reducing agent, in situ reduction of the dye was observed, generating a purple film containing PDI radical anions. The PDI radical anions formed within the films were rather stable, persisting for several minutes in the presence of atmospheric oxygen. Atomic force microscopy (AFM) images showed that the film surface was rather smooth and pinhole-free.

A new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of the cascade-blue-labeled lipid through complex formation with a membrane-impermeable cyclen-tetranaphthalenethiourea synthetic receptor for this dye. The high affinity of the receptor to cascade-blue label allows the use of micromolar concentrations of this receptor during the experiment. At these low concentrations, the receptor does not interfere with the membrane integrity and, therefore, renders this new methodology less invasive to the model and cell membranes than commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Unlike with the NBD-dithionite assay, where the fluorescence quenching of the NBD group is achieved through its chemical modification, this new assay relies on the noncovalent interactions between cascade-blue label and the receptor. Therefore, the quenching can be reverted by either competitive displacement of the lipid-attached label with a water-soluble substrate or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. We demonstrate that this new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments.

Tricarballylate, a citrate analogue, is considered the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Although the normal rumen flora cannot catabolize tricarballylate, the Gram-negative enterobacterium Salmonella enterica can. An operon dedicated to tricarballylate utilization (tcuABC) present in this organism encodes all functions required for tricarballylate catabolism. Tricarballylate is converted to the cis-aconitate in a single oxidative step catalyzed by the FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme. We hypothesized that the uncharacterized TcuB protein was required to reoxidize the flavin cofactor in vivo. Here, we report the initial biochemical characterization of TcuB. TcuB is associated with the cell membrane and contains two 4Fe-4S clusters and heme. Site-directed mutagenesis of cysteinyl residues putatively required as ligands of the 4Fe-4S clusters completely inactivated TcuB function. TcuB greatly increased the Vmax of the TcuA reaction from 69 +/- 2 to 8200 +/- 470 nmol min-1 mg-1; the Km of TcuA for tricarballylate was unaffected. Inhibition of TcuB activity by an inhibitor of ubiquinone oxidation, 2,5-dibromo-3-methyl-6-isoproylbenzoquinone (DBMIB), implicated the quinone pool as the ultimate acceptor of electrons from FADH2. We propose a model for the electron flow from FADH2, to the 4Fe-4S clusters, to the heme, and finally to the quinone pool.

A field study was conducted to evaluate the performance of a ferrous iron based in situ redox zone for the treatment of a dissolved phase Cr(VI) plume at a former industrial site. The ferrous iron based in situ redox zone was created by injecting a blend of 0.2 M ferrous sulfate and 0.2 M sodium dithionite into the path of a dissolved Cr(VI) plume within a shallow medium to fine sand unconfined aquifer formation. Monitoring data collected over a period of 1020 days after more than 100 m of linear groundwater flow through the treatment zone indicated sustained treatment of dissolved phase Cr(VI) from initial concentrations between 4 and 8 mg/L to less than 0.015 mg/L. Sustained treatment is assumed to be primarily due to the reduction of Cr(VI) to Cr(III) by ferrous iron adsorbed to, precipitated on, and/or incorporated into aquifer iron (hydr)oxide solid surfaces within the treatment zone. Precipitated phases likely include FeCO3 and FeS based on saturation index considerations and SEM/EDS analysis. The detection of solid phase sulfites and thiosulfates in aquifer sediments collected from the treatment zone more than 2 years following injection suggests dithionite decomposition products may also play a significant role in the long-term treatment of the dissolved phase Cr(VI).

Mössbauer and EPR spectroscopies were used to study the electronic structure of the A-cluster from recombinant acetyl-CoA synthase (the alpha subunit of the alpha2beta2 acetyl-CoA synthase/CO dehydrogenase). Once activated with Ni, these subunits have properties mimicking those associated with the alpha2beta2 tetramer, including structural heterogeneities. The Fe4S4 portion of the A-cluster in oxidized, methylated, and acetylated states was in the 2+ core oxidation state. Upon reduction with dithionite or Ti3+ citrate, samples of Ni-activated alpha developed the ability to accept a methyl group. Corresponding Mössbauer spectra exhibited two populations of A-clusters; roughly, 70% contained [Fe4S4]1+ cubanes, while approximately 30% contained [Fe4S4]2+ cubanes, suggesting an extremely low [Fe4S4](1+/2+) reduction potential for the 30% portion (perhaps <-800 mV vs NHE). The same population ratio was observed when Ni-free unactivated alpha was used. The 70% fraction exhibited paramagnetic hyperfine structure in the absence of an applied magnetic field, excluding the possibility that it represents an [Fe4S4]1+ cluster coupled to a (proximal) Ni(p)1+. EPR spectra of dithionite-reduced, Ni-activated alpha exhibited features at g = 5.8 and g(ave) approximately 1.93, consistent with a physical mixture of {S = 3/2; S = 1/2} spin-states for A-clusters containing [Fe4S4]1+ clusters. Incubation of Ni-activated alpha with dithionite and CO converted 25% of alpha subunits into the S = 1/2 A(red)-CO state. Previous correlation of this state to functional A-clusters suggests that only the 30% fraction not reduced by dithionite or Ti3+ citrate represents functional A-clusters. Comparison of spin states in oxidized and methylated states suggests that two electrons are required for reductive activation, starting from the oxidized state containing Ni(p)2+. Refitting published activity-vs-potential data supports an n = 2 reductive activation. Enzyme starting in the methylated state exhibited catalytic activity in the absence of an external reductant, suggesting that the two electrons used in reductive activation are retained by the enzyme after each catalytic cycle and that the enzyme does not have to pass through the A(red)-CO state during catalysis. Taken together, our results suggest that a Ni(p)0 state may form upon reductive activation and reform after each catalytic cycle.

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique in that heme is also required for maximal activity, although the function of heme in this enzyme is presently unclear. The study presented herein reveals that the heme of human CBS undergoes a coordination change upon reduction at elevated temperatures. We have termed this new species "CBS424" and demonstrate that its formation is likely irreversible when pH 9 Fe(III) CBS is reduced at moderately elevated temperatures (approximately 40 degrees C and higher) or when pH 9 Fe(II) CBS is heated to similar temperatures. Spectroscopic techniques, including resonance Raman, electronic absorption, and variable temperature/variable field magnetic circular dichroism spectroscopy, provide strong evidence that CBS424 is coordinated by two neutral donor ligands. It appears likely that the native cysteine(thiolate) heme ligand is displaced by an endogenous neutral donor upon conversion to CBS424. This behavior is consistent with other six-coordinate, cysteine(thiolate)-ligated heme centers, which seek to avoid this coordination structure in the Fe(II) state. Functional assays show that CBS424 is inactive and suggest that the ligand switch is responsible for eliminating enzyme activity. When this investigation is taken together with other functional studies of CBS, it provides strong evidence that coordination of Cys52 to the heme iron is crucial for full activity in this enzyme. We hypothesize that cysteine displacement may serve as a mechanism for CBS inactivation and that second-sphere interactions of the Cys52 thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site.

We investigated a method for delivering ferrous iron into the subsurface to enhance chemical reduction of Cr(VI) in chromite ore processing solid waste (COPSW) derived from the production of ferrochrome alloy. The COPSW is characterized by high pH (8.5-11.5) and high Cr(VI) concentrations in the solid phase (up to 550 mg kg(-1)) and dissolved phase (3-57 mg L(-1)). The dominant solid-phase minerals are forsterite (Mg2SiO4), brucite (Mg-(OH)2), and hydrocalumite [Ca4(Al, Fe)2(OH)12X x 6H2O), X = (OH)2(2-), SO4(2-), CrO4(2-)]. The method utilizes FeSO4 in combination with Na2S2O4 to inhibit oxidation and precipitation of the ferrous iron, thereby preventing well and formation clogging. Laboratory batch tests using a 0.05 M FeSO4 + 0.05 M Na2S2O4 solution indicated effective treatment of both dissolved and solid-phase Cr(VI). Contrary to treatments with FeSO4 and FeCl2 alone, the combination resulted in both complete removal of Cr(VI) from solution and sustained Fe(ll) concentrations in solution after a 24 h period. A field test involving injection of 5700 L of a 0.07 M FeSO4 + 0.07 M Na2S2O4 solution into a COPSW saturated zone (pH 11.5) indicated no well and formation clogging during injection. Examination of a core collected 0.46 m from the injection well following injection indicated effective treatment of the solid phase Cr(VI) based on analysis of water, phosphate solution, and high temperature alkaline extracts. The combined reductant solution also imparted a residual treatment capacity to the COPSW allowing for subsequent treatment of dissolved phase Cr(VI); however, dissemination of the iron in the highly alkaline environment appeared to be impeded by the inability to sufficiently lower the pH with distance from the injection well to avoid precipitation of Fe(OH)2 and likely also FeCO3. Injection of a 0.2 M FeSO4 + 0.2 M Na2S2O4 solution into another COPSW saturated zone (pH 9) indicated much more effective dissemination of the injected iron.

The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.

Augmenter of liver regeneration (ALR; hepatopoietin) is a recently discovered enigmatic flavin-linked sulfhydryl oxidase. An N-terminal His-tagged construct of the short form of the human protein has been overexpressed in Escherichia coli. Several lines of evidence suggest that, contrary to a recent report, human ALR is a disulfide-bridged dimer (linked via C15-C124) with two free cysteine residues (C74 and 85) per monomer. The C15-124 disulfides are not critical for dimer formation and have insignificant impact on the dithiothreitol (DTT) oxidase activity of ALR. Although the crystal structure of rat ALR shows a proximal disulfide (C62-C65) poised to interact with the FAD prosthetic group [Wu, C. K., Dailey, T. A., Dailey, H. A., Wang, B. C., and Rose, J. P. (2003) Protein Sci. 12, 1109-1118], only flavin reduction is evident during redox titrations of the enzyme. ALR forms large amounts of neutral semiquinone during aerobic turnover with DTT. This semiquinone arises, in part, by comproportionation between flavin centers within the dimer. Surprisingly, cytochrome c is about a 100-fold better electron acceptor for ALR than oxygen when DTT is the reducing substrate. These data suggest that this poorly understood flavoenzyme may not function as a sulfhydryl oxidase within the mitochondrial intermembrane space but may communicate with the respiratory chain via the mediation of cytochrome c.

Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.

In situ chemical reduction of aquifer sediments is currently being used for chromate and TCE remediation by forming a permeable reactive barrier. The chemical and physical processes that occur during abiotic reduction of natural sediments during flow by sodium dithionite were investigated. In different aquifer sediments, 10-22% of amorphous and crystalline FeIII-oxides were dissolved/reduced, which produced primarily adsorbed FeII, and some siderite. Sediment oxidation showed predominantly one FeII phase, with a second phase being oxidized more slowly. The sediment reduction rate (3.3 h batch half-life) was chemically controlled (58 kJ mol(-1)), with some additional diffusion control during reduction in sediment columns (8.0 h half-life). It was necessary to maintain neutral to high pH to maintain reduction efficiency and prevent iron mobilization, as reduction generated H+. Sequential extractions on reduced sediment showed that adsorbed ferrous iron controlled TCE reactivity. The mass and rate of field-scale reduction of aquifer sediments were generally predicted with laboratory data using a single reduction reaction.

Clay-vesicle systems exhibit a potential for environmental applications, such as herbicide formulations for reduced leaching. Clay-vesicle interactions were addressed by combining adsorption and XRD measurements with fluorescence studies for didodecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDOB), and montmorillonite. XRD and adsorption data indicated that the adsorbing vesicles were transformed after 3 days into paraffinic and bilayer structures. Fluorescence studies revealed that adsorption was almost complete within 5 min for a loading below the cation exchange capacity (CEC). Aggregation and sedimentation of clay-surfactant particles occurred within several minutes. Fluorescent measurements of supernatants indicated decomposition of vesicles at a high clay/surfactant ratio due to rapidly adsorbing cationic monomers. The kinetics of energy transfer between vesicles labeled by NBD-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) and montmorillonite labeled by rhodamine-B follows that of aggregation of surfactant-clay particles and structural changes of the vesicles at times of minutes to hours. Experiments following the reduction of NBD fluorescence by addition of dithionite indicate faster permeabilization of DDOB than DDAB vesicles, which was confirmed by leakage experiments. The faster permeabilization of DDOB vesicles in the presence of clay was correlated with their inferior suitability for the preparation of clay-based formulations of anionic herbicides for slow release.

Benzoyl-CoA reductase (BCR) from the denitrifying bacterium Thauera aromatica catalyzes the ATP driven two-electron reduction of the aromatic moiety of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene (2 ATP/2 e(-)). The enzyme contains two similar but nonidentical ATP-binding sites of the acetate kinase/sugar kinase/Hsp70/actin family. To obtain further insights into the overall catalytic cycle of BCR, the binding affinities and stoichiometries of all substrates as well as their effects on reduction kinetics were studied by stopped-flow UV/vis spectroscopy, freeze-quench EPR spectroscopy, and equilibrium dialysis. BCR bound maximally two nucleotides and a single BCoA. The binding of a single nucleotide induced a molecular switch (BCR --> BCR) as evidenced as follows: (i) the reduction rate of BCR by sulfoxide radical anion was significantly decreased in the nucleotide-bound state, (ii) the binding of BCoA to the reduced enzyme strictly depended on bound nucleotides, and (iii) the nucleotide binding affinities increased up to 60-fold compared to the steady-state values. The "ATP-binding switch" is distinguished from the previously described "low-spin/high-spin switch" of a [4Fe-4S] cluster which strictly depends on ATP hydrolysis. The two nucleotide binding sites were occupied sequentially; binding constants of the two sites differed by a factor of 10-40. The kinetic data obtained suggest that the ATP-binding switch is a rather fast process (>100 s(-)(1)) with a switch equilibrium constant of 54 +/- 10. In contrast, the reverse switch back of the MgADP-bound enzyme (BCR --> BCR) is considered rate-limiting in the overall catalytic cycle of BCR (4 +/- 1 s(-)(1)). The binding of nucleotides did not affect the redox potential of the [4Fe-4S](+1/+2) clusters; the switch is rather considered to alter the kinetics of internal electron transfer. Implications for the overall catalytic cycle of benzoyl-CoA reductase are discussed and compared with other ATP-hydrolyzing enzymes.

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be approximately 100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.

Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H(4)B) or tetrahydrobiopterin analogues (5-methyl-H(4)B and 4-amino-H(4)B), and the ability of bound 5-methyl-H(4)B and 4-amino-H(4)B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H(4)B forms a very stable radical, 5-methyl-H(3)B(*), that accumulates in the arginine reaction to approximately 60% of the HDiNOS concentration and decays approximately 400-fold more slowly than H(3)B(*) (0.0003 vs 0.12 s(-1)). The amount of radical (5-methyl-H(3)B(*) or H(3)B(*)) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H(4)B-saturated FLiNOS and HDiNOS is similar to that when H(4)B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and (*)NO. A pterin radical was not observed with 4-amino-H(4)B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H(4)B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H(4)B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO(-) rather than (*)NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H(4)B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.

Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes [Schütz et al. (2000) J. Mol. Biol. 300, 663-675] indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees. An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data. The cytochrome bc(1) complex from A. aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex. Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods. Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex. The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail. The pool quinone of A. aeolicus was determined to be naphthoquinone. The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes. The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far. An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed.

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.

Cholate esters with phenylurea groups at the 7alpha- and 12alpha-positions are highly effective promoters of phosphatidylcholine translocation across vesicle and cell membranes. The urea groups are essential for strong binding of the highly polar phosphate portion of the phosphocholine headgroup and apparently cannot be replaced by simple amide, alcohol, or amine moieties. NMR and UV studies show that these synthetic translocases have very weak affinity for phosphatidylethanolamine and phosphatidylserine.

The thermodynamics and dynamics of the Cys21-Cys48 disulfide "S" if "R" conformational isomerism in the three-iron, single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) have been characterized by (1)H NMR spectroscopy in both water and water/methanol mixed solvents. The mean interconversion rate at 25 degrees C is 3 x 10(3) s(-1) and DeltaG(298) = -0.2 kcal/mol [DeltaH = 4.0 kcal/mol; DeltaS = 14 cal/(mol.K)], with the S orientation as the more stable form at low temperature (< 0 degrees C) but the R orientation predominating at >100 degrees C, where the organism thrives. The distinct pattern of ligated Cys beta-proton contact shifts for the resolved signals and their characteristic temperature behavior for the forms of the 3Fe Fd with alternate disulfide orientations have been analyzed to determine the influences of disulfide orientation and methanol cosolvent on the topology of the inter-iron spin coupling in the 3Fe cluster. The Cys21-Cys48 disulfide orientation influences primarily the spin couplings involving the iron ligated to Cys17, whose carbonyl oxygen is a hydrogen bond acceptor to the Cys21 peptide proton. Comparison of the Cys beta-proton contact shift pattern for the alternate disulfide orientations with the pattern exhibited upon cleaving the disulfide bridge confirms an earlier [Wang, P.-L., Calzolai, L., Bren, K. L., Teng, Q., Jenney, F. E., Jr., Brereton, P. S., Howard, J. B., Adams, M. W. W., and La Mar, G. N. (1999) Biochemistry 38, 8167-8178] proposal that the structure of the same Fd with the R disulfide orientation resembles that of the Fd upon cleaving the disulfide bond.

Soret-excited resonance Raman (RR) spectra of the spinach cytochrome b6f complex (cyt b6f) are reported for the oxidized, native, ascorbate-reduced, and dithionite-reduced forms. Using excitations at 441.6, 413.1, and 406.7 nm, RR contributions of chlorophyll a, beta-carotene, the c-type heme of cytochrome f, and the b-type hemes of cytochrome b6 of the b6f complex were identified and the data compared to those previously obtained for the Rhodospirillum rubrum bc1 complex [Le Moigne, C., Schoepp, B., Othman, S., Verméglio, A., and Desbois, A. (1999) Biochemistry 38, 1066-1076]. RR bands arising from the b(6)f-associated chlorophyll a and beta-carotene pigments were found to be particularly intense in the spectra excited at 441.6 nm. The frequencies of the phorbin skeleton of chlorophyll a at 1606, 1552, and 1525 cm(-1) are typical of a Mg atom with a single axial ligand. Strong RR bands corresponding to stretching or deformation modes of beta-carotene were detected at 1137, 1157, 1191, 1216, and 1531 cm(-1) in the different forms of cyt b6f. This set of frequencies is assigned to an all-trans configuration of the polyene chain. The redox titrations of the b(6)f complex allow the characterization of RR bands of the three hemes. The nu10, nu2, nu3, and nu8 modes of reduced cyt f are detected at 1619, 1591, 1492, and 356 cm(-1), respectively. From this set of frequencies, one can conclude that the particular histidine/amine heme coordination found in the truncated soluble domain of cyt f is a specific feature of the entire cyt f included in the b6f complex. The frequencies of the nu2, nu8, and nu10 marker modes are consistent with different conformations for the two b-type hemes of cyt b6f. One of these hemes is strongly distorted (nu2, nu8, and nu10 at 1581, 351, and 1610 cm(-1), respectively), while the other one is planar (1586, 345, and 1618 cm(-1), respectively). Largely different structures for the b-type hemes appear to be a common property for the bc1/b6f complexes.

The reductive dechlorination of perchloroethylene (PCE) in homogeneous solutions of dithionite and at the surfaces of dithionite-citrate-bicarbonate (DCB) treated ferruginous smectite and Na-montmorillonite was studied. Transformation products of PCE identified in dosed dithionite-treated samples included TCE, DCE, 1,1,2-trichloroethane (TCA), 1,1-dichloroethane (DCA), chloroacetylene, acetylene, ethene, and ethane. The decomposition of dithionite to sulfate yielded both protons and electrons necessary for hydrodechlorination (hydrogenolysis) of PCE. Dithionite treatment of the Fe-poor Na-montmorillonite enhanced reductive dechlorination of PCE relative to dithionite-treated Fe-rich ferruginous smectite, within the range of 11.5-137.8 mM dithionite. For the same dithionite concentration, the kinetics of the heterogeneous reactions of PCE was generally faster than that of the homogeneous reaction, and higher concentrations of TCE were measured in the heterogeneous reactions. Interestingly, increases in the mass of the clay minerals used, the Fe2+ content in the clay mineral structure, or the dithionite concentration used did not necessarily enhance the abiotic transformation of PCE, as would otherwise be predicted. The most efficient reductive dechlorination of PCE was observed with 0.5% clay (m/v) treated with 34.5 mM dithionite buffered at pH 8.5. The solid-state transfer of electrons to surfaces and edges, rather than the redox capacity, limited the dechlorination of PCE by reduced ferruginous smectite and/or suspensions containing a higher clay mass. The greater reactivity of dithionite-reduced montmorillonite than similarly treated ferruginous smectite is attributed to (i) the well-documented layer collapse and aggregation of chemically reduced clays that increases with the clay's iron content, (ii) the location of solid-phase Fe2+ in the reduced clay mineral and whether it is accessible or inaccessible for reaction with PCE at the mineral edges and surfaces where the reactions are thought to occur, and (iii) the greater swellability of montmorillonite versus ferruginous smectite. The faster dechlorination rate of PCE observed with dithionite-reduced Fe-poor montmorillonite than similarly reduced iron-rich ferruginous smectite suggests that the use of dithionite barriers for in-situ treatment of chlorinated solvent plumes should not be limited to aquifers with Fe-rich sediments.

Lysine 2,3-aminomutase (KAM) belongs to a class of enzymes that use FeS clusters and S-adenosyl-L-methionine to initiate radical-dependent chemistry. Selenium K-edge X-ray absorption spectroscopic analysis of KAM poised at various stages of catalysis, in the presence of selenomethionine or Se-adenosyl-L-selenomethionine, reveals that the cofactor is cleaved only in the presence of dithionite and the substrate analogue trans-4,5-dehydrolysine. A new Fourier transform peak at 2.7 A, assigned as a Se-Fe interaction, appears concomitant with this cleavage. This is the first demonstration of a direct interaction of S-adenosyl-L-methionine, or its cleavage products, with the FeS cluster in this class of enzymes.

To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.

Cytochrome P450BM-3 is a self-sufficient bacterial protein containing three naturally fused domains which bind either heme, FMN, or FAD. Resolution of protein and FMN from the isolated FMN-containing domain of cytochrome P450Betamicro-3 was accomplished using trichloroacetic acid. The apoprotein thus prepared was shown to rebind FMN to regenerate the original holoprotein as indicated by both spectroscopy and activity measurements. To better understand how the protein/flavin interaction might contribute to reactivity, the association process was studied in detail. Fluorescence quenching was used to measure a dissociation constant of the flavin-protein complex of 31 nM, comparable to FMN-containing proteins of similar reactivity and higher than that of flavodoxins. Stopped-flow kinetics were performed, and a multistep binding process was indicated, with an initial k(on) value of 1.72 x 10(5) M(-)(1) s(-)(1). Preparation of the apoprotein allowed substitution of flavin analogues for the native FMN cofactor using 8-chloro-FMN and 8-amino-FMN. Both were found to bind efficiently to the protein with only minor variations in affinity. Reductive titrations established that, as in the native FMN-containing FMN-binding domain, the 8-amino-FMN-substituted domain does not produce a stable one-electron-reduced species during titration with sodium dithionite. The 8-chloro-FMN-substituted domain, however, had sufficiently altered redox properties to form a stable red anionic semiquinone. The 8-chloro-FMN-substituted FMN-binding domain was shown in reconstituted systems to retain most of the cytochrome c reductase activity of the native domain but only a very small amount of palmitic acid hydroxylase activity. The 8-amino-FMN-substituted FMN-binding domain showed no palmitic acid hydroxylase activity and only 30% of the native cytochrome c reductase activity, demonstrating the importance of thermodynamics to the mechanism of this protein.

Electron transfer in reaction center core (RCC) complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum was studied by measuring flash-induced absorbance changes. The first preparation contained approximately three iron-sulfur centers, indicating that the three putative electron acceptors F(X), F(A), and F(B) were present; the Chl. tepidum complex contained on the average only one. In the RCC complex of Ptc. aestuarii at 277 K essentially all of the oxidized primary donor (P840(+)) created by a flash was rereduced in several seconds by N-methylphenazonium methosulfate. In RCC complexes of Chl. tepidum two decay components, one of 0.7 ms and a smaller one of about 2 s, with identical absorbance difference spectra were observed. The fast component might be due to a back reaction of P840(+) with a reduced electron acceptor, in agreement with the notion that the terminal electron acceptors, F(A) and F(B), were lost in most of the Chl. tepidum complexes. In both complexes the terminal electron acceptor (F(A) or F(B)) could be reduced by dithionite, yielding a back reaction of 170 ms with P840(+). At 10 K in the RCC complexes of both species P840(+) was rereduced in 40 ms, presumably by a back reaction with F(X)(-). In addition, a 350 micros component occurred that can be ascribed to decay of the triplet of P840, formed in part of the complexes. For P840(+) rereduction a pronounced temperature dependence was observed, indicating that electron transfer is blocked after F(X) at temperatures below 200 K.

Described herein is a study of the reductive alkylation chemistry of mitosene antitumor agents. We employed a 13C-enriched electrophilic center to probe the fate of the iminium ion resulting from reductive activation. The 13C-labeled center permitted the identification of complex products resulting from alkylation reactions. In the case of DNA reductive alkylation, the type and number of alkylation sites were readily assessed by 13C NMR. Although there has been much excellent work done in the area of mitosene chemistry and biochemistry, the present study provides a number of new findings: (1) The major fate of the iminium ion is head-to-tail polymerization, even in dilute solutions. (2) Dithionite reductive activation results in the formation of mitosene sulfite esters as well as the previously observed sulfonate adducts. (3) The mitosene iminium ion alkylates the adenosine 6-amino group as well as the guanosine 2-amino group. The identification of the latter adduct was greatly facilitated by the 13C-label at the electrophilic center. (4) The mitosene iminium ion alkylates DNA at both nitrogen and oxygen centers without any apparent base selectivity. The complexity of mitosene reductive alkylation of DNA will require continued adduct isolation studies.

Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.

The (57)Fe-enriched cytochrome bf complex has been isolated from hydrocultures of spinach. It has been studied at different redox states by optical, EPR, and Mössbauer spectroscopy. The Mössbauer spectrum of the native complex at 190 K with all iron centers in the oxidized state reveals the presence of four different iron sites: low-spin ferric iron in cytochrome b [with an isomer shift (delta) of 0.20 mm/s, a quadrupole splitting (DeltaE(Q)) of 1.77 mm/s, and a relative area of 40%], low-spin ferric iron of cytochrome f (delta = 0.26 mm/s, DeltaE(Q) = 1.90 mm/s, and a relative area of 20%), and two high-spin ferric iron sites of the Rieske iron-sulfur protein (ISP) with a bis-cysteine and a bis-histidine ligated iron (delta(1) = 0.15 mm/s, DeltaE(Q1) = 0.70 mm/s, and a relative area of 20%, and delta(2) = 0.25 mm/s, DeltaE(Q2) = 0.90 mm/s, and a relative area of 20%, respectively). EPR and magnetic Mössbauer measurements at low temperatures corroborate these results. A crystal-field analysis of the EPR data and of the magnetic Mössbauer data yields estimates for the g-tensors (g(z)(), g(y)(), and g(x)()) of cytochrome b (3.60, 1.35, and 1.1) and of cytochrome f (3.51, 1.69, and 0.9). Addition of ascorbate reduces not only the iron of cytochrome f to the ferrous low-spin state (delta = 0.43 mm/s, DeltaE(Q) = 1.12 mm/s at 4.2 K) but also the bis-histidine coordinated iron of the Rieske 2Fe-2S center to the ferrous high-spin state (delta(2) = 0.73 mm/s, DeltaE(Q2) = -2.95 mm/s at 4.2 K). At this redox step, the Mössbauer parameters of cytochrome b have not changed, indicating that the redox changes of cytochrome f and the Rieske protein did not change the first ligand sphere of the low-spin ferric iron in cytochrome b. Reduction with dithionite further reduces the two hemes of cytochrome b to the ferrous low-spin state (delta = 0.49 mm/s, DeltaE(Q) = 1.08 mm/s at 4.2 K). The spin Hamiltonian analysis of the magnetic Mössbauer spectra at 4.2 K yields hyperfine parameters of the reduced Rieske 2Fe-2S center in the cytochrome bf complex which are very similar to those reported for the Rieske center from Thermus thermophilus [Fee, J. A., Findling, K. L., Yoshida, T., et al. (1984) J. Biol. Chem. 259, 124-133].

A new quinohemoprotein amine dehydrogenase from Paracoccus denitrificans IFO 12442 was isolated and characterized in views of biochemistry and electrochemistry. This enzyme exists in periplasm and catalyzes the oxidative deamination of primary aliphatic and aromatic amines. n-Butylamine or benzylamine as a carbon and energy source strongly induces the expression of the enzyme. Carbonyl reagents inhibit the enzyme activity irreversibly. This enzyme is a heterodimer constituted of alpha and beta subunits with the molecular mass of 59.5 and 36.5 kDa, respectively. UV-vis and EPR spectroscopy, and the quinone-dependent redox cycling and heme-dependent peroxidative stains of SDS-PAGE bands revealed that the alpha subunit contains one quinonoid cofactor and one heme c per molecule, while the beta subunit has no prosthetic group. The redox potential of the heme c moiety was determined to be 0.192 V vs NHE at pH 7.0 by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique. The analysis of the substrate titration curve allowed the evaluation of the redox potential of the quinone/semiquinone and semiquinone/quinol redox couples as 0.19 and 0.11 V, respectively.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its Mössbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.