Hole-scavenging in photo-driven N2 reduction catalyzed by a CdS-nitrogenase MoFe protein biohybrid system.

The light-driven reduction of dinitrogen (N2) to ammonia (NH3) catalyzed by a cadmium sulfide (CdS) nanocrystal­nitrogenase MoFe protein biohybrid is dependent on a range of different factors, including an appropriate hole-scavenging sacrificial electron donor (SED). Here, the impact of different SEDs on the overall rate of N2 reduction catalyzed by a CdS quantum dot (QD)-MoFe protein system was determined. The selection of SED was guided by several goals: (i) molecules with standard reduction potentials sufficient to reduce the oxidized CdS QD, (ii) molecules that do not absorb the excitation wavelength of the CdS QD, and (iii) molecules that could be readily reduced by sustainable processes. Earlier studies utilized buffer molecules or ascorbic acid as the SED. The effectiveness of ascorbic acid as SED was compared to dithionite (DT), triethanolamine (TEOA), and hydroquinone (HQ) across a range of concentrations in supporting N2 reduction to NH3 in a CdS QD-MoFe protein photocatalytic system. It was found that TEOA supported N2 reduction rates comparable to those observed for dithionite and ascorbic acid. HQ was found to support significantly higher rates of N2 reduction compared to the other SEDs at a concentration of 50 mM. A comparison of the rates of N2 reduction by the biohybrid complex to the standard reduction potential (Eo) of the SEDs reveals that Eo is not the only factor impacting the efficiency of hole-scavenging. These findings reveal the importance of the SED properties for improving the efficiency of hole-scavenging in the light-driven N2 reduction reaction catalyzed by a CdS QD-MoFe protein hybrid.

Dithionite promoted microbial dechlorination of hexachlorobenzene while goethite further accelerated abiotic degradation by sulfidation in paddy soil.

It is of great scientific and practical importance to explore the mechanisms of accelerated degradation of Hexachlorobenzene (HCB) in soil. Both iron oxide and dithionite may promote the reductive dechlorination of HCB, but their effects on the microbial community and the biotic and abiotic mechanisms behind it remain unclear. This study investigated the effects of goethite, dithionite, and their interaction on microbial community composition and structure, and their potential contribution to HCB dechlorination in a paddy soil to reveal the underlying mechanism. The results showed that goethite addition alone did not significantly affect HCB dechlorination because the studied soil lacked iron-reducing bacteria. In contrast, dithionite addition significantly decreased the HCB contents by 44.0-54.9%, while the coexistence of dithionite and goethite further decreased the HCB content by 57.9-69.3%. Random Forest analysis suggested that indicator taxa (Paenibacillus, Acidothermus, Haliagium, G12-WMSP1, and Frankia), Pseudomonas, richness and Shannon's index of microbial community, and immobilized Fe content were dominant driving factors for HCB dechlorination. The dithionite addition, either with or without goethite, accelerated HCB anaerobic dechlorination by increasing microbial diversity and richness as well as the relative abundance of the above specific bacterial genera. When goethite and dithionite coexist, sulfidation of goethite with dithionite could remarkably increase FeS formation and then further promote HCB dechlorination rates. Overall, our results suggested that the combined application of goethite and dithionite could be a practicable strategy for the remediation of HCB contaminated soil.

A Limitation of Using Dithionite Quenching to Determine the Topology of Membrane-inserted Proteins.

Determining the topology of membrane-inserted proteins and peptides often relies upon indirect fluorescent measurements. One such technique uses NBD, an environmentally sensitive fluorophore that can be covalently linked to proteins. Relative to a hydrophilic environment, NBD in a hydrophobic environment shows an increase in emission intensity and a shift to shorter wavelengths. To gain further insight, NBD fluorescence can be chemically quenched using dithionite. As dithionite is an anion, it is only expected to penetrate the outer leaflet interfacial region and should be excluded from the hydrocarbon core, the inner leaflet, and the lumen of LUV. This assumption holds at neutral pH, where a large number of NBD/dithionite experiments are carried out. Here, we report control experiments in which LUV were directly labeled with NBD-PE to assess dithionite quenching in acidic conditions. Results showed that at acidic pH, dithionite moved more freely across the bilayer to quench the inner leaflet. For the buffer conditions used, dithionite exhibited a sharp change in behavior between pH 5.5 and 6.0. Therefore, in acidic conditions, dithionite could not differentiate in which leaflet the NBD resided.

Synthesis of Na2S2O4 mediated cleavable affinity tag for labeling of O-GlcNAc modified proteins via azide-alkyne cycloaddition.

A facile and convergent procedure for the synthesis of azobenzene-based probe was reported, which could selectively release interested proteins conducted with sodium dithionite. Besides, the cleavage efficiency is closely associated with the structural features, in which an ortho-hydroxyl substituent is necessary for reactivity. In addition, the azobenzene tag applied in the Ac4GlcNAz-labled proteins demonstrated high efficiency and selectivity in comparison with Biotin-PEG4-Alkyne, which provides a useful platform for enrichment of any desired bioorthogonal proteomics.

Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2.

All radical S-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1-Dph2, require at least one [4Fe-4S](Cys)3 cluster for activity. It is well-known in the radical-SAM enzyme community that the [4Fe-4S](Cys)3 cluster is extremely air-sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1-Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe-4S] cluster is easily degraded into a [3Fe-4S] cluster. Remarkably, the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. This mechanism to maintain radical-SAM enzyme activity in aerobic environments is likely general, and Dph3-like proteins may exist to keep other radical-SAM enzymes functional in aerobic environments.

Impact of the chemicals, essential for the purification process of strict Fe-hydrogenase, on the corrosion of mild steel.

The influence of additional chemical molecules, necessary for the purification process of [Fe]-hydrogenase from Clostridium acetobutylicum, was studied on the anaerobic corrosion of mild steel. At the end of the purification process, the pure [Fe-Fe]-hydrogenase was recovered in a Tris-HCl medium containing three other chemicals at low concentration: DTT, dithionite and desthiobiotin. Firstly, mild steel coupons were exposed in parallel to a 0.1 M pH7 Tris-HCl medium with or without pure hydrogenase. The results showed that hydrogenase and the additional molecules were in competition, and the electrochemical response could not be attributed solely to hydrogenase. Then, solutions with additional chemicals of different compositions were studied electrochemically. DTT polluted the electrochemical signal by increasing the Eoc by 35 mV 24 h after the injection of 300 µL of control solutions with DTT, whereas it drastically decreased the corrosion rate by increasing the charge transfer resistance (Rct 10 times the initial value). Thus, DTT was shown to have a strong antagonistic effect on corrosion and was removed from the purification process. An optimal composition of the medium was selected (0.5 mM dithionite, 7.5 mM desthiobiotin) that simultaneously allowed a high activity of hydrogenase and a lower impact on the electrochemical response for corrosion tests.

Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis.

Fosfomycin is a broad-spectrum antibiotic that is useful against multi-drug resistant bacteria. Although its biosynthesis was first studied over 40 years ago, characterization of the penultimate methyl transfer reaction has eluded investigators. The enzyme believed to catalyze this reaction, Fom3, has been identified as a radical S-adenosyl-L-methionine (SAM) superfamily member. Radical SAM enzymes use SAM and a four-iron, four-sulfur ([4Fe-4S]) cluster to catalyze complex chemical transformations. Fom3 also belongs to a family of radical SAM enzymes that contain a putative cobalamin-binding motif, suggesting that it uses cobalamin for methylation. Here we describe the first biochemical characterization of Fom3 from Streptomyces wedmorensis. Since recombinant Fom3 is insoluble, we developed a successful refolding and iron-sulfur cluster reconstitution procedure. Spectroscopic analyses demonstrate that Fom3 binds a [4Fe-4S] cluster which undergoes a transition between a +2 "resting" state and a +1 active state characteristic of radical SAM enzymes. Site-directed mutagenesis of the cysteine residues in the radical SAM CxxxCxxC motif indicates that each residue is essential for functional cluster formation. We also provide preliminary evidence that Fom3 adds a methyl group to 2-hydroxyethylphosphonate (2-HEP) to form 2-hydroxypropylphosphonate (2-HPP) in an apparently SAM-, sodium dithionite-, and methylcobalamin-dependent manner.

The hydrogenase activity of the molybdenum/copper-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans.

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H2 has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H2 with a limiting rate constant of 5.3 s(-1) and a dissociation constant Kd of 525 µM; both kred and kred/Kd, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H2, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site (63,65)Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H2 oxidation is proposed which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.

[2Fe-2S] proteins in Chlorosomes: CsmI and CsmJ participate in light-dependent control of energy transfer in Chlorosomes of Chlorobaculum tepidum.

Chlorosomes of Chlorobaculum tepidum are formed from stacks of syn-anti coordinated bacteriochlorophyll c dimers, which form a suprastructure comprised of coaxial nanotubes and are surrounded by a glycolipid monolayer envelope containing 10 proteins. Three of these proteins, CsmI, CsmJ, and CsmX, have sequences very similar in their N-terminal domains to those of [2Fe-2S] ferredoxins of the adrenodoxin/putidaredoxin subfamily. The roles of these proteins in chlorosomes were studied in single-, double-, and triple-mutant strains. In each mutant, only the protein(s) corresponding to the mutated gene(s) was missing, and the amounts of other chlorosome proteins did not vary significantly. Electrophoretic analyses and immunoblotting showed that CsmX was much less abundant than CsmI or CsmJ. The growth rates and the pigment and isoprenoid quinone contents of isolated chlorosomes of the mutants were similar to wild-type values. Quenching and recovery of energy transfer in isolated chlorosomes and intact cells were studied by measuring fluorescence emission after exposure to or removal of oxygen. Oxygen-induced activation of the quencher in isolated chlorosomes or in intact cells was largely independent of CsmI and CsmJ. This may be because oxygen can diffuse across the chlorosome envelope easily and directly reacts with the quencher. However, CsmI and CsmJ were required to restore energy transfer fully after isolated chlorosomes were exposed to oxygen. Studies with intact cells suggested that cells contain both light-dependent and light-independent pathways for reducing the quenching species in chlorosomes and that CsmI and CsmJ are components of a light-dependent pathway.

Oxidative and reductive routes of glycerol and glucose fermentation by Escherichia coli batch cultures and their regulation by oxidizing and reducing reagents at different pHs.

Glycerol and glucose fermentation redox routes by Escherichia coli and their regulation by oxidizing and reducing reagents were investigated at different pHs. Cell growth was followed by decrease of pH and redox potential (E ( h )). During glycerol utilization at pH 7.5 ∆pH, the difference between initial and end pH, was lower compared with glucose fermentation. After 8 h growth, during glycerol utilization E ( h ) dropped down to negative values (-150 mV) but during glucose fermentation it was positive (+50 mV). In case of glycerol H(2) was evolved at the middle log phase while during glucose fermentation H(2) was produced during early log phase. Furthermore, upon glycerol utilization, oxidizer potassium ferricyanide (1 mM) inhibited both cell growth and H(2) formation. Reducing reagents DL-dithiothreitol (3 mM) and dithionite (1 mM) inhibited growth but stimulated H(2) production. The findings point out the importance of reductive conditions for glycerol fermentation and H(2) production by E. coli.

Reaction of human cytochrome P450 3A4 with peroxynitrite: nitrotyrosine formation on the proximal side impairs its interaction with NADPH-cytochrome P450 reductase.

The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.

Radical formation in cytochrome c oxidase.

The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O(2) redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O(2) with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77K at reaction times ranging from 50µs to 6ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO(2)(-) ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O(2) with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H(2)O(2)) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50µs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O(2) and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a(3). This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".

Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors.

Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).

Characterization of an NADH-dependent persulfide reductase from Shewanella loihica PV-4: implications for the mechanism of sulfur respiration via FAD-dependent enzymes.

The NADH-dependent persulfide reductase (Npsr), a recently discovered member of the PNDOR family of flavoproteins that contains both the canonical flavoprotein reductase domain and a rhodanese domain, is proposed to be involved in the dissimilatory reduction of S(0) for Shewanella loihica PV-4. We have previously shown that polysulfide is a substrate for this enzyme, and a recently determined structure of a closely related enzyme (CoADR-Rhod from Bacillus anthracis) suggested the importance of a bound coenzyme A in the mechanism. The work described here shows that the in vivo oxidizing substrates of Npsr are the persulfides of small thiols such as CoA and glutathione. C43S, C531S, and C43,531S mutants were created to determine the role of the flavoprotein domain cysteine (C43) and the rhodanese domain cysteine (C531) in the mechanism. The absolute requirement for C43 in persulfide or DTNB reductase activity shows that this residue is involved in S-S bond breakage. C531 contributes to, but is not required for, catalysis of DTNB reduction, while it is absolutely required for reduction of any persulfide substrates. Titrations of the enzyme with NADH, dithionite, titanium(III), or TCEP demonstrate the presence of a mixed-disulfide between C43 and a tightly bound CoA, and structures of the C43 and C43,531S mutants confirm that this coenzyme A remains tightly bound to the enzyme in the absence of a C43-CoA S-S bond. The structure of Npsr suggests a likely site for binding and reaction with the persulfide substrate on the rhodanese domain. On the basis of kinetic, titration, and structural data, a mechanism for the reduction of persulfides by Npsr is proposed.

The aging-associated enzyme CLK-1 is a member of the carboxylate-bridged diiron family of proteins.

The aging-associated enzyme CLK-1 is proposed to be a member of the carboxylate-bridged diiron family of proteins. To evaluate this hypothesis and characterize the protein, we expressed soluble mouse CLK-1 (MCLK1) in Escherichia coli as a heterologous host. Using Mössbauer and EPR spectroscopy, we established that MCLK1 indeed belongs to this protein family. Biochemical analyses of the in vitro activity of MCLK1 with quinone substrates revealed that NADH can serve directly as a reductant for catalytic activation of dioxygen and substrate oxidation by the enzyme, with no requirement for an additional reductase protein component. The direct reaction of NADH with a diiron-containing oxidase enzyme has not previously been encountered for any member of the protein superfamily.

Kinetics of cryptdin-4 translocation coupled with peptide-induced vesicle leakage.

The antimicrobial peptide cryptdin-4 (Crp4), a member of the alpha-defensin family, is shown to translocate cooperatively across phospholipid bilayers. The cooperativity of the process is manifested by translocation kinetics which vary with the peptide to lipid molar ratio. A simple association model suggests dimerization. Black lipid membrane experiments reveal that Crp4 translocation does not create well-defined aqueous pores, as is often common among peptides exhibiting cooperative translocation. Still, the efflux induced by Crp4 upon its interaction with fluorophore-loaded vesicles is shown to be a direct result of the membrane perturbation resulting from the translocation process. Leakage can be predicted by relating membrane permeability to the fraction of peptide translocated. Crp4 translocation has implications for its antimicrobial activity as internalized peptide would be available to attack intracellular targets.

Tricarballylate catabolism in Salmonella enterica. The TcuB protein uses 4Fe-4S clusters and heme to transfer electrons from FADH2 in the tricarballylate dehydrogenase (TcuA) enzyme to electron acceptors in the cell membrane.

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.

S-nitrosylation-induced conformational change in blackfin tuna myoglobin.

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase.

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.

Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging.

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.