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PubMed2002

Paradoxical redox properties of DsbB and DsbA in the protein disulfide-introducing reaction cascade.

Protein disulfide bond formation in the bacterial periplasm is catalyzed by the Dsb enzymes in conjunction with the respiratory quinone components. Here we characterized redox properties of the redox active sites in DsbB to gain further insights into the catalytic mechanisms of DsbA oxidation. The standard redox potential of DsbB was determined to be -0.21 V for Cys41/Cys44 in the N-terminal periplasmic region (P1) and -0.25 V for Cys104/Cys130 in the C-terminal periplasmic region (P2), while that of Cys30/Cys33 in DsbA was -0.12 V. To our surprise, DsbB, an oxidant for DsbA, is intrinsically more reducing than DsbA. Ubiquinone anomalously affected the apparent redox property of the P1 domain, and mutational alterations of the P1 region significantly lowered the catalytic turnover. It is inferred that ubiquinone, a high redox potential compound, drives the electron flow by interacting with the P1 region with the Cys41/Cys44 active site. Thus, DsbB can mediate electron flow from DsbA to ubiquinone irrespective of the intrinsic redox potential of the Cys residues involved.

Amino Acid Motifs↗

PubMed1982

Chemical modification of spinach plastocyanin: separation and characterization of four different forms.

Spinach plastocyanin has been chemically modified by using a water-soluble carbodiimide to form an amide bond between a protein carboxyl group and one amino group of ethylenediamine. This reaction produced four distinct species of chemically modified plastocyanin which could be separated either by polyacrylamide gel electrophoresis (in the absence of sodium dodecyl sulfate) or by ion-exchange chromatography on DEAE-Sephadex. The four forms contained 2.1, 3.2, 4.1, and 6.3 mol of ethylenediamine per mol of plastocyanin and were labeled fractions IV, III, II, and I, respectively. All four fractions showed a shift in midpoint redox potential of greater than +40 mV. In contrast, the Km for P700+ reduction decreased with increasing extents of modification. The addition of Mg2+ ions decreased the Km for the negatively charged fractions II, III, and IV but increased it for the positively charged fraction I. Fraction III showed an increased incorporation of ethylenediamine into residues 42-45 which are located approximately 18 A distant from the copper. This fraction also showed a maximal increase in redox potential. Potential calculations showed that the electrostatic effect of incorporating two positive charges at this distance was sufficient to produce a shift in redox potential of +40 mV. However, an electrostatically induced conformational change which would affect the environment of the copper cannot be ruled out.

Carbodiimides↗

PubMed2006

Role of protein frame and solvent for the redox properties of azurin from Pseudomonas aeruginosa.

We have coupled hybrid quantum mechanics (density functional theory; Car-Parrinello)/molecular mechanics molecular dynamics simulations to a grand-canonical scheme, to calculate the in situ redox potential of the Cu(2+) + e(-) --> Cu(+) half reaction in azurin from Pseudomonas aeruginosa. An accurate description at atomistic level of the environment surrounding the metal-binding site and finite-temperature fluctuations of the protein structure are both essential for a correct quantitative description of the electronic properties of this system. We report a redox potential shift with respect to copper in water of 0.2 eV (experimental 0.16 eV) and a reorganization free energy lambda = 0.76 eV (experimental 0.6-0.8 eV). The electrostatic field of the protein plays a crucial role in fine tuning the redox potential and determining the structure of the solvent. The inner-sphere contribution to the reorganization energy is negligible. The overall small value is mainly due to solvent rearrangement at the protein surface.

Azurin↗

PubMed2000

Copper--ligand interactions and the physiological free radical processes. Part 3. Influence of histidine, salicylic acid and anthranilic acid on copper-driven Fenton chemistry in vitro.

With a view to the possible use of copper(II)-*OH inactivating ligand (OIL) complexes as regulators of inflammation, the reactivity of the copper(II)-ascorbate system with hydrogen peroxide has been investigated in the presence of three key substances: histidine (the main copper(II) low molecular mass ligand in extracellular fluid), salicylic acid (the well-known nonsteroidal antiinflammatory drug, previously shown to be potentiated by copper(II) in animal models of inflammation), and anthranilic acid (an inactive substance by itself, known to be activated by copper(II) in the same models) at physiological pH (7.4) and inflammatory pH (5.5). Such substances may affect the amount of TBARS detected in solution following copper-mediated Fenton-like reactions through three distinct mechanisms: (i) by decreasing the Cu(II)/Cu(I) redox potential, i.e. at the expense of *OH radical production, (ii) by scavenging *OH radicals in the body of the solution, and/or (iii) by acting as a true OIL, i.e. at the expense of *OH detection. Redox potential measurements of initial solutions have been performed in parallel to TBARS determinations to help discriminate between different ligand influences. Computer-aided speciation has been used to understand the role of copper(II) distribution on the ligand effects characterised. Contrary to previous interpretations, histidine has been found to mainly affect *OH production by lowering the redox potential of the Cu(II)/Cu(I) couple. Salicylate, which has no effect on *OH production, has been confirmed to mainly scavenge *OH radicals in the body of the solution. Anthranilate, which both increases *OH production and decreases *OH detection, behaves as a potential OIL. These results tend to confirm our previous hypothesis that copper potentiation of antiinflammatory substances is indirect, i.e. independent of any interaction between metal and drug, whereas copper activation of substances that are inactive by themselves results from specific metal-substance interactions taking place at inflammatory sites.

Copper↗

PubMed1998

Ingredient supplementation effects on viability of probiotic bacteria in yogurt.

The present investigation studied the effects of cysteine, whey powder, whey protein concentrate, acid casein hydrolysates, or tryptone on the viability of Streptococcus thermophilus, Lactobacillus acidophilus, and bifidobacteria. Changes in pH, titratable acidity, redox potential, and viability of bacteria were monitored during 24 h of fermentation and refrigerated storage (4 degrees C) of yogurt for 35 d. The incubation time that was needed to reach pH 4.5 was considerably affected by the added ingredients. Also, the drop in pH or the increase in acidity and redox potential was dependent on the added ingredients. The addition of cysteine, whey protein concentrate, acid casein hydrolysates, or tryptone improved the viability of bifidobacteria to a variable extent, but whey powder failed to improve their viability. The morphology of S. thermophilus, as shown by electron microscopy, was affected by cysteine at 500 mg/L, possibly as a result of reduced redox potential. Sodium dodecyl sulfate-PAGE and amino acid analyses suggested that the nitrogen source in the form of peptides and amino acids improved the viability of bifidobacteria in yogurt made with a commercial ABT (Lactobacillus acidophilus, bifidobacteria, and Streptococcus thermophilus) starter culture, which showed a dramatic decline in the counts of this organism in previous studies.

Bacteria↗

PubMed2004

Perfluorocarbon emulsion improves cerebral oxygenation and mitochondrial function after fluid percussion brain injury in rats.

OBJECTIVE: Cerebral ischemia is a common secondary sequela of traumatic brain injury (TBI). Experimental models of stroke have demonstrated reductions in ischemia after perfluorocarbon (PFC) administration; however, there are no published reports of PFC efficacy after TBI. The current study analyzed the effect of the PFC emulsion Oxygent (AF0144; Alliance Pharmaceutical Corp., San Diego, CA) on cerebral oxygenation, mitochondrial redox potential, and free radical formation after lateral fluid percussion injury. METHODS: After fluid percussion injury, five 2.25 ml/kg doses of PFC or saline were administered to rats breathing 100% O(2), and oxygen tension was recorded. In a second experiment, a single bolus (11.25 ml/kg) of PFC or saline was given after injury, and redox potential and free radical formation were measured at 1 or 4 hours with Alamar blue dye and dihydrorhodamine 123, respectively. RESULTS: Cerebral oxygen tension was significantly increased in both injured and sham animals treated with 11.25 ml/kg of PFC as compared with saline (P < 0.05). Likewise, PFC significantly increased mitochondrial redox potential as compared with saline at 4 hours after injury (P < 0.01). Mitochondrial peroxynitrite and peroxide production also increased with the administration of PFC (P < 0.05). CONCLUSION: The current study demonstrates that a PFC emulsion can significantly increase cerebral oxygenation after TBI and enhance mitochondrial function at 4 hours after injury as compared with saline. This study demonstrates a new therapeutic potential for PFC to enhance cerebral oxygenation and aerobic metabolism after TBI. However, the increased free radical formation with high-dose PFCs suggests the need for further studies combining PFCs with free radical scavengers.

Animals↗

PubMed1999

Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin in the alcohol oxidase respiratory chain of Pseudomonas putida HK5.

A blue copper protein was purified together with a type II quinohemoprotein alcohol dehydrogenase (ADH IIB) from the soluble fraction of Pseudomonas putida HK5 grown on n-butanol. The purified blue copper protein was shown to be azurin, on the basis of several properties such as its absorption maximum (623 nm), its low molecular mass (17 500 Da), its acidic nature (pI of 4.1), its relatively high redox potential (306 mV), the presence of an intramolecular disulfide bond, and N-terminal amino acid sequence homology with respect to azurins from other sources, especially from P. putida NCIB 9869 and Pseudomonas fluorescens. Direct electron transfer from ADH IIB to azurin was shown to occur at a rate of 48-70 s-1. The apparent Km value of ADH IIB for azurin, determined by steady-state kinetics, was decreased several-fold by increasing the ionic strength. Furthermore, the extent of fluorescence quenching of ADH IIB due to the interaction with azurin was increased by increasing the ionic strength, but the binding constant for binding between ADH IIB and azurin was unchanged. The redox potential of azurin was increased 12 mV by incubation with ADH but not vice versa. Furthermore, the redox potential gap between ADH and azurin was increased from 102 to 126 mV by increasing the ionic strength. It is conceivable that a hydrophobic interaction is involved in the electron transfer between both proteins, and it is also suggested that the electron transfer may occur by a freely reversible on and off binding process but may not be related to the global binding process of both proteins. Thus, the results presented here strongly suggest that azurin works as an electron-transfer mediator in a PQQ-dependent alcohol oxidase respiratory chain in P. putida HK5.

Alcohol Dehydrogenase↗

PubMed1988

DT-diaphorase-catalyzed two-electron reduction of various p-benzoquinone- and 1,4-naphthoquinone epoxides.

The oxidation of various quinones by H2O2 results in quinone epoxide formation. The yield of epoxidation is inversely related to the degree of methyl substitution of the quinone and seems not to be dependent on the redox potential of the quinones studied. The following order of H2O2-mediated epoxidation of quinones was found: p-benzoquinone greater than or equal to 1,4-naphthoquinone greater than 2-methyl-p-benzoquinone greater than 2,6-dimethyl-p-benzoquinone greater than or equal to 2-methyl-1,4-naphthoquinone greater than 2,3-dimethyl-1,4-naphthoquinone. DT-Diaphorase reduces several quinone epoxides at different rates. The rate of quinone epoxide reduction cannot be related to either the redox potential of the quinone epoxide (as reflected by the half-wave potential calculated from the corresponding hydrodynamic voltamograms) or the degree of substitution of the quinone epoxide. It appears, however, that a quinone epoxide redox potential more negative than -0.5 to -0.6 volts settles a threshold for the electron transfer reaction. This does not exclude that specificity requirements, i.e. the formation of the quinone epoxide substrate-enzyme complex may chiefly determine the rate of reduction of quinone epoxides by DT-diaphorase. DT-diaphorase-catalyzed two-electron transfer to quinone epoxides--resulting in epoxide ring opening--yields 2-OH-p-benzohydroquinone or 2-OH-1,4-naphthohydroquinone products. These hydroxy-derivatives show a higher rate of autoxidation than do the parent hydroquinones lacking the OH substituent.

Benzoquinones↗

PubMed2002

Behavior of hydrogen peroxide in electrolyzed anode water.

Oxygen electrodes and spectrophotometric analysis have been used to evaluate the contribution of H2O2, in addition to available chlorine, to the high redox potential of electrolyzed anode water (EAW) with potassium chloride as an electrolyte. H2O2 was added externally to EAW, and the reaction between H2O2 and the available chlorine in the water was examined. EAW has a low pH (2.5), a high concentration of dissolved oxygen, and extremely high redox potentials (19 mg/l and 1,319 mV) when the available chlorine is at the concentration of about 580 microM. The addition of H2O2 to EAW led to H2O2 decomposition, and the amount of oxygen produced was equivalent to the amount of available chlorine. Oxygen production was reduced by ascorbic acid, and completely inhibited by 600 microM ascorbate. The rate of oxygen production was much affected by pH, and was slowest at or near pH 5.0. Rates were particularly high in alkaline solution. Absorbance at 235 nm (pH 3.0 and 5.0) and 292 nm (pH 10.0) decreased when H2O2 was added to the EAW at these pHs, and the extent of decrease was similar pH dependency to that of the oxygen production rate. Oxygen was not produced after H2O2 was added to EAW at pH 2.6 when available chlorine was absent, but oxygen was produced after potassium hypochlorite was added to such EAW. The oxygen production rates in EAW without available chlorine at pH 5.0 and 2.0, pH adjustment with KOH and HCl, respectively, were faster than the rate at pH 2.6, and fastest at pH 2.0. These results suggest that H2O2 or hydroxyl radicals derived from Fenton's reaction did not contribute to the high redox potential of EAW prepared with chlorine compounds as an electrolyte, so that the decomposition of H2O2 occurred rapidly with the reactions of chlorine and hypochlorite ions in EAW.

Ascorbic Acid↗

PubMed2004

Prediction of pKa and redox properties in the thioredoxin superfamily.

Electrostatic interactions play important roles in diverse biological phenomena controlling the function of many proteins. Polar molecules can be studied with the FDPB method solving the Poisson-Boltzmann equation on a finite difference grid. A method for the prediction of pK(a)s and redox potentials in the thioredoxin superfamily is introduced. The results are compared with experimental pK(a) data where available, and predictions are made for members lacking such data. Studying CxxC motif variation in the context of different background structures permits analysis of contributions to cysteine DeltapK(a)s. The motif itself and the overall framework regulate pK(a) variation. The reported method includes generation of multiple side-chain rotamers for the CxxC motif and is an effective predictive tool for functional pK(a) variation across the superfamily. Redox potential follows the trend in cysteine pK(a) variation, but some residual discrepancy indicates that a pH-independent factor plays a role in determining redox potentials for at least some members of the superfamily. A possible molecular basis for this feature is discussed.

Amino Acid Motifs↗

PubMed2004

New equations for redox and nano-signal transduction.

Cells maintain redox potentials (Eh) in intracellular compartments, sometimes referred to as redox environments. These potentials are often very reducing, for example in the cytoplasm, but throughout the cell different potentials are maintained, commensurate with the functionality of that particular part of the cell. Furthermore, within a simple cellular compartment, "hot-spots" of redox poise may be maintained. However, despite this complexity, the quantification of such redox potentials has been attempted, and there is indeed a need to accurately assess such potentials, and to monitor how they might change with time. Changes in intracellular potentials may control the oxidation or reduction of protein residues, such as cysteine, which would alter the conformation of those proteins and so modulate their function. Although there are several methods for estimating the intracellular redox potential, the most accessible technique is the measurement of intracellular concentrations of GSH and GSSG, and the calculation of Eh using the Nernst equation. However, using this equation shows that the Eh imposed by the glutathione couple is dependent on the total concentration of glutathione present, and therefore values of Eh obtained may be erroneous. Here, we suggest new equations that can be used to calculate the redox environments of cells.

Animals↗

PubMed1999

Density Functional and Electrostatic Calculations of Manganese Superoxide Dismutase Active Site Complexes in Protein Environments.

Density functional and electrostatic methods have been applied to calculate active site geometries and the redox potential of manganese superoxide dismutase (MnSOD). The initial active site clusters were built up by including only first-shell side chain ligands and then augmented by second-shell ligands. The density functional optimized Mn-ligand bond lengths for the reduced complexes in general compared fairly well with protein crystallography data; however, large deviations for calculated Mn-OH distances were found for the oxidized active site clusters. Our calculations suggest that this deviation can be attributed to the redox heterogeneity of the oxidized protein in X-ray crystallography studies. The redox potential was calculated by treating the protein environment and the solvent bulk by a semimacroscopic electrostatic model. The protein structures were taken from the Thermus thermophilus enzyme. The calculated coupled redox potentials converge toward experimental values with increasing size of the active site cluster models, and the final calculated value was +0.06 V, compared to experimental values of +0.26 V determined for Bacillus stearothermophilus and +0.31 V in Escherichia coli enzymes. Using an energy decomposition scheme, the effects of the second-shell ligands and the protein and reaction fields have been analyzed.

Journal Article↗

PubMed2001

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli.

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

Catalytic Domain↗

PubMed2003

[Effect of technological process in bottled water production on its redox properties].

The redox potential was measured in selected natural mineral and spring waters of different origin and chemical composition. To analyse the effect of the technological process the samples to be studied were collected at different stages of the bottling process: directly at the intake, at the beginning of the pipeline, after the deironing and/or demanganisation, after filtration, at the end of the pipeline and at the end of the process from the freshly filled bottles. Along with the redox potential measurements, some physico-chemical parameters of the samples studied were determined: colour, turbidity, conductivity, pH value, susceptibility to oxidation, contents of iron, manganese, antimony and arsenic. The potentiometric method applied to measure the redox potential was found suitable for the purpose of the study.

Consumer Product Safety↗

PubMed1996

Multiple wavelength anomalous diffraction (MAD) crystal structure of rusticyanin: a highly oxidizing cupredoxin with extreme acid stability.

The X-ray crystal structure of the oxidized form of the extremely stable and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans has been determined by the method of multiwavelength anomalous diffraction (MAD) and refined to 1.9 A resolution. Like other cupredoxins, rusticyanin is a copper-containing metalloprotein, which is composed of a core beta-sandwich fold. In rusticyanin the beta-sandwich is composed of a six- and a seven-stranded beta-sheet. Also like other cupredoxins, the copper ion is coordinated by a cluster of four conserved residues (His85, Cys138, His143, Met148) arranged in a distorted tetrahedron. Rusticyanin has a redox potential of 680 mV, roughly twice that of any other cupredoxin, and it is optimally active at pH values < or = 2. By comparison with other cupredoxins, the three-dimensional structure of rusticyanin reveals several possible sources of the chemical differences, including more ordered secondary structure and more intersheet connectivity than other cupredoxins. The acid stability and redox potential of rusticyanin may also be enhanced over other cupredoxins by a more extensive internal hydrogen bonding network and by more extensive hydrophobic interactions surrounding the copper binding site. Finally, reduction in the number of charged residues surrounding the active site may also make a major contribution to acid stability. We propose that the resulting rigid copper binding site, which is constrained by the surrounding hydrophobic environment, structurally and electronically favours Cu(I). We propose that the two extreme chemical properties of rusticyanin are interrelated; the same unique structural features that enhance acid stability also lead to elevated redox potential.

Azurin↗

PubMed1985

Potentiometric studies of native and flavin-substituted Old Yellow Enzyme.

We have measured the redox potentials for the flavin cofactor of native Old Yellow Enzyme and for a series of chemically modified flavin derivatives bound to the apoprotein. These flavin derivatives have midpoint potentials ranging from -120 to -300 mV in free solution. For the native enzyme, the midpoint potential of the first one-electron couple EFMNox + e- in equilibrium EFMN-. is E1 = -245 +/- 5 mV and that for the second one-electron couple EFMN-. + e- in equilibrium EFMN red is E2 = -215 +/- 5 mV in 0.1 M phosphate buffer at pH 7.0 and 25 degrees C. Thus, the apoprotein lowers the two-electron midpoint potential of FMN below its value in free solution (Emid,bound = -230 mV, Emid,free = -210 mV). A similar effect on the two-electron midpoint potentials of most of the chemically modified flavins is observed upon binding to the apoprotein. Therefore, the relative order of the respective midpoint potentials of this series of flavins is maintained upon binding to the apoenzyme. However, the effect of the apoprotein on the separation between E1 and E2 varies considerably over this series of flavin derivatives, resulting in quite different levels of thermodynamic stability for the one-electron-reduced (semiquinone) forms of the flavin-substituted enzymes. The optical absorption spectra of these flavin-substituted enzymes were determined in the presence of several phenolic compounds which are known to bind to the native enzyme and to give characteristic long wavelength transitions. We have found that the positions of the wavelength maxima of these transitions are shifted to longer wavelengths as the measured redox potentials of the enzyme-bound flavins are increased. The relationship between the energy of these long wavelength transitions (expressed as vCT = 1/lambda max) and the redox potential of the enzyme-bound flavin is interpreted as further evidence that these transitions arise from charge-transfer complexes between the phenolic compounds and the flavin.

Dithionite↗

PubMed1991

The role of the internal hydrogen bond network in first-order protein electron transfer between Saccharomyces cerevisiae iso-1-cytochrome c and bovine microsomal cytochrome b5.

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.

Animals↗

PubMed2004

Inhibitory mechanism of store-operated Ca2+ channels by zinc.

Capacitative calcium influx plays an important role in shaping the Ca(2+) response of various tissues and cell types. Inhibition by heavy metals is a hallmark of store-operated calcium channel (SOCC) activity. Paradoxically, although zinc is the only potentially physiological relevant ion, it is the least investigated in terms of inhibitory mechanism. In the present study, we characterize the inhibitory mechanism of the SOCC by Zn(2+) in the human salivary cell line, HSY, and rat salivary submandibular ducts and acini by monitoring SOCC activity using fluorescence imaging. Analysis of Zn(2+) inhibition indicated that Zn(2+) acts as a competitive inhibitor of Ca(2+) influx but does not permeate through the SOCC, suggesting that Zn(2+) interacts with an extracellular site of SOCC. Application of the reducing agents, dithiothreitol (DTT) and beta-mercaptoethanol, totally eliminated Zn(2+) and Cd(2+) inhibition of SOCC, suggesting that cysteines are part of the Zn(2+) and Cd(2+) binding site. Interestingly, reducing conditions failed to eliminate the inhibition of SOCC by La(3+) and Gd(3+), indicating that the Zn(2+) and lanthanides binding sites are distinct. Finally, we show that changes in redox potential and Zn(2+) are regulating, via SOCC activity, the agonist-induced Ca(2+) response in salivary ducts. The presence of a specific Zn(2+) site, responsive to physiological Zn(2+) and redox potential, may not only be instrumental for future structural studies of various SOCC candidates but may also reveal novel physiological aspects of the interaction between zinc, redox potential, and cellular Ca(2+) homeostasis.

Calcium↗