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PubMed1978

EPR determination of the oxidation-reduction potentials of the hemes in cytochrome c3 from Desulfovibrio vulgaris.

EPR spectroscopy in conjunction with oxidation-reduction potentiometry has been used to determine the half-reduction potentials of the four hemes of cytochrome c3. As predicted, the four hemes of cytochrome c3 have different mid-point potentials. The Em values are: Heme I,--284 mV; Heme II,--310 mV; Heme III,--324 mV and Heme IV,--319 mV. The n-values in each case was near one.

Cytochrome c Group↗
PubMed2003

New polyvinyl chloride membrane electrode without inner reference solution for the determination of methadone.

A PVC membrane electrode, without inner reference solution, based on an ion association extraction system responding to methadone (MD) is described. It incorporates a methadone-tetrakis(4-chlorophenyl)borate ion-pair complex in 2-nitrophenyloctyl ether. The prepared electrode exhibits a near Nernstian response (59.5 mV per decade) over the concentration range 2.5 x 10(-5)-10(-1) M MD in solutions of pH 2.0-9.0. The reproducibility of the electrode potentials was +/-1 mV by day during at least 6 months. Response time was about 5 s for MD concentrations between 10(-5) and 10(-1) M. Determinations of MD in pharmaceutical preparations (tablets and syrups) by direct potentiometry gave an average recovery of 99.3% (w/w) and a mean S.D. of 1.5% (w/w).

Calibration↗
PubMed2003

Polymeric matrix membrane sensors for sensitive potentiometric determination of some beta-blockers in pharmaceutical preparations.

Five poly(vinyl chloride) matrix membrane sensors responsive to some beta-blockers (atenolol, bisoprolol, metoprolol, propranolol and timolol) are described and characterized. The sensors are based on the use of the ion-association complexes of the beta-blocker cations with tungstophosphate anion as electroactive materials. The performance characteristics of these sensors, evaluated according to IUPAC recommendations, reveal fast, stable and near-Nernstian response for 10(-2)-2 x 10(-7) mol l(-1) of different beta-blockers over the pH range 2-9. Many inorganic and organic cations as well as drug excipients and diluents normally used in drug formulations do not interfere. The sensors are used for direct potentiometry of beta-blockers in some pharmaceutical preparations. Validation of the method according to the quality assurance standards shows suitability of the proposed sensors for use in the quality control assessment of these drugs. Results with an average recovery of 99.1% and a mean standard deviation of +/- 1.3% of the nominal are obtained which compare fairly well with data obtained using the British Pharmacopoeia method.

Adrenergic beta-Antagonists↗
PubMed1999

Potentiometric sensors for the selective determination of sulbutiamine.

Five novel polyvinyl chloride (PVC) matrix membrane sensors for the selective determination of sulbutiamine (SBA) cation are described. These sensors are based on molybdate, tetraphenylborate, reineckate, phosphotun gestate and phosphomolybdate, as possible ion-pairing agents. These sensors display rapid near-Nernstian stable response over a relatively wide concentration range 1x10(-2)-1x10(-6) M of sulbutiamine, with calibration slopes 28 32.6 mV decade(-1) over a reasonable pH range 2-6. The proposed sensors proved to have a good selectivity for SBA over some inorganic and organic cations. The five potentiometric sensors were applied successfully in the determination of SBA in a pharmaceutical preparation (arcalion-200) using both direct potentiometry and potentiometric titration. Direct potentiometric determination of microgram quantities of SBA gave average recoveries of 99.4 and 99.3 with mean standard deviation of 0.7 and 0.3 for pure SBA and arcalion-200 formulation respectively. Potentiometric titration of milligram quantities of SBA gave average recoveries of 99.3 and 98.7% with mean standard deviation of 0.7 and 1.2 for pure SBA and arcalion-200 formulation, respectively.

Biosensing Techniques↗
PubMed1999

In vitro investigation of the liberation of fluoride ions from toothpaste compounds in a permeation model.

The most important requirement for effective topical fluoride prophylaxis from toothpaste containing fluoride is that the active fluoride agent must be chemically free, and the rapid spread of the dissolved fluoride ions over the tooth surface. Abrasive compounds in the toothpastes and the brief residence time at the site of action, the oral cavity and tooth surface must not prevent the liberation. Using a two-chamber diffusion cell and an ion-selective fluoride electrode, the content of fluoride ions in five different fluoride-containing toothpastes was determined by direct potentiometry as a function of time and the different abrasive compounds employed. The investigation has demonstrated that a reduction of the release rate of fluoride ions by nearly 50% is seen when calcium carbonate and calcium hydrogen phosphate dihydrate are used as abrasive compounds and combined with sodium fluoride.

Fluorides↗
PubMed2003

A new nuclear magnetic resonance algorithm to determine equilibrium constants of the species in the B(III)-H2O system.

Several efforts have been attempted to study species formation by Nuclear Magnetic Resonance (NMR) in systems with several chemical equilibria present. The majority of these are qualitative and only a few have tried to relate component fractions of a distribution diagram with experimental area fractions determined from NMR spectra to obtain equilibrium constants values. In this work we present a new focus that attempts to relate the species concentration fractions in the system with area fractions beneath NMR peaks to achieve this task. 11B-NMR data of B(III)-H2O systems have been processed with the aid of formation constant values (-log *beta) obtained by potentiometry which are 9.17+/-0.01 for B(OH)3, 9.79+/-0.08 for B2O(OH)5-, 19.90+/-0.09 for B3O3(OH)4- and 38.50+/-0.04 for B5O6(OH)4-, form B(III)-H2O systems with 0.075 M< or = [B(III)]total< or = 0.700 M, in agreement with previous reports and NMR behavior. The treatment of NMR data developed in this work gives a new methodology to obtain formation constants and suggests the possibility to establish a generalization of Beer's law to NMR spectroscopy.

Algorithms↗
PubMed2004

Spectroscopy study of 5-amino-1,10-phenanthroline.

Acidity constants for the 5-amino-1,10-phenanthroline (5-Aphen) were determined in aqueous media, using SQUAD and SUPERQUAD programs. Spectrophotometry and potentiometry data were fitted to the best model to enable correlation of the following acidity equilibria: 5-AphenH = 5-Aphen + H+ (-log K = 5.78 +/= 0.03) and 5-AphenH2 = 5-AphenH2 = 5-Aphen + 2H+ (-log K = 6.89 +/= 0.07). UV absorptivity coefficients obtained suggest that the first protonation takes place on the nitrogens of the heterocycle ring and the second protonation could take place on the amino group. As expected, the electrochemical evidence of the 5-Aphen species depends on the degree of protonation.

Electrochemistry↗
PubMed2006

Calcium pulstrodes with 10-fold enhanced sensitivity for measurements in the physiological concentration range.

Ion-selective electrodes ideally operate on the basis of the Nernst equation, which predicts less than 60- and 30-mV potential change for a 10-fold activity change of monovalent and divalent ions measured at room temperature, respectively. Typical concentration ranges in extracellular fluids are quite narrow for the electrolytes of key importance. A range of 2.2-2.6 mM for calcium ions, for instance, translates into just a 2.2-mV potential change. The direct potentiometric measurement of physiological electrolytes is certainly possible with direct potentiometry and is done routinely in clinical analyzers and handheld measuring devices. It places, however, strong demands on the precision of the reference electrode and requires careful temperature control and frequent calibration runs. In this paper, a robust 10-20-fold sensitivity enhancement for calcium measurements is attained by departing from the classical response mechanism and operating in a non-Nernstian response mode. Stable and reproducible super-Nernstian responses of these so-called pulstrodes in a narrow calcium activity range can be controlled by instrumental means in good agreement with theory. The potentials may be measured during a galvanostatic excitation pulse (mode I) or immediately after it (mode II), under open-circuit conditions. Subtraction of the potentials, sampled at different times during a single pulse, allows one to obtain a sensitive differential peak-shaped signal at a critical and fully adjustable analyte activity range. Calcium pulstrodes based on the diamide ionophore AU-1 were characterized and applied to the measurement in model physiological liquids. Super-Nernstian responses exceeding 700 mV/decade were observed in a physiological range of calcium concentration. Such remarkable sensitivity of the pulstrodes, complemented with the well-documented high selectivity of these potentiometric sensors, may provide a significant increase in the accuracy and precision of electrolyte measurements in clinical analysis.

Biosensing Techniques↗
PubMed2006

Determination of the acid dissociation constant of the biosurfactant monorhamnolipid in aqueous solution by potentiometric and spectroscopic methods.

The acid dissociation constant in water for a monorhamnolipid mixture extracted from Pseudomonas aeruginosa ATCC 9027 has been determined using potentiometry and two spectroscopic approaches at concentrations below and above the critical micelle concentration (cmc). Potentiometric titrations resulted in pKa values ranging from 4.28 +/- 0.16 to 5.50 +/- 0.06 depending on concentration. 1H NMR spectrochemical titrations at concentrations below the cmc revealed a pKa value of 4.39 +/- 0.06. ATR-FT-IR spectrochemical titrations on solutions well above the cmc gave a pKa value of 4.84 +/- 0.05. The value of 4.28 for the free rhamnolipid molecule for concentrations below the cmc differs markedly from that reported previously. However, the pKa of 5.50 for surface-adsorbed and solution aggregates correlates closely to that previously reported. Differences in these pKa values are rationalized in terms of the pH- and concentration-dependent aggregation behavior of rhamnolipids in aqueous solution.

Absorption↗
PubMed1999

Automated flow injection gradient technique for binding studies of micromolecules to proteins using potentiometric sensors: application to bovine serum albumin with anilinonaphthalenesulfonate probe and drugs.

An automated flow injection (FI) gradient technique is described for the binding study of the potentiometric probe 1-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA). Using a single-channel FI system with a mixing chamber and a flow ANS electrode, the binding parameters (binding constant and number of binding sites) were calculated using the Scatchard model. The concentration gradient was calibrated by injecting ANS in the stream, and the binding experiment was performed by injecting ANS-BSA solution in the carrier solution of equal albumin concentration. The equations describing the concentration gradient and the corresponding electrode potential curve are presented. A systematic study of the factors affecting the complexation equilibrium and the electrode response was performed. For the ANS binding to BSA, two binding classes were determined with binding constants of (2.1 +/- 0.3) x 10(5) and (3.3 +/- 0.8) x 10(3) M-1 and 3.8 +/- 0.6 and 10 +/- 2 binding sites per class, respectively, at 27 +/- 1 degrees C, in 0.10 M phosphate pH 7.4. Competitive binding experiments of sulfamethoxazole, salicylate, azapropazone, ketoprofen, and tolmetin to albumin were also performed by monitoring ANS binding inhibition (decrease of apparent binding constant). This technique takes advantage of FI gradients and direct potentiometry and utilizes the total information contained in FI peaks, providing fast and accurate binding information in a wide range of concentration ratios.

Anilino Naphthalenesulfonates↗
PubMed2000

Self-assembly of heme A and heme B in a designed four-helix bundle: implications for a cytochrome c oxidase maquette.

Heme A, a prosthetic group of cytochrome c oxidase [EC 1.9.3.1], has been introduced into two de novo designed four helix bundle proteins, [H10A24](2) and [H10H24](2), known to bind 2-4 equiv of heme B, respectively [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A., J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425-432]. [H10A24](2), [Ac-CGGGELWKL x HEELLKK x FEELLKL x AEERLKK x L-CONH(2)](2)(2), binds two heme A molecules per four-helix unit via bis-histidine ligation at the 10,10' positions with measured K(d) values of <0.1 and 5 nM, values much lower than those measured for heme B (K(d) values of 50 and 800 nM). The heme A-protein complex, [heme A-H10A24](2), exhibits well-defined absorption spectra in both the ferric and ferrous states, and an electron paramagnetic resonance spectrum characteristic of a low spin heme in the ferric form. A single midpoint redox potential (E(m8)) was determined for [heme A-H10A24](2) at -45 mV (vs SHE), which is significantly higher than that of the protein bound heme B (-130 and -200 mV). The observation of a single midpoint redox potential for [heme A-H10A24](2) and a pair of midpoints for [heme B-H10A24](2) indicates that the di-alpha-helical monomers are oriented in an anti topology (disulfides on opposite sides of bundle) in the former (lacking heme-heme electrostatic interaction) and syn in the latter. A mixture of global topologies was indicated by the potentiometric titration of the related [heme A-H10H24](2) which possess two distinct reduction potentials of +41 (31%) and -65 mV (69%). Self-assembly of the mixed cofactor heme A-heme B-[H10A24](2) was accomplished by addition of a single equivalent of each heme A and heme B to [H10A24](2). The single midpoint redox potential of heme B, E(m8) = -200 mV, together with the split midpoint redox potential of heme A in heme A-heme B-[H10A24](2), E(m8) = +28 mV (33%) and -65 mV (67%), indicated the existence of both syn and anti topologies of the two di-alpha-helical monomers in this four helix bundle. Synthesis of the mixed cofactor [heme A-heme B-H10H24](2) was accomplished by addition of a 2 equiv of each heme A and heme B to [H10H24](2) and potentiometry indicated the pair of hemes B resided in the 10,10' sites and heme A occupied the 24,24' sites. The results indicate that heme peripheral structure controls the orientation of the di-alpha-helical monomers in the four-helix bundle which are interchangeable between syn and anti topologies. In the reduced form, [heme A-H10A24](2), reacts quantitatively to form [carbonmonoxy-heme A-H10A24](2) as evidenced by optical spectroscopy. The synthetic [heme A-H10A24](2) can be enzymatically reduced by NAD(P)H with natural reductases under anaerobic conditions, and reversibly oxidized by dioxygen to the ferric form.

Animals↗
PubMed1986

Calcium-proton and calcium-magnesium antagonisms in calmodulin: microcalorimetric and potentiometric analyses.

Microcalorimetry, pH potentiometry, and direct binding studies by equilibrium dialysis or gel filtration were performed to determine the thermodynamic functions delta Ho, delta Go, and delta So guiding the interactions of Ca2+, Mg2+, and H+ with bovine brain calmodulin. At pH 7.5, Ca2+ and Mg2+ binding are both endothermic with enthalpy changes of 19.5 and 72.8 kJ X (mol of calmodulin)-1, respectively. These enthalpy changes are identical for each of the four ion-binding domains. The affinity constants also are identical with intrinsic values of 10(5) M-1 for Ca2+ and 140 M-1 for Mg2+. Ca2+ and Mg2+ do not compete for the same binding sites: at high concentrations of both ions, a calmodulin-Ca4-Mg4 species is formed with an enthalpy value of 24.4 kJ X mol-1 with respect to calmodulin-Ca4 and -28.8 kJ X mol-1 with respect to calmodulin-Mg4. Moreover, in the presence of high concentrations of Ca2+, the affinity of each of the four ion-binding domains in calmodulin for Mg2+ is decreased by a factor of 4 and vice versa, indicative of negative free-energy coupling between Ca2+ and Mg2+ binding. Protons antagonize Ca2+ and Mg2+ binding in a different manner. Ca2+-H+ antagonism is identical in each of the four Ca2+-binding domains in the pH range 7.5-5.2. Our analyses suggest that three chemical geometries, probably carboxyl-carboxylate interactions, are responsible for this antagonism with ionization constants of 10(6.2) M-1 in the metal-free protein. Mg2+-H+ antagonism also is identical for each of the Mg2+-binding sites but is qualitatively different from Ca2+-H+ antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Oxidation-reduction potentials of flavin and Mo-pterin centers in assimilatory nitrate reductase: variation with pH.

Potentiometric titrations of assimilatory nitrate reductase from Chlorella vulgaris were performed within the pH range 6.0-9.0. Mo(V) was measured by room temperature EPR spectroscopy while the reduction state of FAD was monitored by CD spectroscopy. Between pH 6 and 8.5, the line shape of the Mo(V) EPR signal was constant, exhibiting superhyperfine coupling to a single, exchangeable proton. Potentiometric titrations indicated the Em values for the Mo(VI)/Mo(V) (+61 mV, pH 6) and Mo(V)/Mo(IV) (+35 mV, pH 6) couples decreased with increasing pH by approximately -59 mV/pH unit, consistent with the uptake of a single proton upon reduction of Mo(VI) to Mo(V) and Mo(V) to Mo(IV). The pKa values for the dissociation of these redox-coupled protons appeared to lie outside the pH range studied: pKo(MoVI), pKo(MoV) less than 5.5; pKr(MoV), pKr(MoIV) greater than 9. The Em (n = 2) for FAD (-250 mV, pH 7) varied by approximately -30 mV/pH unit within the pH range 6.0-9.0. Low-temperature EPR potentiometry at the extreme pH values indicated less than 0.5% conversion of FAD to the semiquinone form at the midpoint of the titrations. In contrast, NADH-reduced enzyme exhibited approximately 3-5% of the FAD in the semiquinone form, present as the anionic (FAD.-) species, the spectrum characterized by a line width of 1.3 mT at both pH 6.0 and 9.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorella↗
PubMed1981

On the function of N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine in transfer ribonucleic acid. Metal ion binding studies.

The unusual transfer ribonucleic acid (RNA) anticodon adjacent modified nucleoside N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine, t6A, and its N6-methyl (mt6A) and 5'-phosphate (pt6A) derivatives are efficient ligands for magnesium and manganese ions, as demonstrated by potentiometry and nuclear magnetic resonance spectroscopy. Analysis of the 1H and 13C NMR spectra of t6A in the presence of paramagnetic Mn (II) at 32-34 degrees C has shown that the metal ion binds to the carboxyl group and probably N6 of the side chain as well as the N1 or N7 atom of the adenine ring. A more specific and stronger metal-ligand complex between Mn(II) and pt6A is evident from the 1H, 13C, and 31P NMR data. In this case, the metal forms a complex involving phosphate, N7 of the adenine and the carboxyl group, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparisup, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparisup, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparison between them as well as a comparison of the binding between Mg(II) and Mn(II). The stability constants (log K) for 1:1 metal-ligand complexes between Mg(II) and t6A, mt6A, and pt6A are respectively 5.5, 4.3, and 7.1. For Mn(II), the respective values are 6.0, 4.5, and 7.9. In the case of pt6A, the stability constants are about 5 log K units larger than those obtained for Mg(II) and Mn(II) binding to 5'-AMP [Khan, M. M. T., & Martell, A. E. (1967) J. Am. Chem. Soc. 89, 5585]. Thus the threonine side chain is an important determinant in the interaction between the modified nucleosides and metal ions, and these results are supportive of the idea that a facet of the function of this type of unusual nucleoside in transfer RNA is as a specific ligand for magnesium ion, a postulate promulgated earlier [Miller, J. P., Hussain, Z., & Schweizer, M. P. (1976) Nucleic Acids Res. 3, 1185].

Adenosine↗
PubMed2002

Properties of a soluble domain of subunit C of a bacterial nitric oxide reductase.

Bacterial nitric oxide reductases are integral membrane proteins that catalyze the reduction of two molecules of nitric oxide to nitrous oxide and water. They are diverged members of the superfamily of heme/copper oxidases. The enzyme from Paracoccus denitrificans (NorBC) contains two subunits; NorB comprises the membrane-integrated active site, which harbors a heme iron/non-heme iron dinuclear center. NorC is a membrane-anchored c-type cytochrome and presumably the site of electron uptake. A DNA construct encoding the water-soluble domain of NorC (NorC(sol)) was coexpressed with the cytochrome c maturation genes in Escherichia coli. Using redox potentiometry, electronic absorption, circular dichroism (CD), magnetic CD (MCD), nuclear magnetic resonance, and electron paramagnetic resonance (EPR) spectroscopy the following observations were made: (i) NorC(sol) was folded into a alpha-helical structure. (ii) The low-spin heme iron was coordinated by histidine and methionine in both redox states. (iii) The midpoint redox potential of the NorC(sol) heme was 183 mV, much lower than the corresponding value of 275 mV in the NorBC complex. This points to an increased solvent exposure of the NorC(sol) heme compared to in the native NorBC complex and shows that the electronic properties of NorC are modulated by NorB in the complex. (iv) The EPR and MCD spectra of NorC(sol) were considered alongside the spectra of NorBC, which has helped to resolve the contribution that different redox centers make in the holo-enzyme complex.

Bacterial Proteins↗
PubMed2002

Characterization of the redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum.

Dimethyl sulfide dehydrogenase from the purple phototrophic bacterium Rhodovulum sulfidophilum catalyzes the oxidation of dimethyl sulfide to dimethyl sulfoxide. Recent DNA sequence analysis of the ddh operon, encoding dimethyl sulfide dehydrogenase (ddhABC), and biochemical analysis (1) have revealed that it is a member of the DMSO reductase family of molybdenum enzymes and is closely related to respiratory nitrate reductase (NarGHI). Variable temperature X-band EPR spectra (120-122 K) of purified heterotrimeric dimethyl sulfide dehydrogenase showed resonances arising from multiple redox centers, Mo(V), [3Fe-4S](+), [4Fe-4S](+), and a b-type heme. A pH-dependent EPR study of the Mo(V) center in (1)H(2)O and (2)H(2)O revealed the presence of three Mo(V) species in equilibrium, Mo(V)-OH(2), Mo(V)-anion, and Mo(V)-OH. Above pH 8.2 the dominant species was Mo(V)-OH. The maximum specific activity occurred at pH 9.27. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other molybdenum enzymes of the DMSO reductase family showed that it was most similar to the low-pH nitrite spectrum of Escherichia coli nitrate reductase (NarGHI), consistent with previous sequence analysis of DdhA and NarG. A sequence comparison of DdhB and NarH has predicted the presence of four [Fe-S] clusters in DdhB. A [3Fe-4S](+) cluster was identified in dimethyl sulfide dehydrogenase whose properties resembled those of center 2 of NarH. A [4Fe-4S](+) cluster was also identified with unusual spin Hamiltonian parameters, suggesting that one of the iron atoms may have a fifth non-sulfur ligand. The g matrix for this cluster is very similar to that found for the minor conformation of center 1 in NarH [Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P. (1992) Eur. J. Biochem. 307, 63-68]. Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethyl sulfide dehydrogenase. Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are a histidine and methionine, consistent with predictions from protein sequence analysis. Redox potentiometry showed that the b-type cytochrome has a high midpoint redox potential (E degrees = +315 mV, pH 8).

Alcohol Oxidoreductases↗
PubMed2004

The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe-4S] cluster with a high-spin ground state.

We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the [4Fe-4S] cluster (FS0) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). FS0 is clearly visible in the crystal structure of NarGHI [Bertero, M. G., et al. (2003) Nat. Struct. Biol. 10, 681-687] but has novel coordination comprising one His residue and three Cys residues. At low temperatures (<15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556. We have assigned these features to a [4Fe-4S](+) cluster with an S = (3)/(2) ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = (1)/(2) and DeltaS = (3)/(2) transitions, respectively. Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI. The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands. On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.

Amino Acid Sequence↗
PubMed2005

Herbicide-induced changes in charge recombination and redox potential of Q(A) in the T4 mutant of Blastochloris viridis.

To gain new insights into the function of photosystem II (PSII) herbicides DCMU (a urea herbicide) and bromoxynil (a phenolic herbicide), we have studied their effects in a better understood system, the bacterial photosynthetic reaction center of the terbutryn-resistant mutant T4 of Blastochloris (Bl.) viridis. This mutant is uniquely sensitive to these herbicides. We have used redox potentiometry and time-resolved absorption spectroscopy in the nanosecond and microsecond time scale. At room temperature the P(+)(*)Q(A)(-)(*) charge recombination in the presence of bromoxynil was faster than in the presence of DCMU. Two phases of P(+)(*)Q(A)(-)(*) recombination were observed. In accordance with the literature, the two phases were attributed to two different populations of reaction centers. Although the herbicides did induce small differences in the activation barriers of the charge recombination reactions, these did not explain the large herbicide-induced differences in the kinetics at ambient temperature. Instead, these were attributed to a change in the relative amplitude of the phases, with the fast:slow ratio being approximately 3:1 with bromoxynil and approximately 1:2 with DCMU at 300 K. Redox titrations of Q(A) were performed with and without herbicides at pH 6.5. The E(m) was shifted by approximately -75 mV by bromoxynil and by approximately +55 mV by DCMU. As the titrations were done over a time range that is assumed to be much longer than that for the transition between the two different populations, the potentials measured are considered to be a weighted average of two potentials for Q(A). The influence of the herbicides can thus be considered to be on the equilibrium of the two reaction center forms. This may also be the case in photosystem II.

Bacterial Proteins↗