Oxidative Titration Research Articles

How to Quantify Electrons in Plasmonic Colloidal Metal Oxide Nanocrystals

Distinct from noble metal nanoparticles, doped metal oxide nanocrystals (NCs) exhibit localized surface plasmon resonance (LSPR) in the infrared region that can be tuned by changing the free electron concentration through both synthetic and postsynthetic doping. Redox reagents have commonly been used to postsynthetically modulate the LSPR, but to understand the relationship between the electron transfer processes and the resulting optical changes, it is imperative to quantify electrons in the NCs. Titration and LSPR peak fitting analysis are the most common methods used for quantifying electrons; however, a comparison between these methods has previously revealed discrepancies up to an order of magnitude without a clear explanation. Here, we apply these electron quantification techniques concurrently to Sn-doped In2O3 NCs with varying size, doping concentration, and extent of postsynthetic reduction. We find that oxidative titration consistently overestimates the number of electrons per NC, owing to the failure of the assumed stoichiometric equivalents between moles of oxidant added and moles of free electrons extracted from the NCs. The NC characteristics we examine strongly influence the driving force for the oxidation process, affecting the relative agreement between oxidative titration and LSPR fitting; the two methods more closely agree when the electron transfer driving force is larger. Overall, these analyses inform best practices for quantifying electrons in plasmonic semiconductor NCs and reveal how the accuracy is affected by NC characteristics.

Impact of benzimidazole functional groups on the n-doping properties of benzimidazole derivatives

Abstract n-Dopants play a crucial role in improving organic electronic devices through controlled doping of organic semiconductors. Benzimidazoline-based dopants have been reported as one of the best solution-processed n-type dopant precursors. In this study, two benzimidazoline-based dopants (BIBDTO and BBIBDTO) were prepared using benzo[1,2-b:4,5-b′]dithiophene as the 2-Ar unit, and their n-doping properties on the fullerene derivative PTEG-2 as the host material were carried out. For BIBDTO and BBIBDTO, respectively, the temperature at which 5% weight loss was achieved was 229 and 265°C. By comparing the ultraviolet-visible absorption spectroscopy, cyclic voltammetry, and density functional theory calculated data, it is found that BBIBDTO has a higher energy level, which is more favorable for charge transfer. Additionally, both the oxidative titration experiments and conductivity characterization of the dopants showed that BBIBDTO was more advantageous at low doping concentrations, and the BBIBDTO-doped PTEG-2 films obtained a conductivity of 0.15 S cm−1 at 10 mol% doping concentration. However, at high dopant concentrations, the dopant volume increases, potentially disrupting the microstructure. The highest conductivity of 0.29 S cm–1 was obtained at a BIBDTO doping concentration of 15 mol%. This study delves into the effect of benzimidazole functional groups on the doping performance of benzimidazoline-based dopant molecules, providing insight into designing novel efficient n-type dopant molecules and further selecting the type of dopant for various doping systems.

Reactions of amines with ozone and chlorine: Two novel oxidative methods to evaluate the N-DBP formation potential from dissolved organic nitrogen

The composition of oxidant-reactive dissolved organic nitrogen (DON) is poorly characterized, although its ozonation is likely to form a great variety of disinfection by-products containing a nitrogen-oxygen bond (N-DBPs). In this study, two chemical oxidation procedures were developed: continuous ozonation at pH 7.0 and free available chlorine (FAC) titrations at pH 9.2. The formation of two oxidation products (nitrate (NO3−) and chloramines, respectively) was used to quantify and characterize oxidant-reactive nitrogenous moieties in DON. In addition, batch experiments were conducted to study the NO3− yields of 30 selected nitrogenous model compounds upon ozonation. The NO3− yields of 12 primary and secondary amines were highly variable (17–100%, specific ozone dose of 20 molO3/molN), 7 amino acids had high NO3− yields (≥90%), and tertiary amines as well as pyrrole, acetamide and urea had low NO3− yields (≤15%). The mechanisms of NO3− formation were further examined with benzylamine and N-methylbenzylamine as model compounds. Our results show that nitroalkanes are the last intermediate products before the formation of NO3−, both for primary and secondary amines. The presence of an electron-withdrawing group in the vicinity of the N-atom facilitates the formation of NO3− from nitroalkanes. Therefore, the formation of NO3− is attributed to amino acids and activated primary and secondary amines. In contrast, all primary and secondary amines were transformed to chloramines upon chlorination, which was determined by a novel oxidative titration with chlorine. To further support the selectivity of this assay, it was demonstrated by derivatization of amine moieties that chloramine formation could be inhibited. 13–45% of the DON of 4 dissolved organic matter isolates and 2 wastewater effluents formed NO3− and 0–39% formed chloramines, indicating that the potential for N-DBP formation is high (µMN/mgC-level). From differences in the formation of NO3− and chloramines the nature of the precursors can be hypothesized (e.g., activated or non-activated primary and secondary amines, partially oxidized nitrogenous compounds). This study highlights the capacity of two novel methods to characterize the oxidant-reactive DON fraction. Our results suggest that this fraction is significant and could form a variety of potentially toxic N-DBPs.

Oxidant-reactive carbonous moieties in dissolved organic matter: Selective quantification by oxidative titration using chlorine dioxide and ozone

The application of oxidants for disinfection or micropollutant abatement during drinking water and wastewater treatment is accompanied by oxidation of matrix components such as dissolved organic matter (DOM). To improve predictions of the efficiency of oxidation processes and the formation of oxidation products, methods to determine concentrations of oxidant-reactive phenolic, olefinic or amine-type DOM moieties are critical.Here, a novel selective oxidative titration approach is presented, which is based on reaction kinetics of oxidation reactions towards certain DOM moieties. Phenolic moieties were determined by oxidative titration with ClO2 and O3 for five DOM isolates and two secondary wastewater effluent samples. The determined concentrations of phenolic moieties correlated with the electron-donating capacity (EDC) and the formation of inorganic ClO2-byproducts (HOCl, ClO2−, ClO3−). ClO2-byproduct yields from phenol and DOM isolates and changes due to the application of molecular tagging for phenols revealed a better understanding of oxidant-reactive structures within DOM.Overall, oxidative titrations with ClO2 and O3 provide a novel and promising tool to quantify oxidant-reactive moieties in complex mixtures such as DOM and can be expanded to other matrices or oxidants.

Electron-Transfer-Induced Self-Assembly of a Molecular Tweezer Platform.

The design and synthesis of a new molecular tweezer (T-tmp) with electron-rich pincers are reported. The stable monocationic radicals and self-assembled dimeric radicals of this molecular tweezer platform were prepared by chemical oxidative titration. With the aid of DFT calculations, it was found that the dimeric radicals with syn-syn-syn conformer has the most stable structure, with the hole primarily delocalized between parallel stacked pyrenyl groups.

Diarylamine-Fused Subporphyrins: Proof of Twisted Intramolecular Charge Transfer (TICT) Mechanism.

Diarylamine-fused subporphyrins 1 a and 1 b were synthesized from β,β-diiodo-meso-chloro subporphyrin 2 through a one-pot procedure involving nucleophilic aromatic substitution and SRN 1-type intramolecular fusion reaction as the first example of meso-nitrogen-embedded subporphyrin. While non-fused counterparts 3 b and 4 b display effective fluorescence quenching due to twisted intramolecular charge transfer (TICT) in CH2 Cl2 at room temperature, 1 b emits fluorescence with ΦF =0.18 under the same conditions, because conformational twisting is not allowed due to the fused structure. In addition, the oxidative titration with tris(4-bromophenyl)ammoniumyl hexachloroantimonate gave cation radical 7 for 1 a and cation radical and dication for 1 b. Actually, cation radical 7 was isolated through separation over silica gel column but was found to slowly decompose in concentrated solutions.

Nature of the Infrared Transition of Colloidal Indium Nitride Nanocrystals: Nonparabolicity Effects on the Plasmonic Behavior of Doped Semiconductor Nanomaterials

As-synthesized colloidal indium nitride (InN) nanocrystals are degenerately doped with carrier densities large enough to lead to strong localized surface plasmon resonances (LSPR) in the infrared. Intriguingly, the LSPR energy is almost independent of carrier density, which premises that simple classical models that are often used to describe metallic systems inadequately describe the plasmonic response of InN nanoparticles. Here, an oxidative titration approach is used to directly quantify carrier densities in colloidal InN nanocrystals, eliminating the need to rely on any specific model. A size-independent carrier density value of (7.4 ± 0.4) × 1020 cm–3 is obtained for diameters varying between 4 and 9 nm, corresponding to about 30 to 300 electrons per nanocrystal, depending on size. Upon oxidation with nitrosonium salts, the carrier density in InN nanocrystals can be reduced to (3.9 ± 0.3) × 1020 cm–3, also independent of size. The unusual plasmonic signatures of colloidal InN nanocrystals are shown t...

Lateral Extension of π Conjugation along the Bay Regions of Bisanthene through a Diels–Alder Cycloaddition Reaction

Diels-Alder cycloaddition reactions at the bay regions of bisanthene (1) with dienophiles such as 1,4-naphthoquinone have been investigated. The products were submitted to nucleophilic addition followed by reductive aromatization reactions to afford the laterally extended bisanthene derivatives 2 and 3. Attempted synthesis of a larger expanded bisanthene 4 revealed an unexpected hydrogenation reaction at the last reductive aromatization step. Unusual Michael addition was observed on quinone 14, which was obtained by Diels-Alder reaction between 1 and 1,4-anthraquinone. Compounds 1-3 exhibited near-infrared (NIR) absorption and emission with high-to-moderate fluorescent quantum yields. Their structures and absorption spectra were studied by density function theory and non-planar twisted structures were calculated for 2 and 3. All compounds showed amphoteric redox behavior with multiple oxidation/reduction waves. Oxidative titration with SbCl(5) gave stable radical cations, and the process was followed by UV/Vis/NIR spectroscopic measurements. Their photostability was measured and correlated to their different geometries and electronic structures.

Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.

Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes

Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Q p and Q d. In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme b H, E m = + 65 mV, heme b L, E m = − 95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme b L, and with a pronounced delay of reoxidation. When the specific inhibitor 2- n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Q d in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Q p site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Q p or Q d, either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Q p depended on the redox state of the hemes. When both hemes were reduced, and Q d was blocked by HQNO, quinone-mediated communication via the Q p site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.

Surface peculiarities of detonation nanodiamonds in dependence of fabrication and purification methods

A variety of post-treatments, such as thermal annealing and liquid phase oxidation, were used to determine the surface properties of six blasting synthesized nanodiamond powders, produced by various purification conditions. Methods of FTIR-spectroscopy, oxidative titration and pH-measurement were applied for comparing the surface functional groups. All spectra exhibited the characteristic IR-bands of detonation diamond in the regions 3400–2700 cm − 1 and 1000–400 cm − 1. The differences were revealed by the dissimilarity of the bands in the region 2000–1000 cm − 1. The nanodiamond powder annealing up to 600 °C in air most fully revealed the specificity of the attached groups on diamond surface. The chemical oxidation with H 2O 2 modified nanodiamond surface groups and prevented them from further oxidative attack during thermal treatment.

Bcl-2-mediated potentiation of neocarzinostatin-induced apoptosis: requirement for caspase-3, sulfhydryl groups, and cleavable Bcl-2

Overexpression of antiapoptotic Bcl-2 family members is thought to contribute to chemotherapeutic resistance of neural crest tumors. Paradoxical potentiation by Bcl-2 of apoptosis induced by the antineoplastic prodrug, neocarzinostatin (NCS), has been observed in PC12 pheochromocytoma cells. Prior studies have indicated that the cleavage of Bcl-2 to its proapoptotic counterpart mediated by caspase-3 is responsible for this potentiation of apoptosis. This has led to the hypothesis that induction of caspase-3 expression in bcl-2-transfected, caspase-3-deficient MCF-7 cells, will result in Bcl-2 cleavage and Bcl-2-dependent potentiation of NCS-induced apoptosis. These studies have further led to the hypothesis that both cleavable Bcl-2 and sulfhydryl groups are required for the activity of caspase-3 in this regard. As hypothesized, co-transfection of bcl-2-transfected MCF-7 cells with a caspase-3 expression construct results in cleavage of Bcl-2 and potentiation of dose-dependent, NCS-mediated cell death. Furthermore, PC12 cells transfected with an expression construct for cleavage-resistant Bcl-2 demonstrated attenuated potentiation of apoptosis relative to their counterparts transfected with wild-type bcl-2. Finally, irreversible oxidative titration of sulfhydryl groups resulted in concentration-dependent attenuation of apoptosis in PC12 cells, along with prevention of caspase-3 activation and Bcl-2 cleavage. These results definitively demonstrate the requirement for caspase-3, cleavable Bcl-2, and available sulfhydryl groups (separate from those required for NCS activation) in potentiation of NCS-induced apoptosis by Bcl-2.

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH 2) through the cytochrome b 6 f complex (Cyt b 6 f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light–dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700 + and PC + was recorded as leaf transmission measured differentially at 810–950 nm. The signal was deconvoluted into PC + and P700 + components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1–15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225–234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b 6 f to the PSI donors. A significant down-regulation of Cyt b 6 f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b 6 f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light–dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e − transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.

Reductive titration of photosystem I and differential extinction coefficient of P700 + at 810–950 nm in leaves

We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e −) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e − carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) [V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1–15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch ( Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e − generated in PSII by the flash was measured as four times O 2 evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700 + and PC + components using the abovementioned oxidative titration method. The P700 + component was related to the absolute number of e − that reduced the P700 + to calculate the extinction coefficient. The effective differential extinction coefficient of P700 + at 810–950 nm was 0.40±0.06 (S.D.)% of transmittance change per μmol P700 + m −2 or 17.6±2.4 mM −1 cm −1. The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (±14% S.D.) is mainly caused by light-scattering properties of the leaf.

Titrimetric method based on potentiometric titration to evaluate redox couples in wine and polyphenols

Polyphenols are electroactive compounds, each molecule having reductive (Rd) and oxidative forms (Ox) due to the presence of several phenolic hydroxyls. Direct study of Rd/Ox forms was conducted using potentiometric titration in two steps: First, a complete reduction of the polyphenol Rd/Ox couples by titanium III chloride TiCl 3 (100 % Rd), and then, oxidative titration by dichlorophenolindophenol DCPIP (100 % Ox). The second curve represents the totality of polyphenol couples titration with, for each couple, a characteristic E 0 point representing the equilibrium between the concentration of Rd and Ox forms (Rd/Ox = 1). This value was typical for each polyphenol. We conducted several preliminary experiments with a model polyphenol (catechin) to establish the analysis conditions. The titration solution concentration was N/10 in HCl N and N/20 in water for TiCl 3 and DCPIP respectively. The concentration of this solution diminished very rapidly, i.e. within 24 h for TiCl 3 and DCPIP (under nitrogen and in darkness) and regularly needed a fresh preparation. To control the consistency and precision of the method we performed a few tests on pure products (e.g. hydroquinone/quihydrone, Fe III, Fe II, Cu II) permitting comparison between theoretical and experimental E 0 values. Our result indicated less than 5 % variation and curves with a high reproducibility.

Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior.

Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center. Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1. Reproducible hysteresis is demonstrated in the redox titrations. In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV. However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2. A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated. In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1). The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.

Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus. Redox properties and molecular interaction with a [2Fe-2S] ferredoxin.

A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe-2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.

Detection of EPR signals assigned to the 1-equiv-oxidized P-clusters of the nitrogenase MoFe-protein from Azotobacter vinelandii

Component 1 (the MoFe protein) of conventional nitrogenase from Azotobacter vinelandii contains two types of metal clusters, called M-centers and P-clusters. The M-centers, or FeMo-co, are paramagnetic in the as-isolated form of the protein, exhibiting an EPR signal typical of a rhombic S=3/2 system. The P-clusters are diamagnetic in the as-isolated form of the protein but become paramagnetic upon chemical or potentiometric oxidation. We have undertaken a controlled oxidative titration of component 1 with thionine solution and observed EPR signals believed to originate from paramagnetic P-clusters. We present EPR spectra associated with half-integer S=5/2 and S=1/2 spin states and propose that these spectra arise from the 1-equiv-oxidized form of the P-cluster

Purification and characterization of a novel dimeric ferredoxin (FdIII) from Rhodobacter capsulatus

A new ferredoxin, called FdIII, has been isolated and purified from the photosynthetic bacterium Rhodobacter capsulatus. Its complete amino acid sequence has been determined. The FdIII polypeptide consists of 100 residues, including 9 cysteines and has a calculated molecular mass of 10,688 Da, which was confirmed by electrospray mass spectrometry. In its native form, FdIII is a homodimer as deduced from molecular sieve chromatography and non-denaturing polyacrylamide gel electrophoresis, as well as cross-linking experiments. The dimeric ferredoxin was found to contain 15.2 +/- 0.6 iron atoms and 13 +/- 1 inorganic sulfur atoms, consistent with the presence of four [4Fe-4S] clusters/molecule. The UV visible absorption spectrum of oxidized FdIII exhibited maxima at 282 and at 386 nm and a shoulder near 314 nm. FdIII was fully reduced by excess dithionite at pH 8.0 or photochemically using 5-deazaflavin. Anaerobic oxidative titration of reduced FdIII with thionin indicated that each FdIII monomer exchanges two electrons. Exposure of FdIII to air resulted in a rapid and irreversible oxidative denaturation of the Fe-S clusters. The EPR spectrum of fully reduced FdIII showed a broad signal with an average g value of 1.94 that integrated to about two spins/monomer. EPR analysis of partially reduced FdIII (approximately 20% reduction) revealed a complex set of signals which was interpreted as being the resulting sum of the contribution of two distinct paramagnetic centres. Based on its biochemical and spectroscopic properties, it is concluded that FdIII is a dimeric ferredoxin containing four [4Fe-4S] clusters. The synthesis of FdIII occurs only under growth conditions allowing the derepression of nif genes. Results of in vitro electron transfer assays indicate that FdIII cannot serve as an electron donor to nitrogenase.

Reactions of the membrane-bound cytochrome bo terminal oxidase of Escherichia coli with carbon monoxide and oxygen

The cytochrome ‘ bo’ quinol oxidase of Escherichia coli contains 2 mol of haem, one or both of which are ‘haem O’. One of the haems forms, with the single copper present, a binuclear site for ligand binding and oxygen reduction. Cytoplasmic membranes from a strain of E. coli lacking the alternative cytochrome bd quinol oxidase, and having amplified levels of cytochrome bo, were used to study oxygen and carbon monoxide reactivity with this oxidase. The high-spin ligand-binding haem was identified from its contribution to the Soret region and the shift in midpoint potential from +211 to +477 mV in the presence of CO. Oxidative titration of a CO-liganded sample was accompanied by a decrease in the contribution from a photodissociable CO-binding haem. The photodissociation spectrum was typical of a high-spin haem. Photolysis of CO-liganded, reduced membranes in the presence of O 2 at sub-zero temperatures revealed O 2 binding and cytochrome oxidation characterized by differential absorbance changes in the α-spectral region. Monitoring by epr spectroscopy of the same reaction sequence at −80°C revealed a slight increase in g = 6 signal intensity immediately after photolysis attributable to cytochrome o oxidation prior to Cu oxidation. Subsequent decline in the g = 6 signal and appearance of a g = 3 signal indicated sequential electron flow from low-spin to high-spin haems and copper oxidation, suggesting that a second haem carries electrons from ubiquinol to the binuclear centre.