Oxidation-reduction potentials of respiratory chain components in ThiobacillusA2

(1) Cells of ThiobacillusA₂ grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a₃-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (E_m,7.0) and spectral maxima of c-, b-, a- and a₃-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a₃ complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.     

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents

The kinetics of the oxidation-reduction reactions of cytochrome c₁ with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c₁ by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c₁ with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reaction rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c₁ with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4·10⁴, 3.2·10³, 3.8·10⁴ and 1.3·10⁸ M^?1·s^?1 (pH 7.0, I = 0, 10°C), respectively. Application of the Debye-Hückel theory to the the ionic-strength-dependence studies of these redox reactions of cytochrome c₁ yielded for ferrocytochrome c₁ and ferricytochrome c₁ a net charge of ?5 and ?4, respectively. The latter value is close to that of ?3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c₁ governs the reaction rate with small redox molecules.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a³⁺a²⁺₃-CO) complex and the mixed-valence carboxycytochrome c oxidase (a³⁺a²⁺₃-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a²⁺₃-CO complex. This would confirm that the spectral properties of cytochrome a₃ are not affected by the redox state of cytochrome a and Cu_A. When the absorbance changes after photolysis of cytochrome a²⁺₃-CO with a laser flash were followed on a time scale from 1 μs to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7·10³s^?1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a₃. Concomitantly, a decrease of the 830 nm band was observed due to reduction of Cu_A. This demonstrates that in the partially reduced enzyme a pathway is present between Cu_A and the cytochrome a₃-Cu_B pair, via which electrons are transferred rapidly.     

Urea hydrolysis in some tea soils

The influence of application of hexachlorocyclohexane (HCH=gamma BHC), to a submerged tropical field soil at rates equivalent to recommended field rates (1–2.5kg a.i./ha) and twice this level, upon the rhizosphere soil nitrogenase, nitrogen fixers, and soil redox potential (Eh) was investigated. The rhizosphere soil from HCH-treated field exhibited significantly higher nitrogenase activity than that from untreated fields. HCH retarded the drop in redox potential of the field soil upto 80 days after transplantation under submerged conditions. Populations of nitrogen-fixingAzospirillum sp. and Azotobacter, to a greater extent, and anaerobic organisms, to a lesser extent, were stimulated in HCH-treated soils. Results indicate the stimulation of heterotrophic nitrogen-fixing bacteria by HCH in submerged paddy soils.     

Electrostatics of the FeS clusters in respiratory complex I

Respiratory complex I couples the transfer of electrons from NADH to ubiquinone and the translocation of protons across the mitochondrial membrane. A detailed understanding of the midpoint reduction potentials (E_m) of each redox center and the factors which influence those potentials are critical in the elucidation of the mechanism of electron transfer in this enzyme. We present accurate electrostatic interaction energies for the iron-sulfur (FeS) clusters of complex I to facilitate the development of models and the interpretation of experiments in connection to electron transfer (ET) in this enzyme. To calculate redox titration curves for the FeS clusters it is necessary to include interactions between clusters, which in turn can be used to refine E_m values and validate spectroscopic assignments of each cluster. Calculated titration curves for clusters N4, N5, and N6a are discussed. Furthermore, we present some initial findings on the electrostatics of the redox centers of complex I under the influence of externally applied membrane potentials. A means of determining the location of the FeS cofactors within the holo-complex based on electrostatic arguments is proposed. A simple electrostatic model of the protein/membrane system is examined to illustrate the viability of our hypothesis.     

Organometallic amphiphiles: novel mechanisms for redox chemical control of aggregation behavior

Many natural and synthetic organic amphiphiles have been discovered, prepared, and/or characterized. Aggregation behavior has been studied for many of these monomers. Very few of these many amphiphiles are organometallic compounds and almost nothing is known about this novel group of amphiphiles. Under appropriate circumstances, aggregation of certain organometallic amphiphile monomers can be controlled by use of redox switching. Compounds which are not amphiphilic can be made so and amphiphilic compounds can be deprived of this property by appropriate alterations in the metal ion's oxidation state. Redox-switched aggregation-deaggregation behavior is described for bis(hexadecycloxy)-1,10-phenanthrolinium perchlorate (2), its nickel complex (3), [CH₃(CH₂)₁₅NH₂]₂Ag⁺AcO⁻ (4) and bis(hexadecylamine)copper dichloride (5), chloride acetate (6) and diacetate (7), all of the general formula: [CH₃(CH₂)₁₅NH₂]₂Cu²⁺X₂². Two completely new switching mechanisms are disclosed for the first time which involve electron transfer from dilute aqueous sugar solutions or from solid zinc metal which are effective in collapsing [CH₃(CH₂)₁₅NH₂]₂Cu²⁺X₂²⁻ vesicles.     

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L⁻¹ l-lysine from mannitol at a yield of 0.26 mol mol⁻¹ and a maximum productivity of 2.1 g L⁻¹ h⁻¹. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol⁻¹ using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol⁻¹. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.     

Genoprotection and genotoxicity of green tea (Camellia sinensis): Are they two sides of the same redox coin?

Abstract

Objectives

Regular intake of green tea associates with lower DNA damage and increased resistance of DNA to oxidant challenge. However, in vitro pro-oxidant effects of green tea have been reported. Both effects could be mediated by hydrogen peroxide (H₂O₂) which is generated by autoxidation of tea catechins. In large amounts, H₂O₂ is genotoxic, but low concentrations could activate the redox-sensitive antioxidant response element (ARE) via the Keap-1/Nrf2 redox switch, inducing genoprotective adaptations. Our objective was to test this hypothesis.

Methods

Peripheral lymphocytes from healthy volunteers were incubated for 30 minutes at 37°C in freshly prepared tea solutions (0.005, 0.01, 0.05%w/v (7, 14, 71 µmol/l total catechins) in phosphate buffered saline (PBS), with PBS as control) in the presence and absence of catalase (CAT). H₂O₂ in tea was measured colorimetrically. Oxidation-induced DNA lesions were measured by the Fpg-assisted comet assay.

Results

H₂O₂ concentrations in 0.005, 0.01, and 0.05% green tea after 30 minutes at 37°C were, respectively, ～3, ～7, and ～52 µmol/l. Cells incubated in 0.005 and 0.01% tea showed less (P < 0.001) DNA damage compared to control cells. Cells treated with 0.05% green tea showed ～50% (P < 0.001) more DNA damage. The presence of CAT prevented this damage, but did not remove the genoprotective effects of low-dose tea. No significant changes in expression of ARE-associated genes (HMOX1, NRF2, KEAP1, BACH1, and hOGG1) were seen in cells treated with tea or tea + CAT.

Conclusion

Genoprotection by low-dose green tea could be due to direct antioxidant protection by green tea polyphenols, or to H₂O₂-independent signalling pathways.