Characterization of electron transfer reactions of microperoxidase assembled at short-chain thiol-monolayers on gold

In this study thiol-monolayers were used in order to modify gold and provide suitable chemical functionalities for the immobilization of the small redox-active haem-containing peptide, microperoxidase (MP-11). Initially, we assembled a variety of thiol-containing species on the gold electrodes and measured a series of electron transfer reactions, each characteristic of the surface-modifier. Using suitable immobilization strategies, we subsequently covalently bound MP-11 to appropriate monolayers and found two characteristic electrochemical responses (i.e. using MP-11 bound to mercaptopropionic acid, redox peaks were seen at E^0′ = −315 mV and at +179 mV versus Ag|AgCl, with the former being attributed to the haem and the latter with the thiol monolayer). Exposure of the peptide-thiol surface to UV irradiation resulted in cleavage of the Au---S bond, leading to a decrease in the magnitude of both responses. Our work is supported by corroborative evidence showing the immobilization of the peptide, obtained using both X-ray photoelectron and reflectance Fourier transform infra-red spectroscopies. We hypothesize that differences in the ionic charges on the protein backbone account for the shift in E^0′ for MP-11, as observed in our system, when compared to that found for MP-11 immobilized by different strategies.     

Mitochondria transfer into mouse ova by microinjection

A method for mitochondria isolation and interspecific transfer of mitochondria was developed in mice. Mitochondria were isolated from Mus spretus liver samples for microinjection into fertilized ova obtained from superovulated M. musculus domesticus females. Electron microscopic observations of mitochondria preparations used for microinjection demonstrated intact mitochondrial vesicles with little microsomal contamination. Species-specific nested PCR primers complementary to sequence differences in the mitochondrial DNA D-loop region revealed high rates of successful transfer of foreign mitochondria after isolation and injection into zygotes cultured through the blastocyst stage of embryonic development. Of 217 zygotes, 67 survived mitochondria injection and 23 out of 37 zygotes developed were at the blastocyst-stage of embryonic development after 4.5 days of in vitro culture. All 23 of these blastocysts contained detectable levels of foreign mitochondria. These results represent an initial step in developing a model system to study mitochondrial dynamics and development of therapeutic strategies for human metabolic diseases affected by aberrations in mitochondrial function or mutation     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Purification and characterization of two essential cytochromes of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus A2 (Thiobacillus versutus)

Four c-type cytochromes were purified by several procedures including chromatography on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Sephadex G-75, G-100 and G-200 and chromatofocusing. Cytochrome c-551 had a pI value of 5.2 and an M_r of 260 000 consisting of six non-covalently bound polypeptides each with an M_r of 43 000, and contained four to five haems. Cytochrome c-552.5 had a pI value of 4.8 and an M_r of 56 000 consisting of two polypeptides with the same M_r 29 000, and contained two haems. Cytochromes c-551 and c-552.5 were reduced by ascorbate to about 70 and 60% of the fully dithionite-reduced values, respectively, and both were essential components in the thiosulphate-oxidizing multi-enzyme system (other components of the system were ‘enzyme A’, ‘enzyme B’ and sulphite: cytochrome c oxidoreductase). These two cytochromes functioned as electron carriers and effectors in the oxidation of thiosulphate. Some evidence suggested that cytochrome c-551 might be a specialized electron transfer component for sulphonate-sulphur oxidation. Both cytochromes could be reduced by thiosulphate in the presence of enzymes A and B. Cytochrome c-550 (basic) and cytochrome c-550 (acidic) were small proteins with M_r 15 000 and 14 000 and pI values of over 8 and 5, respectively. Their physiological role is uncertain.     

Reduction kinetics of the photo-oxidized chlorophyll a+II in the nanosecond range: Measurements of the absorption changes at 688 nm under repetitive flash excitation

The 688 nm absorption changes (ΔA₆₈₈), indicating the photochemical turnover of chlorophyll a_II (Chl a_II) have been investigated under repetitive laser flash excitation conditions in spinach chlorplasts. It was found that under steady state conditions about 50–60% of the photo-oxidized primary donor of Photosystem II (PS II), Chl a⁺_II, becomes re-reduced with a biphasic kinetics in the nanosecond time scale with half-life times of about 50 ns and 400 ns. The remaining Chl a⁺_II becomes re-reduced in the microsecond range.     

Energy conservation during nitrate respiration in Paracoccus denitrificans

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the Y _ATP ^max values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate-and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source the average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively.H⁺/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate-and nitratelimited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate-and nitritelimitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.     

Transference of the high molecular weight carbohydrate sequences of fetuin to the surface of carrot embryo protoplasts

A method is described for the removal of the carbohydrate sequences of glycoproteins, and their covalent attachment to hydrocarbon chains. These synthetic membrane components may then be incorporated into liposome and cell membranes. Pronase-liberated glycopeptides derived from fetuin were linked by a reduced Schiff's base linkage to tetradecyl aldehyde. The resulting glycolipid was incorporated by external addition, into phosphatidylcholine liposomes. Glycolipid transfer to these liposomes rendered them suseptible to agglutination by wheat germ lectin, which binds N-acetylneuraminic acid, the terminal carbohydrate of the high molecular weight fetuin sugar sequence. Sequential removal of the terminal sugars, and subsequent agglutination behaviour towards various lectins, suggests that the carbohydrate sequence had been transfered intact. The glycolipid was incorporated into plant protoplast membranes by incubation with glycolipid-containing liposomes for 2 h at 37°C. These synthetic glycolipids may find a use in the study of carbohydrate-based recognition systems in animal and plant membranes. In addition they may prove useful in the development of cell and membrane tagging and handling techniques, by the insertion of sugar groups not normally present in these membranes.     

Influence of mass transfer limitations on determination of the half saturation constant for hydrogen uptake in a mixed-culture CH(4)-producing enrichment

There is strong evidence in the literature supporting the existence of significant mass transfer limitations on the kinetics of exogenous H(2) consumption by methanogens. The half saturation constant for H (2) uptake by a mixed-culture, CH(4) producing enrichment was measured using an experimental protocol that avoided internal mass transfer limitations. The value obtained was two orders of magnitude smaller than any other previously reported. A mathematical model for acetogenic syntrophic associations was developed to check the capacity of H(2) as electron transporter between syntrophic partners. It was found that H(2) diffusion could account for the rate of transport of electrons between the syntrophic microorganisms and that formate is not a necessary intermediate. The possibility that formate may be an intermediate in this system was not ruled out. A Monod-type kinetic equation was modified to include the observed H(2) threshold effect. This modified equation was used to predict the CH(4)-production rate in a batch-fed digester. The results show that the external and internal H(2) pools are kinetically coupled. (c) 1992 John Wiley & Sons, Inc.