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.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution.

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.     

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     

Localization of the enzymes involved in H2 and formate metabolism inSyntrophospora bryantii

Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacteriumSyntrophospora bryantii contained high hydrogenase activities (8.5–75.8 µmol · min^–1 mg^–1 protein) and relatively low formate dehydrogenase activities (0.04–0.07 µmol · min^–1 mg^–1 protein). The K _M value and threshold value of the hydrogenase for H₂ were 0.21 mM and 18 µM, respectively, whereas the K _M value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 µM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO₂ reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H₂ is formed intracellularly while formate is formed at the outside of the cell.     

‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG

Summary Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D ¹H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain ‘random coil’ amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these ‘random coil’ peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K. (1979) Biopolymers, 18, 285–297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH₂ [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67–81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.     

Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer.

A plasmid-free, non-pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanτ) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, β-galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro-organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.     

Influence of elevated ambient temperature after breeding on plasma corticoids, estradiol and progesterone in gilts

Thirty Yorkshire gilts were used to determine the influence of elevated ambient temperature during days 8 to 16 after breeding on concentrations of progesterone, corticoids and estradiol in plasma. Gilts were mated to a boar between 0800 and 1000 hr of the first day (day 0) and each subsequent day of estrus. On day 5 or 6, 22 gilts were anesthetized and a cannula was placed in the anterior vena cava. Gilts were randomly allotted to either control (23+/-1 C) or hot (35+/-1 C for 12 hr and 32+/-1 C for 12 hr daily) environmental chambers on day 8. Gilts were bled twice daily (0800 and 2000 hr) on days 9 through 16, then once daily until day 28. A third group of noncannulated gilts was bred and assigned to the control chamber. All cannulated gilts were injected intravenously with 25 IU of ACTH on day 16, and frequent blood samples were collected. Concentrations of progesterone in plasma were similar for heat-stressed and control gilts that were pregant. However, progesterone in plasma was reduced during days 13 to 19 in nonpregnant heat-stressed gilts compared to the pregnant and nonpregnant control gilts. Concentrations of estradiol in plasma were greater in nonpregnant heat-stressed gilts than in nonpregnant control and all pregnant gilts on days 10, 11 and 12 after estrus. Concentrations of corticoids and progesterone in plasma after infusion of ACTH on day 16 after breeding were reduced in heat-stressed nonpregnant gilts compared to heat-stressed pregnant, control pregnant and control nonpregnant gilts. These data indicate that reduced reproductive performance which occurs after exposure of gilts to increased ambient temperature during days 8 to 16 after breeding may be related to altered endocrine function.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D⁺Q_AQ^?_B proceeds via the intermediate state D⁺Q^?_AQ_B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k_AD-times the fraction of reaction centers in the Q^?_AQ_B state) with the observed recombination rate, k^obs_{D⁺ →D}. The kinetic measurements were used to obtain the pH dependence (6.1 ? pH ? 11.7) of the free energy difference between the states Q^?_AQ_B and Q_AQ^?_B. At low pH (less than 9) Q_AQ^?_B is stabilized relative to Q^?_AQ_B by 67 meV, whereas at high pH Q^?_AQ_B is energetically favored. Both Q^?_A and Q^?_B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q^?_B provides the driving force for the forward electron transfer.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.