Insect crossbridges, relaxed by spin-labeled nucleotide, show well-ordered 90 degrees state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder.

The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.     

A sequential electron transfer from hydrogenases to cytochromes in sulfate-reducing bacteria

A central step in the energy metabolism of sulfate-reducing bacteria is the oxidation of molecular hydrogen, catalyzed by a periplasmic hydrogenase. The resulting electrons are then transferred to various electron transport chains and used for cytoplasmic sulfate reduction. The complex formation between [NiFeSe] hydrogenase and the soluble periplasmic polyheme cytochromes from Desulfomicrobium norvegicum was characterized by cross-linking experiments, BIAcore and kinetics analysis. Analysis of electron transfer between [NiFeSe] hydrogenase and octaheme cytochrome c(3) (M(r) 26? omitted?000) pointed out that this cytochrome is reduced faster in the presence of catalytic amounts of tetraheme cytochrome c(3) (M(r) 13? omitted?000) isolated from the same organism. The activation of the hydrogenase-dependent reduction of polyheme cytochromes by cytochrome c(3) (M(r) 13? omitted?000), which is now described in both Desulfovibrio and Desulfomicrobium, is proposed as a general mechanism. During this process, cytochrome c(3) (M(r) 13? omitted?000) would act as an electron shuttle in between hydrogenase and the polyheme cytochromes and its conductivity appears to be an important factor.     

HISTOCHEMICAL LOCALIZATION AT THE ELECTRON MICROSCOPE LEVEL

Albersheim, Peter, and Ursula Killias. (Harvard U., Cambridge, Mass.) Histochemical localization at the electron microsco pe level. Amer. Jour. Bot. 50(7): 732–745. Illus. 1963.—This paper discusses the use of chemical reactions for specifically locating cellular constituents with the electron microscope. Presented in detail are results obtained by using alkaline hydroxylamine and ferric chloride as an electron stain for pectin. This treatment also results in the staining of certain cytoplasmic and nucleolar particles of unknown nature. Also described is the use of bismuth as an electron stain for nucleic acids. A series of micrographs is presented depicting bismuth stained onion root tip cells in various stages of mitosis.     

Comparing risk factors for population extinction

Extinction risk of natural populations of animals and plants is enhanced by many different processes, including habitat size reduction and toxic chemical exposure. We develop a method to evaluate different risk factors in terms of the decrease in the mean extinction time. We choose a population model with logistic growth, environmental and demographic stochasticities with three parameters (intrinsic growth rate r, carrying capacity K, and environmental noise sigma(2)(e)). The reduction in the habitat size decreases carrying capacity K only, whilst toxic chemical exposure decreases survivorship (or fertility) and in effect reduces both r and K. We derived a formula for the reduction in habitat size that decrease the mean extinction time by the same magnitude as a given level of toxic chemical exposure. In a large population (large K) or in a slowly growing population (small r), a small decrease in survivorship can cause the extinction risk increase corresponding to a significant reduction in the habitat size. This conclusion depends also on the nonlinearity of dose-effect relationship. To illustrate the method, we analyse a freshwater fish, Japanese crucian carp (Carassius auratus subsp.) in Lake Biwa.     

Influences of growth substrates and oxygen on the electron transport system in Acinetobacter sp. HO1-N.

The electron transport system of Acinetobacter sp. HO1-N was studied to determine the specific cytochromes and to measure changes in the composition of the respiratory system due to growth in various concentrations of oxygen or types of growth substrates. Spectrophotometric analysis revealed that the quantity and types of cytochromes changed in response to growth under various concentrations of oxygen. Growth on alkane and nonalkane substrates resulted in only minor differences in cytochrome composition or oxidase activities. Membranes prepared from cells grown under oxygen-limiting conditions contained at least one b-type cytochrome, cytochrome o, cytochrome d, and slight traces of cytochrome a1, whereas membranes prepared from cells grown in the presence of high oxygen concentrations contained only low levels of cytochromes b and o. Polarographic measurements, electron transport inhibitor studies, and photoaction spectrum analyses indicated that cytochromes o, a1, and d were potentially capable of functioning as terminal oxidases in this organism. These experiments also revealed that all three cytochromes may be involved in the oxidation of reduced nicotinamide adenine dinucleotide, succinate, or N,N,N',N'-tetramethyl-p-phenylenediamine.     

Regulation of electron transport in microalgae

Unicellular algae are characterized by an extreme flexibility with respect to their responses to environmental constraints. This flexibility probably explains why microalgae show a very high biomass yield, constitute one of the major contributors to primary productivity in the oceans and are considered a promising choice for biotechnological applications. Flexibility results from a combination of several factors including fast changes in the light-harvesting apparatus and a strong interaction between different metabolic processes (e.g. respiration and photosynthesis), which all take place within the same cell. Microalgae are also capable of modifying their photosynthetic electron flow capacity, by changing its maximum rate and/or by diverting photogenerated electrons towards different sinks depending on their growth status. In this review, we will focus on the occurrence and regulation of alternative electron flows in unicellular algae and compare data obtained in these systems with those available in vascular plants. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Purification and properties of the alkaline phosphatase ofSerratia marcescens

1. A hypothesis based on the Hill-Bendall-model of photosynthetic electron transport is proposed to explain positive and negative photo-phobotaxis inPhormidium uncinatum. In the non-cyclic electron chain a pool is located into which photosystem II (e. g. by absorption by C-phycoerythrin, 561 nm) feeds electrons while photosystem I (e.g. 723 nm) drains electrons out of it.
2. Interruption of the electron flow into the pool causes a sudden decrease of the pool size and thus a positive phobic response. This happens e.g. when an organism leaves a trap which is illuminated by a wavelength absorbed by photosystem II pigments (e. g. 561 nm).
3. A negative reaction takes place when electrons are suddenly drained out of the pool; again the pool size decreases. This is the case when an organism enters a light trap illuminated by photosystem I light (723 nm).
4. The net flow of electrons into or out of the pool—and thus the reaction sense—can be manipulated by the relative excitation of the two photosystems or by blocking the electron influx by DCMU.

     

Cloning, characterization, and expression in Escherichia coli of the genes encoding the cytochrome d oxidase complex from Azotobacter vinelandii.

Azotobacter vinelandii is a free-living nitrogen-fixing bacterium that has one of the highest respiratory rates of all aerobic organisms. Based on various physiological studies, a d-type cytochrome has been postulated to be the terminal oxidase of a vigorously respiring but apparently uncoupled branch of the electron transport system in the membranes of this organism. We cloned and characterized the structural genes of the two subunits of this oxidase. The deduced amino acid sequences of both subunits of the A. vinelandii oxidase have extensive regions of homology with those of the two subunits of the Escherichia coli cytochrome d complex. Most notably, the histidine residues proposed to be the axial ligands for the b hemes of the E. coli oxidase and an 11-amino-acid stretch proposed to be part of the ubiquinone binding site are all conserved in subunit I of the A. vinelandii oxidase. The A. vinelandii cytochrome d was expressed in a spectrally and functionally active form in the membranes of E. coli, under the control of the lac or tac promoter. The spectral features of the A. vinelandii cytochrome d expressed in E. coli are very similar to those of the E. coli cytochrome d. The expressed oxidase was active as a quinol oxidase and could reconstitute an NADH to oxygen electron transport chain.     

Identification of the ligand-exchange process in the alkaline transition of horse heart cytochrome c.

Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c in the presence of 1-methylimidazole have also been examined. These studies strongly suggest that histidine-18, the proximal ligand of the haem, is the ionizing group that triggers the alkaline transition. Low-temperature m.c.d. and e.p.r. spectra are also reported for Pseudomonas aeruginosa cytochrome c551. It is shown that no ligand exchange takes place at the haem in this species over the pH range 6.0-11.3.     

Inhibition of chloroplast electron transport reactions by trifluralin and diallate

The herbicides trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N, N-dipropyl-p-toluidine) and diallate (S-[2,3-dichloroallyl] diisopropylthiocarbamate) inhibit electron transport, ATP synthesis, and cytochrome f reduction by isolated spinach (Spinacia oleracea L.) chloroplasts. Both compounds inhibit noncyclic electron transport from H(2)O to ferricyanide more than 90% in coupled chloroplasts at concentrations less than 50 mum. Neither herbicide inhibits electron transport in assays utilizing only photosystem I activity, and the photosystem II reaction elicited by addition of oxidized p-phenylenediamine or 2,5-dimethylquinone is only partially inhibited by herbicide concentrations which block electron flow from H(2)O to ferricyanide. Inhibition of ATP synthesis parallels inhibition of electron flow in all noncyclic assay systems, and cyclic ATP synthesis catalyzed by either diaminodurene or phenazine metho-sulfate is susceptible to inhibition by both herbicides. These results indicate that trifluralin and diallate both inhibit electron flow in isolated chloroplasts at a point in the electron transport chain between the two photosystems.     

Mechanism of electron transfer between myoglobin derivatives and ferricytochrome C

Progress in the studies of the electron transport mechanism in biological systems is greatly hindered by the lack of detailed structural information about the components of these systems. That is why a study of electron transfer between protein molecules with the known spatial organization in model reactions in vitro is of great importance. In this respect the MbO2--Cyt C oxidation-reduction reaction offers unique possibilities. Studies of the effects of pH and ionic strength of the medium on the kinetics of this reaction in combination with chemical modification of single amino acid residues of Mb and Cyt C enabled us to identify those parts of the surface of haemoproteins where the molecules come into "active contact". A variation in the number or/and the arrangement of the charged groups at the "active sites" of the molecules induced by both changing the medium pH and chemical modification of some of these groups lowers markedly the probability of electron transfer in the system (e.g. His GH1 and His A10 in Mb) or blocks it entirely (acylation of Lys 72 (73) or Tyr 74 in Cyt C). Based on the results obtained and on the data of Mb and Cyt C X-ray analysis, the figures of spatial arrangement of the groups at the "active sites" of these molecules are presented.     

Scanning electron microscopy of dairy equipment surfaces contaminated by two milk-borne micro-organisms

Ethanol dehydration followed by argon replacement induced drying (ARID) was found to be a suitable method for the preparation of glass, stainless steel and rubber surfaces which had been in contact with inoculated milk and which were to be examined using scanning electron microscopy (SEM). This technique was used to examine samples of all three materials which had been subjected to both single and repeated inoculation with whole milk containing a Pseudomonas sp. or a Micrococcus sp. and incubated for various periods. Some samples were also prepared for SEM using a cryofixation technique. The Pseudomonas sp. was found to proliferate on glass and stainless steel surfaces but not on rubber. Due to the clumping tendency of the Micrococcus sp. proliferation of this organism was more difficult to assess accurately. In general there was no difference in results obtained between single and repeated inoculation. Various factors which may have aided attachment of micro-organisms to surfaces were identified viz. , surface channels present in stainless steel, milk deposits and the production of extracellular material. The value of using both the cryofixation and chemical preparatory techniques for the identification of artifacts is discussed.     

Effects of protein-protein interactions on electron transfer: docking and electron transfer calculations for complexes between flavodoxin and c-type cytochromes

 Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c ₅₅₃ and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c ₅₅₃ this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases. Received: 29 December 1998 / Accepted: 22 March 1999     

Metabolomic analyses show that electron donor and acceptor ratios control anaerobic electron transfer pathways in Shewanella oneidensis

This study investigated the physiological impact of changing electron donor–acceptor ratios on electron transfer pathways in the metabolically flexible subsurface bacterium Shewanella oneidensis, using batch and chemostat cultures, with an azo dye (ramazol black B) as the model electron acceptor. Altering the growth rate did result in changes in biomass yield, but not in other key physiological parameters including the total cytochrome content of the cells, the production of extracellular flavin redox shuttles or the potential of the organism to reduce the azo dye. Dramatic increases in the ability to reduce the dye were noted when cells were grown under conditions of electron acceptor (fumarate) limitation, although the yields of extracellular redox mediators (flavins) were similar under conditions of electron donor (lactate) or acceptor limitation. FT-IR spectroscopy confirmed shifts in the metabolic fingerprints of cells grown under these contrasting conditions, while spectrophotometric analyses supported a critical role for c-type cytochromes, expressed at maximal concentrations under conditions of electron acceptor limitation. Finally, key intracellular metabolites were quantified in batch experiments at various electron donor and acceptor ratios and analysed using discriminant analysis and a Bayesian network to construct a central metabolic pathway model for cells grown under conditions of electron donor or acceptor limitation. These results have identified key mechanisms involved in controlling electron transfer in Shewanella species, and have highlighted strategies to maximise reductive activity for a range of bioprocesses.     

Electron paramagnetic resonance studies on spin-labelling of pepsin: effects of temperature, pH and urea on its conformation.

Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra.     

L-malate oxidation by the electron transport fraction of Azotobacter vinelandii

The membrane-bound l-malate oxidoreductase of Azotobacter vinelandii strain O was found to be a flavoprotein-dependent enzyme associated with the electron transport system (R(3)) of this organism. The particulate R(3) fraction, which possessed the l-malate oxidoreductase, carried out the cyanide-sensitive oxidation of l-malate, d-lactate, reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, succinate, cytochrome c, tetramethyl-p-phenylenediamine, and p-phenylenediamine, with molecular O(2) as the terminal electron acceptor. d-Malate was not oxidized, but l-malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K(3), K(3)Fe(CN)(6), nitro blue tetrazolium, and dichloroindophenol all served as good terminal electron acceptors for the l-malate oxidoreductase. Cytochrome c was a poor electron acceptor. Extensive studies on the l-malate oxidase and PMS and K(3) reductases revealed that all were stimulated specifically by flavine adenine dinucleotide and nonspecifically by di- or trivalent cations, i.e., Ca(++), Ba(++), Mn(++), Mg(++), Fe(+++), Ni(++), and Al(+++). All these activities were markedly sensitive to ethylenediaminetetraacetate (EDTA). The V(max) values for the l-malate oxidase, PMS, and vitamin K(3) reductases were, respectively, 3.4, 15.1, and 45.5 mumoles of substrate oxidized per min per mg of protein at 37 C. Spectral studies revealed that the Azotobacter R(3) flavoprotein and cytochromes (a(2), a(1), b(1), c(4), and c(5)) were reduced by l-malate. l-Malate oxidase activity was sensitive to various inhibitors of the electron transport system, namely, p-chloromercuriphenylsulfonic acid, chlorpromazine, 2-n-heptyl-4-hydroxyquinoline-N-oxide, antimycin A, and KCN. Minor inhibitory effects were noted with the inhibitors 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, rotenone, and Amytal.     

Sterilization of plastic containers using electron beam irradiation directed through the opening

AIMS: Studies were performed to demonstrate the efficacy of a novel electron beam irradiation system for the on-line sterilization of polymeric containers using energetic electrons directed through the container opening. METHODS AND RESULTS: The distribution of dosage delivered during conveyance beneath the electron beam treatment system was determined for two sizes (8 or 16 ounces) of high-density polyethylene (HDPE) blow-moulded bottles. The biological effects of treatment were then determined using Bacillus pumilus ATCC 27142 spores inoculated as 10 microl droplets dried (i) onto the surface of flat coupons of the high-density polyethylene bottle material, and (ii) onto the region of lowest delivered dose (the bottom side wall) within each bottle. The inactivation obtained was determined by examining the level of survival after swab recovery of the inoculated spores, with reference to the level recovered from untreated control samples. CONCLUSIONS: The inactivation of B. pumilus spores during treatment was reproducible and proportional to the applied dose. In each instance, the logarithm of the surviving fraction of spores was well fitted by linear regression models and in good general agreement with values reported for inactivation of the same organism using gamma irradiation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this work demonstrate the potential of a new physical method capable of high throughput for in-line sterilization of polymeric containers. The ability of the process to eliminate the washing step involved with traditional chemical sterilants, such as hydrogen peroxide, greatly reduces the size and environmental impact of these systems.     

Evaluation by electron microscopy of the integrity of iodinated plasmalipoproteins.

Iodination of proteins and lipoproteins is a widely used "in vitro" labelling procedure in metabolic, autoradiographic and various other studies. However, all available iodination techniques have involved the possible damage to the proteins by self-irradiation, oxidizing agents, the alkaline milieu or by the introduction of iodine into the molecular structure itself. To evaluate the integrity of iodinated lipoprotiens, we observed the electron microscopic appearance of normal and iodinated rabbit very low density lipoproteins (VLDL) by negative staining with phosphotungstic acid. Iodination up to a molar iodine/protein ratio of 2.89 did not results in any change of shape, size or aggregating tendency of the particles. No stacks or disk-like particles like those of various hyperlipoproteinemic states were found. We conclude that electron microscopy is a valuable tool in assessing the morphological appearance of lipoprotein iodination, but it should be complemented by other techniques.     

Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells

The surfaces of flocculent and nonflocculent yeast cells have been examined by electron microscopy. Nonextractive preparative procedures for scanning electron microscopy allow comparison in which sharp or softened images of surface details (scars, etc.) are the criteria for relative abundance of flocculum material. Asexually flocculent budding-yeast cells cannot be distinguished from nonflocculent budding-yeast cells in scanning electron micrographs because the scar details of both are well resolved, being hard and sharp. On the other hand, flocculent fission-yeast cells are readily distinguished from nonflocculent cells because fission scars are mostly soft or obscured on flocculent cells, but sharp on nonflocculent cells. Sexually and asexually flocculent fission-yeast cells cannot be distinguished from one another as both are heavily clad in "mucilaginous" or "hairy" coverings. Examination of lightly extracted and heavily extracted flocculent fission-yeast cells by transmission electron microscopy provides micrographs consistent with the scanning electron micrographs.