Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans.

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Photographic microdensitometry for evaluation of acid phosphatase activity at the electron microscope level

Summary The dry mass of reaction products in ultrathin sections was determined using electron micrographs of polystyrene spheres of known weight deposited on Formvar membranes and evaluating the negatives photometrically. This method was applied to the quantification of the final reaction product of the acid phosphatase reaction in a model system in which enzyme was incorporated in gelatin. The enzyme activity was demonstrated by the lead precipitation method and quantified by direct microphotometry at the light microscope level. Models were then embedded and sectioned for electron microscopy. Microphotometric values afforded by the electron negatives were in linear correlation with incubation times and enzyme concentration. Section thickness and its possible variations due to deformation or contamination under the electron beam were also evaluated. Measurements of lysosomal acid phosphatase activity in rat kidney sections served to illustrate the application of the technique.     

The involvement of iron and ubiquinone in electron transfer reactions mediated by reaction centers from photosynthetic bacteria.

Reaction centers from Rhodopseudomonas sphaeroides strain R-26 were prepared with varying Fe and ubiquinone (Q) contents. The photooxidation of P-870 to P-870+ was found to occur with the same quantum yield in Fe-depleted reaction centers as in control samples. The kinetics of electron transfer from the initial electron acceptor (I) to Q also were unchanged upon Fe removal. We conclude that Fe has no measurable role in the primary photochemical reaction. The extent of secondary reaction from the first quinone acceptor (QA) to the second quinone acceptor (QB) was monitored by the decay kinetics of P-870+ after excitation of reaction centers with single flashes in the absence of electron donors, and by the amount of P-870 photooxidation that occurred on the second flash in the presence of electron donors. In reaction centers with nearly one iron and between 1 and 2 ubiquinones per reaction center, the amount of secondary electron transfer is proportional to the ubiquinone content above one per reaction center. In reaction centers treated with LiClO4 and o-phenanthroline to remove Fe, the amount of secondary reaction is decreased and is proportional to Fe content. Fe seems to be required for the secondary reaction. In reaction centers depleted of Fe by treatment with SDS and EDTA, the correlation between Fe content and secondary activity is not as good as that found using LiClO4. This is probably due in part to a loss of primary photochemical activity in samples treated with SDS; but the correlation is still not perfect after correction for this effect. The nature of the back reaction between P-870+ and Q-B was investigated using stopped flow techniques. Reaction centers in the P-870+ Q-B state decay with a 1-s half-time in both the presence and absence of o-phenanthroline, an inhibitor of electron transfer between Q-B and QB. This indicates that the back reaction between P-870+ and Q-A is direct, rather than proceeding via thermal repopulation of Q-A. The P-870+ Q-B state is calculated to lie at least 100 mV in free energy below the P-870+ Q-A state.     

Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.     

Facilitated ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase in peripheral nerve

p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.     

Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods were able to demonstrate both retrograde labeling of motor neurons and transganglionic anterograde transport into the dorsal horn. However, there were differences in the appearance of the various reaction products under the electron microscope. In addition, differences in the distribution of the reaction products were observed by both light and electron microscopy. The largest distribution of reaction product was observed with TMB. BDHC and o-tolidine were next, followed by the DAB procedures and PPD-PC. The TMB, BDHC, and o-tolidine reaction products were all found to be suitable for electron microscopy. The TMB reaction product was electron dense and had a very distinctive crystalloid appearance that made identification of HRP-labeled neuronal profiles easy and unequivocal.     

New histochemical techniques for the demonstration of carboxyl groups in mucosubstances

Synopsis The histochemical demonstration of the carboxyl groups of mucosubstances was studied by two methods at the light and electron microscopic levels. Conditions for activating carbohydrate carboxyl groups were elucidated from which a modified carbodiimide reaction procedure was worked out. With this procedure several acid mucosubstances could be demonstrated; some were characterized as sialomucoproteins. The mechanism of the carbodiimide reaction is discussed.A method is also discussed for increasing the electron opacity of Alcian Blue-stained mucosubstances with a sulphide-silver reaction.     

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.     

Quantitative dehydrogenase histochemistry with exogenous electron carriers (PMS,MPMS, MB)

Summary The relative efficiencies of phenazine methosulfate (PMS), 1-methoxy-phenazine methosulfate (MPMS) and Meldola Blue (MB) as electron carriers were determined biochemically (non-enzymic NADH-tetrazolium salt-test) and by quantitative histochemistry (heart and kidney slices; succinate dehydrogenase, SDH; lactate dehydrogenase, LDH). MPMS developed the highest electron transfer velocity in biochemical assays. The reaction was independent of the pH value between 7.0–8.5. PMS and MB always showed a lower transfer ability in biochemical tests which was higher with iodonitrotetrazolium chloride (INT) than with nitro blue tetrazolium chloride (NBT). A distinct pH dependence was demonstrable with MB in this respect, preferentially using INT as tetrazolium salt.Quantitative histochemical results with electron carriers are often at variance with biochemical ones. MPMS leads to somewhat higher demonstrable activities only in the determination of the NAD-dependent LDH, whereas MB results in somewhat higher LDH activity than PMS (reaction medium with agarose). MB and PMS yielded almost equally high activities in the demonstration of the flavoprotein-dependent SDH using a reaction medium with agarose. With an aqueous reaction medium, PMS resulted in higer SDH activities than MB. MPMS always had the lowest efficiency in electron transfer ability using an aqueous or agarose containing reaction medium (SDH). With PVA in the reaction medium (SDH determination) PMS was clearly superior to MPMS. MB showed only a small transfer activity under these conditions because PVA seems to bind MB almost completely. It is concluded that in histochemistry an appropriate electron carrier and electron carrier concentration must be determined for different incubation conditions, tissues, tissue preparations and dehydrogenases studied. General statements about the efficiency or inefficiency of an electron carrier as a result of only one incubation condition does not seem to be justified.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Theoretical study of the effect of several reverse reaction on the kinetics of the primary processes of photosynthesis

A theoretical model of energy migration and electron transport in photosynthesis of higher plants was considered. The set of different equations describing these processes takes into consideration the states of 4 components of electron transport chain and back reactions of electron transfer from the reduced acceptors to the oxidized reaction centres. The numerical integration of these equations was made for various kinetics parameters characterizing the electron transport chain.     

Fluorescent analysis of excess electron transfer through DNA

The DNA base stack provides unique features for the efficient long-range charge transfer. For the purpose of investigating excess electron transfer process through DNA, we developed a new method for fluorescence analysis of excess electron transfer based on reductive cleavage of a disulfide bond and a thiol-specific fluorescent probe. Excess electron transfer was detected by monitoring the fluorescence of emissive pyrene monomer generated by the reaction of pyrene maleimides with the cleaved disulfide bond (thiols). Mechanism of reductive cleavage of disulfides through excess electron transfer and subsequent reaction with the fluorescent probes were discussed. This facile and sensitive detection by fluorescence method can be applied for mechanistic study of excess electron transfer.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Effect of pressure on electron transfer reactions in inorganic and bioinorganic chemistry

Kinetic and thermodynamic studies involving the application of different high-pressure techniques, are very useful in gaining mechanistic information on the basis of volume changes that occur during inorganic and bioinorganic electron transfer reactions. The most fundamental type of electron transfer reaction concerns self-exchange reactions, for which the overall reaction volume is zero, and activation volumes can be measured and discussed. In the case of non-symmetrical electron transfer reactions, intra- and intermolecular processes can be studied and volume profiles can be constructed. Precursor complex formation can in some cases be recognized kinetically in such systems. Typical values of activation and reaction volumes are reviewed for various reversible and irreversible electron transfer reactions. Mechanistic conclusions reached on the basis of these parameters are presented. Volume profiles for electron transfer reactions enable a simplistic presentation of the reaction mechanism on the basis of intrinsic and solvational volume changes along the reaction coordinate.     

Photophosphorylation sensitized by chlorophyll in the adsorbed state]

Products of phosphate oxidation in a weak alkaline medium in the electrolysis and in the reaction, photosensitized by chlorophyll, are studied. It is shown that besides perphosphates, pyrophosphate can be formed under electrochemical phosphate oxidation. Phosphate was used as an electron donor and the air oxygen was an electron acceptor in the red-ox reaction, photosensitized by chlorophyll. The reaction was accompanied by the decrease of inorganic phosphate content in the reaction medium and by the phosphorylation of ADP to ATP. The mechanism of photophosphorylation is discussed on the basis of the data obtained.     

Electron transport and triplet formation in membranes of the photosynthetic bacterium Heliobacterium chlorum

The spectroscopic and thermodynamic properties of the electron-transport components of the photosynthetic bacterium Heliobacterium chlorum were studied by means of absorbance-difference spectroscopy. Upon flash illumination of membranes of H. chlorum photooxidation of the primary electron donor, P-798, was observed. In about 15% of the reaction centers P-798⁺ was reduced by cytochrome c-553, while in the remaining reaction centers P-798⁺ reduction occurred via a back reaction with a reduced electron acceptor. Titration experiments indicated a midpoint potential of −440 mV for the electron acceptor. At low redox potentials the formation of the triplet of P-798 was observed after a flash. The triplet was formed in about 30 ns by a back reaction with a reduced electron acceptor and decayed with a time constant of 35 μs. The yield of triplet formed in a flash was 30%. Upon continuous illumination at low redox potentials the accumulation in the reduced state of an electron acceptor was observed. The difference spectrum of this acceptor indicates that it is an iron-sulfur center. The yield of triplet formation was independent of the redox state of the iron-sulfur center, which indicates that the center is not located in the main electron-transport chain. A scheme with three acceptors in the main electron-transport chain is presented to accomodate our results and those of others.     

The mechanism of quinonediimine acceptor activity in photosynthetic electron transport.

The rates of electron flow catalyzed by a variety of unsubstituted and C- or N-methylated quinonediimine electron acceptors in a reaction requiring photosystem II in KCN-inhibited chloroplasts vary according to the structure of acceptor used. Quinonediimine, but not quinone, electron acceptor activities are inhibited by a variety of uncouplers. Kinetic analysis of this inhibition shows that it is competitive. Low concentrations of aniline also inhibit the activity of C-methylated quinonediimines, but this appears to be due to a chemical reaction between the acceptor and aniline at low pH inside the chloroplast. Light-induced uptake of a quinonediimine, p-phenylenediimine, was shown to occur in a DCMU-sensitive reaction. Methylamine uncoupling inhibits this uptake to the same extent as it inhibits electron flow. Experiments with a lipophobic acceptor, N,N,N',N'-tetramethyl-p-phenylenediimine, indicate that it catalyzes electron flow by the same mechanism as other quinonediimines. A model is proposed to account for quinonediimine-catalyzed electron flow.     

Direct electron transfer catalysed by enzymes: application for biosensor development

The ability to catalyse an electrode reaction via direct (mediatorless) electron transfer has been demonstrated for a number of redox enzymes. In the case of mediatorless electron transfer, the electron is transferred directly from the electrode to the substrate molecule via the active site of the enzyme, or vice versa. The electron itself is the second substrate for the reaction. An important point characterizing bioelectrocatalysis is the catalytic removal of the reaction over-voltage. Therefore the enzyme attached to the electrode is able to catalyse electrode reaction and forms a 'molecular transducer'. The substrate can be detected by potentiometric measurement of the removal of reaction over-voltage. The enzyme laccase is able to catalyse the reaction of oxygen electroreduction. Therefore a laccase molecular layer attached to the electrode surface forms an oxygen transducer. The formation of the layer results in a change of the electrocatalytic feature of the electrode. Laccase label coupled with either ligand or receptor allows the detection of ligand-receptor complex formation/dissociation on the electrode surface. The detection is virtually reagentless. The substrates for the reaction are molecular oxygen and the electron itself. Numerous reagentless immunosensors of different formats (competitive, displacement and sandwich) have been developed, as well as the reagentless detection system for immunofiltration/immunochromatography.     

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.     

The back reaction in the primary electron transfer couple of photosystem II of photosynthesis

Changes of C-550, cytochrome b₅₅₉ and fluorescence yield induced in chloroplasts by single saturating flashes were studied at low temperature. A single saturating flash at −196°C was quite ineffective in reducing C-550, oxidizing cytochrome b₅₅₉ or increasing the fluorescence yield, presumably because most of the charge separation induced by the flash was dissipated by a direct back reaction in the primary electron transfer couple. The back reaction, which competes with the dark reduction of the oxidized primary electron donor by a secondary electron donor, becomes increasingly important as the temperature is lowered because of the temperature coefficient of the reaction with the secondary donor. The effect of the back reaction is to lower the quantum yield for the production of stable photochemical products by steady irradiation. Assuming a quantum yield of unity for the photoreduction of C-550 at room temperature, the quantum yield for the reaction is about 0.40 at −100°C and 0.27 at −196°C.