Electron Flow Pathways Research Articles

Nickel containing CO dehydrogenases and hydrogenases.

The two redox catalysts described here can generate very low potential electrons in one direction and perform chemically difficult reductions in the other. The chemical transformations occur at unusual metal clusters. Spectroscopic, crystallographic, and kinetic analyses are converging on answers to how the metals in these clusters are arranged and how they are involved in the chemical and redox steps. The first structure of CO dehydrogenase, which will appear in the next year, will help define a firm chemical basis for future mechanistic studies. In the immediate future, we hope to learn whether the hydride intermediate in hydrogenase or the carbonyl intermediate in CO dehydrogenase bind to the Ni or Fe subsites in these heterometallic clusters. Or perhaps could they be bridged to two metals? Inter- and intramolecular wires have been proposed that connect the catalytic redox machine to proximal redox centers leading eventually to the ultimate redox partners. Elucidating the pathways of electron flow is a priority for the future. There is evidence for molecular channels delivering substrates to the active sites of these enzymes. In the next few years, these channels will be better defined. The products of CO2 and proton reduction are passed to the active sites of other enzymes and, in the case of H2, even passed from one organism to another. In the future, the mechanism of gas transfer will be uncovered. General principles of how these redox reactions are catalyzed are becoming lucid as the reactions are modeled theoretically and experimentally. Proton and CO2 reduction and the generation of C-C bonds from simple precursors are important reactions in industry. H2 could be the clean fuel of the future. Hopefully, the knowledge gained from studies of hydrogenase, CO dehydrogenase, and acetyl-CoA synthase can be used to improve life on earth.

Biochemical and molecular characterization of the [NiFe] hydrogenase from the hyperthermophilic archaeon, Thermococcus litoralis.

Thermococcus litoralis is a hyperthermophilic archaeon that grows at temperatures up to 98 degrees C by fermentative metabolism and reduces elemental sulfur (S0) to H2S. A [NiFe] hydrogenase, responsible for H2S or H2 production, has been purified and characterized. The enzyme is composed of four subunits with molecular mass 46, 42, 34 and 32 kDa. Elemental analyses gave approximate values of 22 Fe, 22 S and 1 Ni per hydrogenase. EPR spectra at 70 and 5 K indicated the presence of four or five [4Fe-4S] and one [2Fe-2S] type clusters. The optimal temperature for both H2 evolution and oxidation, using artificial electron carriers, was around 80 degrees C. The operon encoding the T. litoralis enzyme is composed of four genes forming one transcriptional unit, and transcription is not regulated by S0. An unusual transcription-initiation site is located 139 bp upstream from the translational start point. Sequence analyses indicated the presence of new putative nucleotide-binding domains. Upstream from the hydrogenase operon, ORFs probably encoding a molybdopterin oxidoreductase enzyme have been identified. Based on sequence, biochemical and biophysical analyses, a model of the enzyme and the pathway of electron flow during catalysis is proposed.

Sap flow, leaf gas exchange and chlorophyll fluorescence of container-grown cashew (Anacardium occidentale L.) trees subjected to repeated cycles of soil drying

Summary. Cashew (Anacardium occidentale L.) is an emerging horticultural crop in tropical northern Australia. Supplementary watering is required during the dry season to achieve high yields but irrigation guidelines are not well defined. As an introduction to large-scale field experiments which will aim to define the irrigation requirements for cashew, this experiment was conducted on small, container-grown cashew trees to examine their response to drying soil and to evaluate a range of techniques for measuring tree water use and photosynthesis with possible application in the proposed field experiments. Measurements of sap flow, leaf chlorophyll fluorescence and leaf gas exchange were made on all trees throughout the experimental period. The water use of trees in drying soil was measured using Granier’s sap flow system. Sap flux density (L/dm2 sapwood area . h) of drying trees declined progressively over a 4-day period to a minimum level that was only 10% of the sap flow in the well watered trees. Measurements of leaf gas exchange showed similarly large reductions in photosynthesis and transpiration which were associated with a low (0.05 mol/m2 . s) stomatal conductance in the drying trees. After rewatering, sap flow and leaf gas exchange recovered to the high levels of the well watered trees over 3–4 days. Similar behaviour was observed during the second drying period. Measurements of the ratio of variable to maximum fluorescence, Fv : Fm, an indicator of photoinhibition, were made on dark-adapted leaves before dawn and during the day. Fv : Fm was in the range 0.65–0.80 with no large or sustained differences between drying and well watered trees. When stomatal conductance and net photosynthetic rate progressively declined during the period following irrigation, the quantum yield of photochemical energy conversion in photosystem II, ΦPSII, remained almost constant. It is possible that by providing a pathway for electron flow as an alternative to CO2 assimilation during this period, photorespiration played an important role in avoiding photoinhibition.

Physiology of Carbohydrate to Solvent Conversion by Clostridia

The solvent-forming clostridia have attracted interest because of their ability to convert a range of carbohydrates to end-products such as acetone, butanol and ethanol. Polymeric substrates such as cellulose, hemicellulose and starch are degraded by extracellular enzymes. The majority of cellulolytic clostridia, typified by Clostridium thermocellum, produce a multi-enzyme cellulase complex in which the organization of components is critical for activity against the crystalline substrate. A variety of enzymes involved in degradation of hemicellulose and starch have been identified in different strains. The products of degradation, and other soluble substrates, are accumulated via membrane-bound transport systems which are generally poorly characterized. It is clear, however, that the phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in solute uptake in several species. Accumulated substrates are converted by intracellular enzymes to end-products characteristic of the organism, with production of ATP to support growth. The metabolic pathways have been described, but understanding of mechanisms of regulation of metabolism is incomplete. Synthesis of extracellular enzymes and membrane-bound transport systems is commonly subject to catabolite repression in the presence of a readily metabolized source of carbon and energy. While many genes encoding cellulases, xylanases and amylases have been cloned and sequenced, little is known of control of their expression. Although the mechanism of catabolite repression in clostridia is not understood, some recent findings implicate a role for the PTS as in other low G-C Gram-positive bacteria. Emphasis has been placed on describing the mechanisms underlying the switch of C. acetobutylicum fermentations from acidogenic to solventogenic metabolism at the end of the growth phase. Factors involved include a lowered pH and accumulation of undissociated butyric acid, intracellular concentration of ATP and reduced pyridine nucleotides, nutrient limitation, and the interplay between pathways of carbon and electron flow. Genes encoding enzymes of solvent pathways have been cloned and sequenced, and their expression correlated with the pattern of end-product formation in fermentations. There is evidence that the initiation of solvent formation may be subject to control mechanisms similar to other stationary-phase phenomena, including sporulation. The application of recently developed techniques for genetic manipulation of the bacterium is improving understanding of the regulatory circuits, but a complete molecular description of the control of solvent formation remains elusive. Experimental manipulation of the pathways of electron flow in other species has been shown to influence the range and yield of fermentation end-products. Acid-forming clostridia can, under appropriate conditions, be induced to form atypical solvents as products. While the mechanisms of regulation of gene expression are not at all understood, the capacity to adapt in this way further illustrates the metabolic flexibility of clostridial strains.

The effect of chloroform on mitochondrial energy transduction.

The effect of chloroform on mitochondrial respiration with succinate was investigated by applying the method of Brand, Chien and Diolez [(1994) Biochem. J. 297, 27-29] to examine whether chloroform causes redox slip (fewer protons pumped per electron transferred) during mitochondrial electron transport. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD), which lowers H+/O (the number of protons pumped to the external medium by the electron transport complexes per oxygen atom consumed) by altering the electron flow pathway, was investigated for comparison. Non-phosphorylating mitochondria that had been treated with 350 microM TMPD or 30 mM chloroform were titrated with malonate in the presence of submaximal concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Linear relations between CCCP-induced extra respiration and protonmotive force were obtained. These results showed that there was no measurable protonmotive force-dependent or rate-dependent slip in mitochondria treated with either TMPD or chloroform. However, both TMPD and chloroform seemed to decrease H+/O in a manner independent of protonmotive force and rate. The relationship between non-phosphorylating respiration and protonmotive force was simulated in mitochondria of which 25% of the total population were assumed to have been broken. The simulation showed that the apparent decrease in H+/O on the addition of TMPD or chloroform to mitochondria could be in principle accounted for by breakage. Assays of mitochondrial breakage (ATP hydrolysis in the presence of atractyloside and oxidation of exogenous NADH) showed that chloroform broke mitochondria but TMPD did not. We conclude that chloroform changes the measured H+/O as an artifact by causing mitochondrial breakage and does not cause measurable redox slip, whereas TMPD genuinely lowers H+/O.

Involvement of anaerobic reductases in the spontaneous lysis of formate by immobilized cells of Escherichia coli

In the lysis of formate by the immobilized formate hydrogen lyase (FHL) of Escherichia coli, generation of hydrogen was dependent on the addition of a small amount of glucose, tetrathionate, thiosulfate, or any one of the dicarboxylic acids such as fumarate, malate, succinate, and oxalocetate. During lysis, activities of anaerobic reductases of E. coli, i.e., fumarate → succinate and tetrathionate → thiosulfate were evident as cells converted all acids and glucose into succinate and tetrathionate into thiosulfate. However, the cells also reduced fumarate and tetrathionate and converted glucose into succinate in the absence of formate. The presence of endogenous reducing substrate in the cell was also indicated by the spontaneous reduction of methylene blue (MB) by the cells which was stimulated by formate. MB reduction was not dependent on FHL activity and the presence of succinate or thiosulfate. It appeared that electron flow by endogenous substrate oxidation could reduce oxidants of high (MB) and low (fumarate and tetrathionate) redox potentials. This pathway is overlapping with the electron flow pathway of FHL probably due to the presence of common or similar electron transport carriers of close redox potentials. Thus, hydrogen production was terminated due to channeling of electrons from formate to the oxidants produced by the endogenous substrate but not to hydrogenase. The presence of excess thiosulfate and succinate prevented the channeling and maintained the flow of electrons for the reduction of hydrogen ion.

Evidence for three separate electron flow pathways through complex I: an inhibitor study

The mammalian mitochondrial electron transport chain catalyzes the oxidation of NADH at pH 8.0 and pH 6.5, and the oxidation of NADPH at pH 6.5. The pH-dependencies of the rate of steady-state oxidation of NADPH and NADH by Complex I as well as by its flavoprotein fraction have been extensively studied by the laboratory of Hatefi. One model to explain these pH-dependent oxidations was proposed by Bakker and Albracht (Biochim. Biophys. Acta 850 (1986) 413–422 and 423–428, modified by Van Belzen and Albracht (Biochim. Biophys Acta 974 (1989) 311–320), which predicts that Complex I is a heterodimer with protomer B, containing FMN and FeS clusters 1–4 in stiochiometric amounts, catalyzing NADH oxidation at pH 8, and Protomer A, containing FMN and FeS clusters 2, 4, catalyzing NAD(P)H oxidation at pH 6.5. A pH-dependent transfer of electrons from protomer A FeS clusters 2, 4 to protomer B FeS clusters 2, 4 is an obligate step in the oxidation of NAD(P)H at low pH. Strict interpretation of this model allows for only three types of inhibitor: one which inhibits all three oxidase activities (type 1); one which inhibits NADH oxidase, pH 8.0 (type 4) and a third which inhibits NAD(P)H oxidase, pH 6.5 (type 5). Another possibility is that there are three separate pathways of oxidation of NAD(P)H, which would allow for a total of seven different types of inhibitor, e.g., the three types above plus type 2 inhibiting NADH oxidase pH 8.0 and pH 6.5; type 3 inhibiting NADH oxidase pH 8.0, and NADPH oxidase pH 6.5; type 6 inhibiting NADH oxidase pH 6.5; and type 7 inhibiting NADPH oxidase pH 6.5. Using a series of thirteen inhibitors of Complex I activity and the chemical modification reagent ethoxyformic anhydride (EFA), four different inhibitor types were found: seven inhibitors of type 1, four inhibitors of type 2, one inhibitor of type 3 and one inhibitor of type 4. Treatment of submitochondrial particles (SMP) with EFA abolished NADH-dependent reduction of coenzyme Q at both pH 8.0 and 6.5, while inhibiting NADPH-dependent reduction of coenzyme Q at pH 6.5 by only 30%. These results do not support the heterodimer model of Complex I electron transport of Bakker and Albracht, but do support three separate electron flow pathways through complex 1 from reduced pyridine nucleotides to coenzyme Q. A new model of electron flow through Complex I based on these finding is proposed.

Ecophysiological adaptations of anaerobic bacteria to low pH: analysis of anaerobic digestion in acidic bog sediments.

The dynamics of anaerobic digestion were examined in the low-pH sediments of Crystal Bog in Wisconsin. The sediments (pH 4.9) contained 71% organic matter and the following concentrations of dissolved gases (micromoles per liter): CO2, 1,140; CH4, 490; and H2, 0.01. The rate of methane production was 6.2 mumol/liter of sediment per h, which is slower than eutrophic, neutral sediments. Microbial metabolic processes displayed the following pH optima: hydrolysis reactions, between 4.2 and 5.6; aceticlastic methanogenesis, 5.2; and hydrogen-consuming reactions, 5.6. The turnover rate constants for key intermediary metabolites were (h-1): glucose, 1.10; lactate, 0.277; acetate, 0.118; and ethanol, 0.089. The populations of anaerobes were low, with hydrolytic groups (10(6)/ml) several orders of magnitude higher than methanogens (10(2)/ml). The addition of carbon electron donors to the sediment resulted in the accumulation of hydrogen, whereas the addition of hydrogen resulted in the accumulation of fatty acids and the inhibition of hydrogen-producing acetogenic reactions. Strains of Lactobacillus, Clostridium, and Sarcina ventriculi were isolated from the bog, and their physiological attributes were characterized in relation to hydrolytic process functions in the sediments. The present studies provide evidence that the pH present in the bog sediments alter anaerobic digestion processes so that total biocatalytic activity is lower but the general carbon and electron flow pathways are similar to those of neutral anoxic sediments.

Molecular aspects of nitrogen fixation by photosynthetic prokaryotes.

The photosynthetic prokaryotes possess diverse metabolic capabilities, both in carrying out different types of photosynthesis and in their other growth modes. The nature of the coupling of these energy-generating processes with the basic metabolic demands of the cell, such as nitrogen fixation, has stimulated research for many years. In addition, nitrogen fixation by photosynthetic prokaryotes exhibits several unique features; the oxygen-evolving cyanobacteria have developed various strategies for protection of the oxygen-labile nitrogenase proteins, and some photosynthetic bacteria have been found to regulate their nitrogenase (N2ase) activity in a rapid response to fixed nitrogen, thus saving substantial amounts of energy. Recent advances in the biochemistry, physiology, and genetics of nitrogen fixation by cyanobacteria and photosynthetic bacteria are reviewed, with special emphasis on the unique features found in these organisms. Several major topics in cyanobacterial nitrogen fixation are reviewed. The isolation and characterization of N2ase and the isolation and sequence of N2ase structural genes have shown a great deal of similarity with other organisms. The possible pathways of electron flow to N2ase, the mechanisms of oxygen protection, and the control of nif expression and heterocyst differentiation will be discussed. Several recent advances in the physiology and biochemistry of nitrogen fixation by the photosynthetic bacteria are reviewed. Photosynthetic bacteria have been found to fix nitrogen microaerobically in darkness. The regulation of nif expression and possible pathways of electron flow to N2ase are discussed. The isolation of N2ase proteins, particularly the covalent modification of the Fe protein, the nature of the modifying group, properties of the activating enzyme, and regulating factors of the inactivation/activation process are reviewed.

Availability of Ferric Iron for Microbial Reduction in Bottom Sediments of the Freshwater Tidal Potomac River

The distribution of Fe(III), its availability for microbial reduction, and factors controlling Fe(III) availability were investigated in sediments from a freshwater site in the Potomac River Estuary. Fe(III) reduction in sediments incubated under anaerobic conditions and depth profiles of oxalate-extractable Fe(III) indicated that Fe(III) reduction was limited to depths of 4 cm or less, with the most intense Fe(III) reduction in the top 1 cm. In incubations of the upper 4 cm of the sediments, Fe(III) reduction was as important as methane production as a pathway for anaerobic electron flow because of the high rates of Fe(III) reduction in the 0- to 0.5-cm interval. Most of the oxalate-extractable Fe(III) in the sediments was not reduced and persisted to a depth of at least 20 cm. The incomplete reduction was not the result of a lack of suitable electron donors. The oxalate-extractable Fe(III) that was preserved in the sediments was considered to be in a form other than amorphous Fe(III) oxyhydroxide, since synthetic amorphous Fe(III) oxyhydroxide, amorphous Fe(III) oxyhydroxide adsorbed onto clay, and amorphous Fe(III) oxyhydroxide saturated with adsorbed phosphate or fulvic acids were all readily reduced. Fe(3)O(4) and the mixed Fe(III)-Fe(II) compound(s) that were produced during the reduction of amorphous Fe(III) oxyhydroxide in an enrichment culture were oxalate extractable but were not reduced, suggesting that mixed Fe(III)-Fe(II) compounds might account for the persistence of oxalate-extractable Fe(III) in the sediments. The availability of microbially reducible Fe(III) in surficial sediments demonstrates that microbial Fe(III) reduction can be important to organic matter decomposition and iron geochemistry. However, the overall extent of microbial Fe(III) reduction is governed by the inability of microorganisms to reduce most of the Fe(III) in the sediment.

Organic Matter Mineralization with Reduction of Ferric Iron in Anaerobic Sediments

The potential for ferric iron reduction with fermentable substrates, fermentation products, and complex organic matter as electron donors was investigated with sediments from freshwater and brackish water sites in the Potomac River Estuary. In enrichments with glucose and hematite, iron reduction was a minor pathway for electron flow, and fermentation products accumulated. The substitution of amorphous ferric oxyhydroxide for hematite in glucose enrichments increased iron reduction 50-fold because the fermentation products could also be metabolized with concomitant iron reduction. Acetate, hydrogen, propionate, butyrate, ethanol, methanol, and trimethylamine stimulated the reduction of amorphous ferric oxyhydroxide in enrichments inoculated with sediments but not in uninoculated or heat-killed controls. The addition of ferric iron inhibited methane production in sediments. The degree of inhibition of methane production by various forms of ferric iron was related to the effectiveness of these ferric compounds as electron acceptors for the metabolism of acetate. The addition of acetate or hydrogen relieved the inhibition of methane production by ferric iron. The decrease of electron equivalents proceeding to methane in sediments supplemented with amorphous ferric oxyhydroxides was compensated for by a corresponding increase of electron equivalents in ferrous iron. These results indicate that iron reduction can outcompete methanogenic food chains for sediment organic matter. Thus, when amorphous ferric oxyhydroxides are available in anaerobic sediments, the transfer of electrons from organic matter to ferric iron can be a major pathway for organic matter decomposition.

Effect of Fall Turnover on Terminal Carbon Metabolism in Lake Mendota Sediments

The carbon and electron flow pathways and the bacterial populations responsible for the transformation of H(2)-CO(2), formate, methanol, methylamine, acetate, ethanol, and lactate were examined in eutrophic sediments collected during summer stratification and fall turnover. The rate of methane formation averaged 1,130 mumol of CH(4) per liter of sediment per day during late-summer stratification versus 433 mumol of CH(4) per liter of sediment per day during the early portion of fall turnover, whereas the rate of sulfate reduction was 280 mumol of sulfate per liter of sediment per day versus 1,840 mumol of sulfate per liter of sediment per day during the same time periods, respectively. The sulfate-reducing population remained constant while the methanogenic population decreased by one to two orders of magnitude during turnover. The acetate concentration increased from 32 to 81 mumol per liter of sediment while the acetate transformation rate constant decreased from 3.22 to 0.70 per h, respectively, during stratification versus turnover. Acetate accounted for nearly 100% of total sedimentary methanogenesis during turnover versus 70% during stratification. The fraction of CO(2) produced from all C-labeled substrates examined was 10 to 40% higher during fall turnover than during stratification. The addition of sulfate, thiosulfate, or sulfur to stratified sediments mimicked fall turnover in that more CO(2) and CH(4) were produced. The addition of Desulfovibrio vulgaris to sulfate-amended sediments greatly enhanced the amount of CO(2) produced from either [C]methanol or [2-C]acetate, suggesting that H(2) consumption by sulfate reducers can alter methanol or acetate transformation by sedimentary methanogens. These data imply that turnover dynamically altered carbon transformation in eutrophic sediments such that sulfate reduction dominated over methanogenesis principally as a consequence of altering hydrogen metabolism.

The role of auxiliary oxidants in the maintenance of a balanced redox poise for photosynthesis in bacteria

Carotenoid absorbance changes were used to monitor the development of membrane potential in intact cell suspensions of Rhodopseudomonas capsulata strain N22. Low concentrations of phenazine methosulphate almost completely inhibited the generation of membrane potential in the light by anaerobic cells. The light-dependent reactions were restored by addition of either trimethylamine N-oxide, dimethylsulphoxide, nitrous oxide, or oxygen. In Rhodopseudomonas capsulata strain N22 DNAR + addition of nitrate was also effective. The inhibition by phenazine methosulphate and restoration by auxiliary oxidant were observed in the presence of sufficient rotenone to block NADH dehydrogenase or with low concentrations of uncoupling agent to dissipate the membrane potential under dark, anaerobic conditions. It is suggested that in intact cells of these organisms there are mechanisms which operate to maintain the electron-transport chain at an optimal redox poise for efficient photosynthesis. Phenazine methosulphate perturbs the optimal redox poise by hastening equilibrium of the photosynthetic electron-transport chain with low-potential couples in the cell. The addition of auxiliary oxidants restores the optimal redox poise. This suggests a role in photosynthesis for the pathways of respiratory electron flow to nitrate, nitrous oxide, trimethylamine N-oxide/dimethylsulphoxide and oxygen.

Oxygenic photoreduction of methyl viologen and nicotinamide adenine dinucleotide phosphate without the involvement of photosystem I during plastid development

Studies on the appearance of various electron transport functions were followed during greening of etiolated cucumber cotyledons. Appearance of dichlorodimethoxy-p-benzoquinone, dimethyl quinone, tetramethyl-p-phenylenediamine, dichlorophenol indophenol and ferricyanide Hill reactions were observed after 8h of greening. However, photoreduction of methyl viologen (MV) and nicotinamide adenine dinucleotide phosphate (NADP) was observed from 2h of greening. Variable fluorescence, which is a direct indication of water-splitting function, was observed from 2h of greening in cotyledons, thylakoid membranes and photosystem II (PSII) particles. The decrease in variable fluorescence in the presence of MV (due to rapid reoxidation of Q −) observed from early stages of greening confirmed the photoreduction of MV by PSII. The early development of water-splitting function was further confirmed by the abolition of variable fluorescence in thylakoid membranes and PSII particles by heat treatment and concomittant loss of light dependent oxygen uptake in the presence of MV in heat treated chloroplasts. However, the photoreduction of MV and NADP was insensitive to intersystem electron transport inhibitors, dichlorophenyl dimethylurea or dibromomethyl isopropyl-p-benzoquinone till 8h of greening. Though the oxidation of intersystem electron carrier cytochrome f was observed from early stages of greening, the reduction of cytochrome f was not observed till 8h of greening. All these observations confirm that during early stages of greening MV and NADP are photoreduced by PSII without the involvement of intersystem electron carriers or the collaboration of PSI. Since these observations are at variance with the currently prevalent concept (Z-Scheme) of the photosynthetic generation of reducing power, which requires definite collaboration of the two photosystems, an alternate electron flow pathway is proposed.

Influence of pH on Terminal Carbon Metabolism in Anoxic Sediments from a Mildly Acidic Lake

The carbon and electron flow pathways and the bacterial populations responsible for transformation of H(2)-CO(2), formate, methanol, methylamine, acetate, glycine, ethanol, and lactate were examined in sediments collected from Knaack Lake, Wis. The sediments were 60% organic matter (pH 6.2) and did not display detectable sulfate-reducing activity, but they contained the following average concentration (in micromoles per liter of sediment) of metabolites and end products: sulfide, 10; methane, 1,540; CO(2), 3,950; formate, 25; acetate, 157; ethanol, 174; and lactate, 138. Methane was produced predominately from acetate, and only 4% of the total CH(4) was derived from CO(2). Methanogenesis was limited by low environmental temperature and sulfide levels and more importantly by low pH. Increasing in vitro pH to neutral values enhanced total methane production rates and the percentage of CO(2) transformed to methane but did not alter the amount of CO(2) produced from [2-C]acetate ( approximately 24%). Analysis of both carbon transformation parameters with C-labeled tracers and bacterial trophic group enumerations indicated that methanogenesis from acetate and both heterolactic- and acetic acid-producing fermentations were important to the anaerobic digestion process.

Chemical modification of the mitochondrial bc1 complex by N,N'-dicyclohexylcarbodiimide inhibits proton translocation.

We report here that N,N'-dicyclohexylcarbodiimide (DCCD) decreases the H/2e stoichiometry of the cytochrome bc1 complex from 3.8 +/- 0.2 (10) to 2.1 +/- 0.1 (8) but has only a minimal effect on the H/2e ratio of cytochrome oxidase under the relatively mild conditions used. The effect on the bc1 complex cannot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage. We conclude that DCCD is an inhibitor of proton translocation within the bc1 complex. There are three possible explanations of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reactions but not electron transport, (c) DCCD could prevent the conduction of the translocated proton to the external phase.

Reassessment of pathways of electron flow to nitrate reductase that are coupled to energy conservation in Paracoccus denitrificans

Electron flow from succinate dehydrogenase, or from non-physiological reductants including ascorbate plus phenazine methosulphate, to nitrate reductase in Paracoccus denitrificans is shown to be linked to energy conservation, contrary to previously accepted schemes for electron transport in this organism. The function of the P. denitrificans nitrate reductase is compared with its counterpart in Escherichia coli.

Energetics of photosynthesis in Zea mays. I. Studies of the flash-induced electrochromic shift and fluorescence induction in bundle sheath cells

Bundle sheath strands free of mesophyll contamination were isolated from 3–4-week-old leaves of maize ( Zea mays L.). Patterns of electron flow in the preparations were studied in the presence of physiological substrates. Relative electron flow rates were estimated from the flash-induced electrochromic band shift changes (P-518) and cytochrome f turnover. Induction of chlorophyll fluorescence was also measured. Little Photosystem II activity was found to be present, the principal pathway of electron flow being Photosystem I-driven cyclic electron transfer. The latter was activated through reductive poising by NADPH, generated via malate decarboxylation or (less efficiently) from dihydroxyacetone phosphate. The actions of these electron donors and of oxygen, nitrite and methyl viologen as electron acceptors in redox poising the Photosystem I-driven cycle were investigated and are discussed in relation to the regulation of photosynthesis in the bundle sheath.

The pathway of electron flow through ubiquinol:cytochrome c oxidoreductase in the respiratory chain Evidence from inhibition studies for a modified ‘Q cycle’

The pathway of electron flow through ubiquinol:cytochrome <i>c</i> oxidoreductase in the respiratory chain Evidence from inhibition studies for a modified ‘Q cycle’

Respiratory control and the basis of light-induced inhibition of respiration in chromatophores from Rhodopseudomonas capsulata

A preparation of chromatophores from Rhodopseudomonas capsulata is described in which NADH oxidase activity is stimulated by uncouplers and during ATP synthesis, and is strongly inhibited upon illumination. The inhibition is completely reversed by addition of a protonophore or the combination of valinomycin, nigericin and K + , thus demonstrating that the proton electro-chemical gradient generated by light-driven cyclic electron flow is responsible for the inhibition of respiration. By analogy it is suggested that light-induced inhibition of respiration in intact cells of photosynthetic bacteria is also controlled by the proton electrochemical gradient. The results show that all chromatophores that possess oxidase activity must also have light-driven cyclic electron flow pathways. This has implications for experiments in which carotenoid band shifts and distributions of permeant anions have been compared as indicators of membrane potentials in chromatophores.