Phototaxis and membrane potential in the photosynthetic bacterium Rhodospirillum rubrum.

Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off" light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.     

Photoresponses in Rhodobacter sphaeroides: role of photosynthetic electron transport.

Rhodobacter sphaeroides responds to a decrease in light intensity by a transient stop followed by adaptation. There is no measurable response to increases in light intensity. We confirmed that photosynthetic electron transport is essential for a photoresponse, as (i) inhibitors of photosynthetic electron transport inhibit photoresponses, (ii) electron transport to oxidases in the presence of oxygen reduces the photoresponse, and (iii) the magnitude of the response is dependent on the photopigment content of the cells. The photoresponses of cells grown in high light, which have lower concentrations of light-harvesting photopigment and reaction centers, saturated at much higher light intensities than the photoresponses of cells grown in low light, which have high concentrations of light-harvesting pigments and reaction centers. We examined whether the primary sensory signal from the photosynthetic electron transport chain was a change in the electrochemical proton gradient or a change in the rate of electron transport itself (probably reflecting redox sensing). R. sphaeroides showed no response to the addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, which decreased the electrochemical proton gradient, although a behavioral response was seen to a reduction in light intensity that caused an equivalent reduction in proton gradient. These results strongly suggest that (i) the photosynthetic apparatus is the primary photoreceptor, (ii) the primary signal is generated by a change in the rate of electron transport, (iii) the change in the electrochemical proton gradient is not the primary photosensory signal, and (iv) stimuli affecting electron transport rates integrate via the electron transport chain.     

Gene mutation eliminating antimycin A-tolerant electron transport in Ustilago maydis

A class of mutants of Ustilago maydis selected on a fungitoxic oxathiin lack of antimycin A-tolerant respiratory system which is present in wild-type cells. This system provides, directly or indirectly, for considerable resistance to antimycin A because growth of mutant cells lacking the system is much more sensitive to the antibiotic than that of the wild type. Antimycin A-sensitive O(2) uptake and growth is found in half of the progeny from crosses of mutant to wild type. All antimycin A-sensitive segregants are somewhat more resistant to oxathiins than the antimycin A-resistant segregants. The respiration of the mutant is strongly inhibited by cyanide and azide at concentrations which stimulate respiration of the wild type. Respiration of both mutant and wild type is about equally inhibited by rotenone. It appears that the mutation alters some component of the respiratory system located between the rotenone inhibition site and the antimycin A inhibition site that permits shift of electron transport to an alternate terminal oxidase when the normal electron transport pathway is blocked.     

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.     

Transport properties of membrane vesicles fromAcholeplasma laidlawii

Membrane vesicles obtained from Acholeplasma laidlawii accumulate glucose as well as maltose and fructose against their concentration gradient in the absence of exogenous energy sources. Glucose uptake by membrane vesicles is inhibited by anaerobiosis and by electron transfer inhibitors, such as rotenone and amytal, but not by 2-heptyl-4-hydroxyquinoline N-oxide, antimycin A, cyanide and azide. Rotenone, cyanide and amytal also produce a rapid efflux of glucose from the membrane vesicles. Arsenate, oligomycin and N,N'-dicyclohexylcarbodimide do not inhibit glucose transport. Transport of glucose is markedly inhibited by proton conductors such as CCCP and pentachlorophenol. It is concluded that glucose transport can be driven by a high-energy state of the membrane or by the membrane potential.     

The interaction of rubroskyrin, a modified bis-anthraquinone pigment fromPenicillium islandicum Sopp, with respiratory chain of liver mitochondria

Summary  Rubroskyrin, a modified bisanthraquinone pigment from an yellow rice moldPenicillium islandicum Sopp, was examined for its redox-interaction with the mitochondrial respiratory chain by using rat liver submitochondrial particles (SMP) and was compared with luteoskyrin and rugulosin. Rubroskyrin showed a redox interaction with the NAD-linked respiratory chain of SMP, promoting NADH oxidase in the presence of rotenone, a specific inhibitor to coupling site I of the respiratory chain. Rubroskyrin-mediated NADH oxidase was not inhibited by antimycin A and cyanide, inhibitors to coupling sites II and III, respectively, indicating a generation of an electron transport shunt from a rotenone-insensitive site of NADH dehydrogenase (complex I) to dissolved oxygen. An electrontransport shunt to cytochromec oxidase from complex I was also observed in the experiment with cytochromec and antimycin A. Rubroskyrin did not interact with succinate-linked respiratory chain. Such enzymatic redox response which generates electron transport shunt was not detected for luteoskyrin and rugulosin in the present study.     

Respiratory System of Rhodotorula glutinis I. Inhibitor Tolerance and Cytochrome Components

Oxygen uptake by the carotenoid-containing yeast, Rhodotorula glutinis was not affected by concentrations of cyanide and antimycin A which completely inhibit the respiration of Saccharomyces cerevisiae. The tolerance of R. glutinis to these inhibitors was somewhat dependent on the age of the cultures. Reduced minus aerated difference spectra of cells revealed spectral changes presumably due to cytochromes and carotenoids. The kinetics of these spectral changes induced by oxygen were followed. Carotenoid deficient cells were prepared by growth in the presence of diphenylamine. Difference spectra of these cells revealed the presence of flavoprotein, and a, b, and c type cytochromes. Growth of R. glutinis was completely inhibited by concentrations of cyanide which did not affect respiration. Oxidation of reduced nicotinamide adenine dinucleotide by sub-cellular fractions was sensitive to cyanide and antimycin A. Although respiration of intact cells is tolerant to these inhibitors, studies with cell-free extracts suggest the presence of a cyanide and antimycin A-sensitive, cytochrome-linked, respiratory chain.     

Electrogenic function of submitochondrial particles at the water-octane interphases]

Studies on submitochondrial particles (SMP) preparation showed that in the sourse of the redox reactions at the octane-water interface, catalyzed by SMP enzymes, the charges are transferred from the aqueous to the octane phase. The effects were detected by a shift of the Volta potential, using the vibrating electrode method. In the presence of 2-N-methyl-amino-1,4-naphthoquinone in octane, acting as electron acceptor, the negative charges were transferred from water to octane following the oxidation of NADH, succinate and ascorbate. The charging of the octane phase was sensitive to the inhibitors of the respiratory chain, e. g. rotenone, antimycin and cyanide. In the presence of 2,4-DNP in octane, acting as a proton acceptor, the oxidation of NADH and succinate by ferricyanide, catalyzed by CMP in the presence of antimycin and cyanide correspondingly, was followed by a transfer of positive charges from water to octane. The positive charging of the octane phase, coupled with NADH oxidation, was found insensitive to rotenone, and that coupled with succinate oxidation, was completely inhibited by antimycin. The positive charging of the octane phase was also observed during the reverse transhydrogenase reaction, catalyzed by SMP at the division of the phases. The effect was inhibited by palmitoyl-CoA.     

Generation of hydrogen peroxide by brain mitochondria: the effect of reoxygenation following postdecapitative ischemia

The hypothesis that mitochondria damaged during complete cerebral ischemia generate increased amounts of superoxide anion radical and hydrogen peroxide (H2O2) upon postischemic reoxygenation has been tested. In rat brain mitochondria, succinate supported H2O2 generation, whereas NADH-linked substrates, malate plus glutamate, did so only in the presence of respiratory chain inhibitors. Succinate-supported H2O2 generation was diminished by rotenone and the uncoupler carbonyl cyanide m-chlorphenylhydrazone and enhanced by antimycin A and increased oxygen tensions. When maximally reduced, the NADH dehydrogenase and the ubiquinone-cytochrome b regions of the electron transport chain are sources of H2O2. These studies suggest that a significant portion of H2O2 generation in brain mitochondria proceeds via the transfer of reducing equivalents from ubiquinone to the NADH dehydrogenase portion of the electron transport chain. Succinate-supported H2O2 generation by mitochondria isolated from rat brain exposed to 15 min of postdecapitative ischemia was 90% lower than that of control preparations. The effect of varying oxygen tensions on H2O2 generation by postischemic mitochondrial preparations was negligible compared with the increased H2O2 generation measured in control preparations. Comparison of the effects of respiratory chain inhibitors and oxygen tension on succinate-supported H2O2 generation suggests that the ability for reversed electron transfer is impaired during ischemia. These data do not support the hypothesis that mitochondrial free radical generation increases during postischemic reoxygenation.     

Effects of selected inhibitors on electron transport in Neisseria gonorrhoeae.

The electron transport system of Neisseria gonorrhoeae was partially characterized by using spectrophotometric, spectroscopic, and oxygen consumption measurements. The effects of selected electron transport inhibitors (amytal, rotenone, 2-heptyl-4-hydroxyquinoline, antimycin A1, and potassium cyanide [KCN]) on electron transfer in whole-cell and sonically treated whole-cell preparations of N. gonorrhoeae were examined. The oxidation of reduced nicotinamide adenine dinucleotide, measured as a decrease in absorbance at 340 nm, was inhibited by each of the compounds tested. Oxygen consumption stimulated by reduced nicotinamide adenine dinucleotide was also inhibited, whereas oxygen uptake stimulated by succinate and malate was inhibited by KCN alone, suggesting the presence of a KCN-sensitive terminal oxidase. Room temperature optical difference spectra indicate an operational electron bypass around the amytal-rotenone-binding site. Difference spectra in the presence of 2-heptyl-4-hydroxyquinoline suggest a possible site of interaction of this compound at the substrate side of cytochrome b. Reduced-minus-oxidized spectra of ascorbate-tetramethyl-p-phenylenediamine suggest the participation of b-, a-, and d-type cytochromes in terminal oxidase activity. Hence, N. gonorrhoeae appears to have an electron transport chain containing cytochrome c, two b-type cytochromes (one of which has an oxidase function), and possibly a- and d-type cytochromes. An abbreviated chain exists through which succinate and malate can be oxidized directly by a KCN-sensitive component.     

Nitric oxide-dependent proton translocation in various denitrifiers.

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.     

Aerotaxis in Salmonella typhimurium: role of electron transport

Sensory transduction in aerotaxis required electron transport, in contrast to chemotaxis, which is independent of electron transport. Assays for aerotaxis were developed by employing spatial and temporal oxygen gradients imposed independently of respiration. By varying the step increase in oxygen concentration in the temporal assay, the dose-response relationship was obtained for aerotaxis in Salmonella typhimurium. A half-maximal response at 0.4 microM oxygen and inhibition by 5 mM KCN suggested that the "receptor" for aerotaxis is cytochrome o. The response was independent of adenosine triphosphate formation via oxidative phosphorylation but did correlate with changes in membrane potential monitored with the fluorescent cyanine dye diS-C3-(5). Nitrate and fumarate, which are alternative electron acceptors for the respiratory chain in S. typhimurium, inhibited aerotaxis when nitrate reductase and fumarate reductase were induced. These results support the hypothesis that taxis to oxygen, nitrate, and fumarate is mediated by the electron transport system and by changes in the proton motive force. Aerotaxis was normal in Escherichia coli mutants that were defective in the tsr, tar, or trg genes; in S. typhimurium, oxygen did not stimulate methylation of the products of these genes. A cheC mutant which shows an inverse response to chemoattractants also gave an inverse response to oxygen. Therefore, aerotaxis is transduced by a distinct and unidentified signally protein but is focused into the common chemosensory pathway before the step involving the cheC product. When S. typhimurium became anaerobic, the decreased proton motive force from glycolysis supported slow swimming but not tumbling, indicating that a minimum proton motive force was required for tumbling. The bacteria rapidly adapted to the anaerobic condition and resumed tumbling after about 3 min. The adaptation period was much shorter when the bacteria had been previously grown anaerobically.     

Electron transport-dependent taxis in Rhodobacter sphaeroides.

Rhodobacter sphaeroides showed chemotaxis to the terminal electron acceptors oxygen and dimethyl sulfoxide, and the responses to these effectors were shown to be influenced by the relative activities of the different electron transport pathways. R. sphaeroides cells tethered by their flagella showed a step-down response to a decrease in the oxygen or dimethyl sulfoxide concentration when using them as terminal acceptors. Bacteria using photosynthetic electron transport, however, showed a step-down response to oxygen addition. Addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone did not cause a transient behavioral response, although it decreased the electrochemical proton gradient (delta p) and increased the rate of electron transport. However, removal of the ionophore, which caused an increase in delta p and a decrease in the electron transport rate, resulted in a step-down response. Together, these data suggest that behavioral responses of R. sphaeroides to electron transport effectors are caused by changes in the rate of electron transport rather than changes in delta p.     

The electric field generated by photosynthetic reaction center induces rapid reversed electron transfer in the bc1 complex

The cytochrome bc(1) complex is the central enzyme of respiratory and photosynthetic electron-transfer chains. It couples the redox work of quinol oxidation and cytochrome reduction to the generation of a proton gradient needed for ATP synthesis. When the quinone processing Q(i)- and Q(o)-sites of the complex are inhibited by both antimycin and myxothiazol, the flash-induced kinetics of the b-heme chain, which transfers electrons between these sites, are also expected to be inhibited. However, we have observed in Rhodobacter sphaeroides chromatophores, that when a fraction of heme b(H) is reduced, flash excitation induces fast (half-time approximately 0.1 ms) oxidation of heme b(H), even in the presence of antimycin and myxothiazol. The sensitivity of this oxidation to ionophores and uncouplers, and the absence of any delay in the onset of this reaction, indicates that it is due to a reversal of electron transfer between b(L) and b(H) hemes, driven by the electrical field generated by the photosynthetic reaction center. In the presence of antimycin A, but absence of myxothiazol, the second and following flashes induce a similar ( approximately 0.1 ms) transient oxidation of approximately 10% of the cytochrome b(H) reduced on the first flash. From the observed amplitude of the field-induced oxidation of heme b(H), we estimate that the equilibrium constant for sharing one electron between hemes b(L) and b(H) is 10-15 at pH 7. The small value of this equilibrium constant modifies our understanding of the thermodynamics of the Q-cycle, especially in the context of a dimeric structure of bc(1) complex.     

The effect of respiration on the phototactic behavior of the purple nonsulfur bacterium Rhodospirillum centenum

The effect of respiration on the positive phototactic movement of swarming agar colonies of the facultative phototroph Rhodospirillum centenum was studied. When the electron flow was blocked at the bc ₁ complex level by myxothiazol, the oriented movement of the colonies was totally blocked. Conversely, inhibition of respiration via the cytochrome c oxidase stimulated the phototactic response. No phototaxis was observed in a photosynthesis deficient mutant (YB707) lacking bacteriochlorophylls. Analyses of the respiratory activities as monitored by a oxygen microelectrode in single agar colonies during light/dark transitions showed a close functional correlation between the photosynthetic and respiratory apparatuses. The respiratory chain of Rsp. centenum was formed by two oxidative pathways: one branch leading to a cytochrome c oxidase inhibited by low cyanide concentrations and a second pathway formed by an oxidase less-sensitive to cyanide that also catalyzes the light-driven respiration. These results were interpreted to indicate that (1) there is a cyclic electron transport, and (2) photoinduced cyclic electron flow is required for the phototactic response of Rsp. centenum. Furthermore, under oxic conditions in the light, reducing equivalents may switch from photosynthetic to respiratory components so as to reduce both the membrane potential and the rate of locomotion. Received: 25 September 1996 / Accepted: 11 November 1996     

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.     

Light-induced oxygen uptake by chromatophores and subchromatophore pigment-protein complexes of Rhodospirillum rubrum]

Chromatophores of R. rubrum incubated with electron donors, e. g. reduced diaminodurene, TMPD, phenazine methosulphate, cytochrome c or ferrocyanide, are able to catalyze O2 uptake upon illumination. This process is inhibited by o-phenanthroline as well as upon extraction of quinones from chromatophores, but not by antimycin A, rotenone or CN-. The O2 uptake sensitive to the action of o-phenanthroline is also observed in the illuminated subchromatophore P870 reaction center complexes and reaction center plus light-harvesting antenna complexes incubated with electron donors, quinones and detergents. The data obtained are in agreement with a suggestion that the photooxidase activity of chromatophores and subchromatophore pigment-protein complexes is due to the interaction of photoreduced ubiquinone with O2.     

The respiratory system of Rhodotorula glutinis. II. Mechanism of inhibitor tolerant respiration

The mechanism of inhibitor-tolerant respiration in Rhodotorula glutinis was studied. This inhibitor-tolerant respiratory pathway was not due to the presence of an excess of cytochrome c oxidase, nor to the operation of an inhibitor-resistant cytochrome c oxidase. Carotenoids do not appear to be involved in this respiratory chain pathway; data are also presented which show that the inhibitors penetrate into the cell. Although the initial rate of oxygen uptake by intact cells was not inhibited in the presence of cyanide or antimycin A, in the presence of these inhibitors the rate of oxygen uptake decreased significantly when the oxygen concentration fell below 100 mum. This change in rate of oxygen uptake as a function of pO(2), suggests that a respiratory chain with a low affinity for oxygen operates in the presence of inhibitors. The characteristics of this alternate pathway are described.     

The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.