Donald L. Keister

Energy-linked reactions in photosynthetic bacteria: I. Succinate-linked ATP-driven NAD+ reduction by Rhodospirillum rubrum chromatophores

Succinate-linked NAD+ reduction by Rhodospirillum rubrum chromatophores has been demonstrated to be driven by ATP and pyrophosphate as well as light. Gramicidin, m-chlorocarbonylcyanide phenylhydrazone, desaspidin, dicumarol, and quiuacrine are well known uncouplers which inhibited both the light and ATP-driven reactions. The electron transport inhibitors, 2-heptyl-4-hydroxyquinolme-N-oxide, antimycin A, and a 3-alkyl hydroxy naphthoquinone, inhibited the light-driven reduction but had no effect on the ATP-driven reaction. Roteunone and amytal inhibited both reactions. The effect of these compounds on NAD+ reduction has been compared with their effect on photophosphorylation and on the energy-linked transhydrogenase. The light-driven NAD+ reduction was stimulated by, but did not require, divalent cations, bovine serum albumin, and oligomycin. The energy-linked NAD+ reduction is proposed to occur by reversed electron flow in the oxidative electron transport chain.

Energy-linked reactions in photosynthetic bacteria: VI. Inorganic pyrophosphate-driven ATP synthesis in Rhodospirillum rubrum☆

Abstract A new reaction, the inorganic pyrophosphate-driven synthesis of ATP in Rhodospirillum rubrum chromatophores, was demonstrated. The effect of inhibitors such as m-Cl-CCP, S-13, desaspidin, and oligomycin indicate that this is an energy-linked reaction. Phosphotransferase and exchange reactions involving the direct transfer of phosphate from PPi to ADP or ATP were very low or nonexistent in chromatophores. Thus, the reaction can be described by the following equations where “~X” represents an energized state or intermediate: PPi ⇌ “~X” + 2 Pi ADP + Pi + “~X” ⇌ ATP The characteristics and requirements for the reaction were elucidated and the interaction between this reaction and other energy-linked reactions, such as NAD+ reduction, transhydrogenation, and ATP-Pi exchange, was demonstrated. The discussion focuses on the advantage of this energy-conserving reaction to the cell.

ATP synthesis driven by inorganic pyrophosphate in Rhodospirillum rubrum chromatophores

Inorganic pyrophosphate was demonstrated to drive the synthesis of ATP from ADP and Pi in Rhodospirillum rubrum chromatophores. The reaction was inhibited by uncouplers and inhibitors of phosphorylation such as Cl-CCP, S-13, and oligomycin, thus illustrating the energy-linked characteristics of the reaction. Exchange reactions involving the direct transfer of phosphate from PPi to ADP or ATP were very low in chromatophores and did not appear to contribute to the ATP synthesis. Fluoride inhibited the inorganic pyrophosphatase and PPi⇋Pi exchange of chromatophores but had no effect on ATP-linked reactions. The PPi driven synthesis of ATP was inhibited by fluoride also, indicatting that the membrane bound inorganic pyrophosphatase may be involved in the energy metabolism in the cell.

Effect of oxygen tension on nitrogenase and on glutamine synthetases I and II in Rhizobium japonicum 61A76

Abstract When grown under aerobic conditions, Rhizobium japonicum 61A76 contains two forms of glutamine synthetase, GSI and GSII, as previously described. In contrast, cells grown under the low O 2 tensions required for nitrogenase synthesis contain only GSI. GSII activity disappears completely at O 2 levels below 0.4%. GSI activity decreases by only 50%, but the enzyme appears to become highly adenylylated under the low O 2 tensions required for nitrogenase synthesis.

Energy-linked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillumrubrum

The energy-linked membrane-bound inorganic pyrophosphatase of Rhodospirollumrubrum, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl2. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PPi and requires Mg2+ for activity as has been found for other PPiases.

ELECTRON TRANSPORT IN RHIZOBIUM JAPONICUM. ISOLATION OF CYTOCHROME c DEFICIENT MUTANTS

Electron transport mutants of Rhizobium japonicum 61A76 were isolated and have been partially characterized spectrally and biochemically. Mutants which are almost totally deficient in cytochromes c and aa 3 and with a reduced amount of cytochrome b grow normally under aerobic conditions. These mutants are symbiotically infective but form ineffective nodules.

K+-independent effects of valinomycin in photosynthetic systems.

With chromatophores ofRhodospirillum rubrum, valinomycin inhibited electron transport in the presence or absence of K+. NH4Cl had no effect on photophosphorylation but uncoupled with valinomycin present. ATPase activity was stimulated by NH4Cl plus valinomycin but not by either alone. K+ partially reversed the inhibition of phosphorylation and the stimulation of ATPase by valinomycin plus NH4Cl.With chloroplasts, valinomycin inhibited coupled but not basal electron transport. The inhibition was only partially reversed by uncouplers. Valinomycin stimulated the light-activated Mg2+-dependent ATPase similar to several uncouplers such as quinacrine, methylamine, and S-13. In addition, valinomycin inhibited delayed light emission and stimulated the H+/e− ratio. These contrasting activities in chloroplasts are not easily explained.

Energy-linked reactions in photosynthetic bacteria: Pi ⇋ HOH oxygen exchange catalyzed by the membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum

Abstract The inorganic phosphate-water oxygen exchange reaction has been studied in chromatophores of Rhodospirillum rubrum . Under appropriate conditions, chromatophores catalyzed this exchange at a rate of up to 150 μatom oxygen/h/mg bacteriochlorophyll. The reaction is largely inhibited by inhibitors of the membrane-bound inorganic pyrophosphatase, fluoride and methylene diphosphonate, and is not inhibited by oligomycin. These results indicate that the P i ⇋ HOH oxygen exchange is almost entirely due to the pyrophosphatase. In the presence of ADP, the exchange reaction was stimulated by about 40% and this portion of the exchange was sensitive to oligomycin, but not to fluoride or methylene diphosphonate. Thus this portion of the exchange can be attributed to the ATP synthese complex. The rates of the oxygen exchange reaction and other chromatophore-catyalyzed reactions are compared.

NITROGEN FIXATION IN PHOTOSYNTHETIC BACTERIA

Publisher Summary This chapter discusses ion gradients across chloroplast membranes as a form of energy storage while keeping in mind that ion fluxes may contribute to the regulation of photosynthetic activity. Major amounts of energy have been observed stored in the form of H+ gradients. The magnitude of the electric gradient (Δψ) associated with H+ uptake in chloroplasts rests primarily on the interpretation of experiments on the light-induced absorbance increase in the region of 515 nm. Thus, H+ uptake in chromatophores and chloroplasts probably generates a membrane potential at a steady state. The magnitude of this potential is probably less in chloroplasts than in chromatophores, possibly because chloroplasts are more permeable to ions such as Cl−. In the initial period of H+ uptake, the magnitude of the membrane potential should be higher. In flashing light, ATP formation can take place, but an H+ concentration gradient is not formed. Presumably, a membrane potential is formed as 518 nm absorption change is observed in flashing light. Thus, the dissipation of a membrane potential under conditions where an H+ concentration gradient cannot be formed may be sufficient to inhibit phosphorylation in chloroplasts.

Energy-linked reactions in photosynthetic bacteria: VII. Inhibition of NAD+ reduction by phenethylbiguanide

Abstract Phenethylbiguanide (DBI) and several other substituted guanidines are effective inhibitors of energy-linked NAD + reduction in Rhodospirillum rubrum chromatophores. Energy (light or ATP) is required for development of the inhibition. The inhibition is pH dependent and is essentially irreversible once developed. Neither photophosphorylation, energy-linked transhydrogenation, nor NADH oxidation were affected at DBI concentrations which strongly inhibited NAD + reduction. The membrane potential in chromatophores is apparently of the wrong polarity to be involved in DBI uptake and the inhibition was not influenced by the ionophores, nigericin or valinomycin, which affect the internal proton concentration. The inhibition was suppressed by divalent cations such as Mg 2+ and Ba 2+ which are stimulatory for NAD + reduction. Thus, it is proposed that the guanidines are site-specific inhibitors which act by virtue of their ability to interfere with essential cationic function in energy-coupling at the NADH dehydrogenase site.