P. John

Measurement by a flow dialysis technique of the steady-state proton-motive force in chromatophores from Rhodospirillum rubrum. Comparison with phosphorylation potential.

1. In the light a transmembrane electrical potential of 100 mV has been estimated to occur in chromatophores from Rhodospirillum rubrum. The potential was determined by measuring the steady-state distribution of the permeant SCN- across the chromatophore membrane using a flow dialysis technique. The potential was not observed in the dark, nor in the presence of antimycin. It was dissipated on the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The potential was reduced by between 15 and 20 mV when ADP and Pi were added. Hydrolysis of ATP by the chromatophores generated a membrane potential of about 80 mV. 2. Using a flow dialysis technique light-dependent uptake of methylamine was observed only in the presence of concentrations of SCN- that were 500-fold higher than were used to measure the membrane potential. It is concluded that the pH gradient across the illuminated chromatophore membrane is insignificant except in the presence of relatively high concentrations of a permeant anion like thiocyanate. Further evidence that a negligible pH gradient was generated by the chromatophores is that addition of K+ and nigericin to illuminated chromatophores did not stimulate uptake of SCN-. 3. In the light of chromatophores established and maintained a phosphorylation potential of up to 14 kcal/mol. If a phosphorylation potential of this magnitude is to be poised against a proton-motive force that comprises solely a membrane potential of approx. 100 mV, then at least five protons must be translocated for each ATP synthesised via a chemiosmotic mechanism.

Selective and reversible inhibition of the ATPase of Micrococcus denitrificans by 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole

Abstract The covalent inhibitor of the beef heart mitochondrial ATPase 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole inhibits the ATPase of phosphorylating particles prepared from Micrococcus denitrificans . Inhibition of both ATP synthesis and ATP hydrolysis occurs at similar rates, with a similar pH dependence, and in each case the inhibition is relieved by treatment with dithiothreitol. These results are compared with those previously obtained with the mitochondrial ATPase.

Continuous monitoring of the electrical potential across energy-transducing membranes using ion-selective electrodes Application to submitochondrial particles and chromatophores

It is now widely believed that a primary result of electron transport in the energy-transducing membranes of mitochondria, chloroplasts and bacteria is the vectorial translocation of protons, leading to the generation of a transmembrane electrochemical proton gradient, the protonmotive force Ap [l] . Ap is composed of both a chemical component ApH and an electrical component A

The ATPase as an irreversible component in electron transport linked ATP synthesis

according to the relationship :

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

The reversibility of the ATPase that participates in oxidative phosphorylation is implicit within the idea that the phosphate potential (energy stored in ATP) comes into equilibrium with the respiratory chain [ 1 ] . This is the basis of an explanation of respiratory control in which ADP stimulated respiration (state 3) continues until this equilibrium is reached, whereupon respiration decreases to a controlled rate (state 4). Our purpose in this paper is to enquire whether this equilibrium view is always correct by considering the properties of the oxidative phosphorylation apparatus of ‘inside out’ phosphorylating membrane vesicles from Puracoccus denitrificans which exhibit respiratory control [2,3,4].

The bioenergetics of paracoccus denitrificans

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300,000. An inhibitor protein in bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised condtions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Ppi is incorporated into the beta and gamma positions of the bound nucleotides, but beta-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.