The bioenergetics of paracoccus denitrificans

This thesis concerns the mechanism of oxidative phosphorylation, with special reference to the bacterium Paracoccus denitrificans. A new method using affinity chromatography is described for the separation of vesicles of different membrane orientation. The working hypothesis is the chemisosmotic theory of Mitchell with its emphasis on the transmembrane proton motive force made up of a membrane potential and a transmembrane pH gradient. The 3 experimental systems used ape bovine heart submitochondrial particles, chromatophores from the photosynthetic bacterium Rhodospirillum rubrum and phosphorylating vesicles from P. denitrificans. From the uncoupling obtained with various ions added to the P. denitrificans particles it is concluded that the transmembrane distribution of weak bases and permeant anions permits a determination of the protonmotive force. A flow-dialysis method has been developed to measure the steady-state transmembrane distribution of weak bases and anions in a determination of the protonmotive force. This method successfully overcomes problems which arise with the techniques previously used for these measurements. Under the experimental conditions used to determine the extent of oxidative phosphorylation (the phosphorylation potential) no transmembrane pH gradient was detectable, and the protonmotive force could be accounted for completely by the membrane potential. A method using ion-selective electrodes was developed to provide a continuous determination of this membrane potential. In all 3 systems the proton motive force determined by the above methods was found to be significantly lower than that required by the chemiosmotic hypothesis. A simple modification of the chemiosmotic hypothesis is proposed. This new concept is based on electrodic theory. It is shown to be useful in explaining results reported in the literature (from both photosynthetic and respiratory systems) which appeared to be at odds with the chemiosmotic theory in its usual form.