The respiratory chain is relatively inaccessible in the experimental manipulation of intact mitochondria

Question

After disrupting mitochondria with ultrasound, however, it is possible to isolate functional submitochondrial particles, which consist of broken cristae that have resealed inside-out into small, closed vesicles. In these vesicles, the components that originally faced the matrix are now exposed to the surrounding medium.
A. How might such an arrangement aid in the study of electron transport and ATP synthesis?
B. Consider an anaerobic preparation of such submitochondrial particles. If a small amount of oxygen is added, do you predict that the preparation will consume oxygen in respiration reactions? Will the medium outside the particles become more acidic or more basic? What, if anything, will change if the flow of protons through ATP synthase is blocked by an inhibitor? Explain your answer.

Answer 1

A. This arrangement of components within the vesicles allows the experimental manipulation of the medium surrounding the vesicles, which permits the consequences of different conditions in the mitochondrial matrix to be examined. The medium can be altered by changing pH, adding electron carriers and oxygen, and providing ADP and Pi, for example. The oxidation of electron carriers, the consumption of oxygen, and the production of ATP can be measured in the medium. By changing the composition of the medium, it should be possible, for example, to identify the electron carriers that can donate electrons from the matrix to the transport chain (the side of the membrane that normally faces the matrix is now on the outside), to assess the redox potentials of various components of the transport chain, and to determine the dependence of ATP synthesis on the pH gradient across the membrane and on the ATP/ADP ratio.
B. Respiration reactions will rapidly consume at least some of the added oxygen. During the anaerobic conditions, the electron carriers in the electron-transport chain were reduced; on the addition of oxygen, electrons will be transferred to oxygen, thereby reducing the oxygen and oxidizing the carriers. Concomitantly with the electron flow, protons will be pumped from the medium into the vesicles, thereby making the medium slightly more basic and the inside of the vesicles acidic. Inhibition of the ATP synthase will not have an immediate effect on oxygen consumption or proton pumping. However, the proton concentration inside the vesicles will quickly become too high to continue the activity of the electron-transport-coupled proton pumping, and thus electron transport and oxygen consumption will cease.