1. MEMBRANE POTENTIALS AND pH GRADIENTS IN MICROSCOPIC SYSTEMS: THE CHEMIOSMOTIC PARADIGM H.R. Kaback

2. A Little History: In , the British scientist Peter Mitchell put forward the heretical postulate that a pH gradient (the so-called Proton-Motive Force) across the mitochondrial or bacterial membrane is the immediate driving force for oxidative phosphorylation, as well as the accumulation of metabolites against a concentration gradient (The Chemiosmotic Hypothesis) At about the same time, Robert Crane of Rutgers University suggested that sodium gradients are responsible for driving glucose accumulation across the intestinal epithelium.
Although Crane’s hypothesis was embraced relatively quickly by electrophysiologists and epithelial physiologists, the biochemical community found Mitchell’s hypothesis not only unacceptable, but repulsive primarily because some of the most well recognized biochemists in the world were convinced that a high-energy phosphate intermediate which could be isolated and identified must be involved in oxidative phosphorylation. Furthermore, rather than use standard physical chemical nomenclature, Mitchell preferred to improvise his own terms which put off biochemists and physiologist alike. Finally, the whole idea that an ion gradient might drive a covalent reaction like ATP synthesis seemed absolutely ridiculous to most.

3. In the late ‘60s, Andre Jagendorf of Cornell University carried out the first experiments that made scientists begin to take Mitchell’s hypothesis seriously. By using thylakoids (the intracellular organelles responsible for photophosphorylation in plants), Jagendorf demonstrated that sudden acidification of the external medium leads to synthesis of ATP. Over the ensuing 10 years, work in many laboratories using a variety of techniques with different experimental systems showed virtually unequivocally that Mitchell’s Chemiosmotic Hypothesis is the paradigm for bioenergetics in energy transducing membranes, and in 1977, Mitchell was awarded the Nobel Prize in Chemistry.

4. Clearly at this level, the major questions are two-fold: a
Clearly at this level, the major questions are two-fold: a. Are membrane potentials and pH gradients present in microscopic systems (too small to be impaled by microelectrodes)? b. If there are membrane potentials and pH gradients in these systems, are they of sufficient magnitude to drive the process in question? In this lecture, the focus is on right-side-out (RSO) membrane vesicles from E. coli and respiration-driven active transport in this well-defined system with the goal of providing a basic intuitive understanding of the Chemiosmotic Paradigm with respect to active transport. We will then see how the principles apply to more complex eucaryotic systems.

5. igure 1. The Chemiosmotic Paradigm
igure 1. The Chemiosmotic Paradigm. The two basic energy sources in living systems--respiration and light--lead to the generation of a proton electrochemical gradient ( ) across the appropriate energy-transducing membrane which is the immediate driving force for a wide range of seemingly unrelated processes like oxidative or photophosphorylation, active transport, transhydrogenation of NADP by NADH and many other cellular phenomena. It is also important to note that the arrows shown point in both directions. Thus, ATP hydrolysis, movement of accumulated substrates down a concentration gradient or reduction of NAD by NADPH can lead to generation of

6. Figure 2. How electron transfer down a membrane-embedded respiratory chain might pump protons. In order to account for this process, Mitchell postulated the existence of “loops” in the respiratory chain. Translated into English, this means alternation of electron/proton carriers with electron carriers across the membrane. More specifically, a flavoprotein hydrogenase on the inner surface of the membrane [(Fp); upon reduction, FAD or FMN accepts electrons and protons] transfers its electron to a non-heme iron sulfur protein (NHFeS) disposed towards the outer surface of the membrane (iron accepts electrons only), and the proton is released into the external medium. The NHFeS then transfers electrons to ubiquinone (Q) on the inner surface of the membrane which picks up protons from the internal solvent. QH2 then transfers electrons to cytochrome b (CYT b) towards the outer surface which accepts only electrons, and 2 protons are released into the external medium. Finally, CYT. B transfers electrons to the terminal oxidase (CYT ox) on the inner surface which reduces oxygen on the inside utilizing another 2 protons to make water.

7. Although there are instances in which loops probably occur in respiratory chains, things are generally more complicated. For example, certain terminal oxidases in purified form pump protons by themselves when reconstituted into artificial membranes. There are also respiratory chains in some bacteria that pump sodium. The point is that loops in the respiratory chain provide a simple conceptual possibility for how electron transfer might be coupled to proton pumping.

8. Figure 3

9. Figure 4

13. From the decrease in the dialyzable [14C]acetate concentration upon addition of ASC-PMS, the quantity of acetate accumulated by the vesicles can be readily calculated per mg of vesicle protein, and knowing the intravesicular volume per mg of vesicle protein, this value can be transformed into the internal concentration of [14C]acetate (in molarity). Since the external concentration of the weak acid in the medium surrounding the vesicles is given directly from the flow dialysis profile, the concentration gradient of [14C]acetate in the presence of ASC-PMS is determined from [acetate]in/[acetate]out and converted in mV by using the constants shown in Figure 1 (2.3RT/F60 at room temperature).