Charge separation and energy transfer in the mitochondrial membrane.

It is clearly emerging from the work of various laboratories that membrane-bound electron transfer chains are anisotropically arranged along an axis perpendicular to the plane of the membrane (1-6). Anisotropic organization of redox chains, composed of hydrogen and electron carriers can lead, as proposed by Lundegardh (7), to generation of transmembrane electrical fields and proton gradients if the proton-electron separation and proton-electron recombination processes occur at opposite sides of the insulating layer of the membrane (see Mitchell, [8]). The respiratory chain of mitochondria is organized in four polymeric units or "complexes." The purpose of this paper is to summarize recent work on the topographical arrangement in the membrane of cytochrome c oxidase (complex IV) and ubiquinol cytochrome c reductase (complex III) and to discuss its relevance with respect to the mechanism of the redox proton pump. As regards complex IV cytochrome c is located at the external side of the inner mitochondrial membrane (see ref. 4 for review). The location of the site where cytochrome oxidase reacts with oxygen and protons and electrons recombine is controversial (4,9-11). This problem has been attacked in our laboratory by analyzing with flow potentiometric and spectrophotometric techniques the kinetic and stoichiometric relationship between aerobic oxidation of the terminal electron carriers and oxygen protonation (5,6, 12). Fast aerobic oxidation of cytochrome oxidase and c cytochromes is accompanied, in intact beef-heart mitochondria, by synchronous proton release in the medium instead of the expected stoichiometric proton consumption (6). In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) oxidation of electron carriers is accompanied by proton disappearance from the medium. This proton uptake, which roughly corresponds to the content of the oxygen-terminal electron carriers, evidently takes place at the inner side of the membrane. It does, in fact, manifest itself to the electrode in the medium only when a high proton conductance is induced in the membrane by FCCP and still detection of proton consumption is delayed with respect to the time of oxidation of electron carriers (5, 6). This conclusion is reinforced by results of experiments in sonic submitochondrial particles. In sonic particles, due to inverted orientation of the mitochondrial mem-