ATP synthase K+- and H+-fluxes drive ATP synthesis and enable mitochondrial K+-‘uniporter’ function: I. Characterization of ion fluxes

ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological pH = 7.2 and K+ = 140 mEq/L conditions. Purely K+-driven ATP synthesis from single F1Fo molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K+ currents by voltage clamp, both blocked by specific Fo inhibitors. In the presence of K+, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K+:H+ stoichiometry. The excellent agreement between the functional data obtained from purified F1Fo single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K+ presence, is entirely consistent with K+ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K+ (harnessing ΔΨm) and H+ (harnessing its chemical potential energy, ΔµH) drive ATP generation during normal physiology.

Inhibition of electron transport and oxidative phosphorylation in plant mitochondria by gladiolic acid and structurally-related aromatic ortho dialdehydes

Gladiolic acid (GA, 4-methoxy-5-methyl-o-phthalaldehyde-3-carboxylic acid), an antifungal aromatic ortho dialdehyde produced by Penicillium gladioli was found to be a potent inhibitor of electron transport and oxidative phosphorylation reactions in sweet potato and mung bean mitochondria. Similar results were also found with the naturally occurring ortho dialdehydes, cyclopaldic acid, quadrilineatin, and flavipin as well as the synthetic dialdehydes, 3-formyl opianic acid and o-phthalaldehyde. Because of their highly reactive ortho-diformyl grouping, GA and structurally related dialdehydes apparently act as multisite inhibitors affecting electron transport and oxidative phosphorylation (at each coupling site). Gladiolic acid has no uncoupling effect like 2,4-dinitrophenol and does not have the same point of interaction in the energy transfer process as oligomycin. Several "partial" reactions of phosphorylation (Mg+2–DNP-stimulated ATPase; ATP–Pi exchange) were strongly inhibited by the various dialdehydes. Flavipin and quadrilineatin are potent inhibitors (80% at a concentration of 25 μM) of site III phosphorylation. Gladiolic acid and related ortho dialdehydes inactivate the catalytic activity of native cytochrome c in vitro. Lysyl ε-NH2 rich cytochrome c may be a major site of GA action in the intact mitochondrion. In view of the high chemical reactivity of the ortho-diformyl group, it is suggested that mitochondrial function may be affected by aromatic ortho dialdehydes through a combination of reactions involving cross-linking of amino groups on membrane polypeptides and monofunctional reaction with free amino groups important for enzyme function, including ε-NH2 groups on cytochrome c. Cross-linking in mitochondrial membrane systems might affect function by interfering with molecular motion in the operation of the terminal portion of the electron-transport chain. The primary toxicological mode of action of GA and related dialdehydes appears to be due to inhibition of mitochondrial function.

The distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin.

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.

The action of digitonin on rat liver mitochondria. Electron microscopy

1. Rat liver mitochondria were examined in the electron microscope by using negative staining in the presence of 0·3m-sucrose. The intact outer membrane does not appear to be freely permeable to the stain. Where the stain penetrated through a tear it was seen that the inner membrane had randomly oriented grooves, many of which contained round structures varying between 200 and 900å in diameter. Laminar structures containing two to five layers of approx. 50å each were found at the periphery. 2. When the outer membrane was removed by treating the mitochondria with digitonin several types of inner-membrane complexes were formed and they showed a general correlation with those observed in sectioned samples of the same preparations. The main types were: (a) a condensed form looking very much like the intact mitochondrion without the outer membrane (this still showed the grooves, some of which contained the round structures, and the laminar whirls at the edges); (b) a more transparent form containing tubules of uniform width and various lengths (some of these appeared to terminate in a hole at the surface of the inner membrane); (c) a large torn sac, probably the inner membrane, containing some tubules and vesicles. 3. When the inner-membrane complex was further treated with digitonin it was disrupted and the resulting material consisted of pieces of membrane, doughnut-shaped units and lamellar structures. Most of these pieces varied in size between 500 and 1000å.