Energy-linked Changes of Hydrogen Ion Concentration in Submitochondrial Particles (1967)

Abstract 1. Submitochondrial particles supplemented with bromthymol blue, found in independent experiments to be bound to the particles, exhibit characteristic decreases of absorbance upon activation of electron transport by addition of substrate or oxygen to the anaerobic material or by addition of adenosine 5'-triphosphate to the sulfide-inhibited material. The responses are reversible upon exhaustion of substrate or oxygen or upon utilization of adenosine 5'-triphosphate. 2. Inhibitors of electron transport (sulfide or antimycin A) reverse the bromthymol blue responses activated by substrate and oxygen. 3. Uncoupling agents, for example, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, rapidly reverse the bromthymol blue responses upon addition of substrate, oxygen, or adenosine 5'-triphosphate. 4. Glass electrode measurements external to the submitochondrial particles confirm an alkalinization outside the particles on activation of electron transport. On the basis of these observations, the decolorization of bromthymol blue upon activation of electron transport is attributed to an intravesicular acidification. 5. The total amount of hydroxyl ion production exterior to the vesicles is 50 to 60 mµmoles of hydroxyl ions per mg of protein, a value roughly 10 times in excess of the maximal content of respiratory carriers of the particles, and several fold greater than the measured calcium content, but considerably less than the magnesium content. 6. In view of the observed cation content of the submitochondrial particles employed and the similarities in the bromthymol blue responses caused by activation of electron transport to those caused by the addition of acetate or ethylene glycol 2-(2-aminoethyl)-tetraacetate, the intravesicular acidification is attributed to the energy-linked movement of bound cations from the inside of the vesicle to the outside medium. 7. The response of bromthymol blue affords a highly sensitive measure of energy conservation; a response to the oxidation of 1 nmole of reduced β-diphosphopyridine nucleotide is readily measurable. 8. The kinetics of activation of the bromthymol blue response and of its inactivation by inhibitors of electron transport and by uncouplers as well suggests that the cation movement is a reversible phenomenon and that a circulation of cations through the vesicular membrane occurs. 9. Much experimental evidence is consistent with a chemical mechanism for cation movements below 0.2 mg of protein per ml, suggesting that the bromthymol blue response is due to a dissociable component of the vesicular membranes, presumably a cation such as calcium. Also, ethylene glycol 2-(2-aminoethyl)-tetraacetic acid and acetate give bromthymol blue responses similar to those induced by activation of electron transport or adenosine 5'-triphosphate. The slow initial rate of hydrogen ion formation is consistent with the chemical mechanism. 10. The data of this paper do not afford support for compulsory movements of electrons in the respiratory chain and of hydrogen ions through the vesicular membrane. The initial rate of hydrogen ion formation is over 1000-fold slower than the initial rate of oxidation of reduced quinone and flavoprotein. The magnitude of the hydrogen ion change is too small by a factor of about 10 to represent a jump due to the oxidation of reduced quinones and flavin. The collapse of the pH gradient across the vesicular membrane occurs nearly as rapidly upon inhibition of electron transport by antimycin A as it does upon addition of an uncoupling agent. 11. A structural model for the movement of cations in a circulating pathway through the vesicular membrane operating according to the chemical mechanism has been proposed and is consistent with the available experimental data. 12. It is concluded that the chemical mechanism for oxidative phosphorylation is worthy of detailed experimental investigation.