The effect of mitochondrial energization on cytochrome c oxidase kinetics as measured at low temperatures. II. The binding and reduction of dioxygen.

The effect of pre-energization of isolated mitochondria by ATP at room temperature upon the kinetics of oxygen intermediates (measured at very low temperatures) of cytochrome c oxidase has been studied. It was found that "energization" of mitochondria at room temperature had dramatic effects on several partial reactions of cytochrome aa3. Thus, in the "energized" frozen state, the rate of O2 binding to ferrous cytochrome a3 and the subsequent formation of the "peroxy" compound B are accelerated, while oxidation of cytochromes c and c1 is inhibited. These effects of ATP are abolished by oligomycin and uncoupling agents and may, therefore, be reflections of the coupling of the mitochondrial ATP synthetase to the respiratory chain at the level of cytochrome c oxidase, which is the basis of the mechanism of coupling respiration to ATP synthesis and respiratory control.

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase.

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa3) is coupled to translocation of H+ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa3 is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational "strain" in the cytochrome aa3 complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.

Effect of inhibitors on the sigmoidicity of the calcium ion transport kinetics in rat liver mitochondria.

The kinetic plot (initial rate of Ca2+ transport versus concentration) of mitochondrial Ca2+ transport is hyperbolic in a sucrose medium. The plot becomes sigmoidal in the presence of competitive inhibitors of Ca2+ binding to low affinity sites of the membrane surface such as Mg2+ and K+. The plot also becomes sigmoidal in the presence of Ba2+. Ba2+ is a competitive inhibitor of both Ca2+ transport and Ca2+ binding to the low affinity sites. The 5i for the inhibition of Ca2+ transport by Ba2+ increases in the presence of K+ and Mg2+, which suggests a competition for the low affinity sites between the cations. The plot is still hyperbolic in the presence of La3+, which inhibits Ca2+ transport competitively. Ruthenium red which is a pure non-competitive inhibitor of mitochondrial Ca2+ transport, does not affect the shape of the kinetic plot. These results indicate that the surface potential, which depends on the ions bound to the low affinity sites, determines whether the kinetics of Ca2+ uptake in mitochondria is sigmoidal or hyperbolic.

Conformational changes in cytochrome aa3 and ATP synthetase of the mitochondrial membrane and their role in mitochondrial energy transduction.

1. The thermodynamics and molecular basis of energy-linked conformational changes in the cytochrome aa3 and ATP synthetase complexes of the mitochondrial membrane have been studied with spectrophotometrical and fluorometrical techniques. 2. Ferric cytochrome aa3 exists in two conformations, high spin and low spin, the equilibrium between these states being controlled by the electrical potential difference across the mitochondrial membrane. The conformational change is brought about by an electrical field-driven binding of one proton per aa3 to the complex. At pH 7.2 the concentration of the two conformations is equal at a membrane potential of 170 mV corresponding to about 4 kcal/mole. 3. The high to low spin transition in ferric aa3 is also induced by hydrolysis of ATP in which case two molecules of aa3 are shifted per ATP molecule hydrolyzed. This is in accordance with translocation of two protons across the mitochondrial membrane coupled to hydrolysis of ATP as proposed in the chemiosmotic theory of oxidative phosphorylation. 4. The conformational transition in cytochrome aa3 is not an expression of the formation of a 'high-energy' intermediate or reversal of the energy-transducing pathway of oxidative phosphorylation, but is presumably the basis of allosteric control of the activity of cytochrome oxidase by the energy state of the mitochondrion. This control is exerted by a regulatory mechanism in which the electrical potential difference controls the conformation and redox properties of the heme centres and thereby the rate of oxygen consumption. 5. The synthesis of one molecule of ATP by oxidative phosphorylation is energetically equivalent to the work done in carrying two electrical charges across the entire mitochondrial membrane. 6. Fluorescence changes of aurovertin bound to ATP synthetase reveal that the electrical membrane potential induces a conformational change in the F1 portion of the enzyme which is probably associated with dissociation of the natural F1 inhibitor protein. This conformational change is energetically equivalent to the work done in carrying one electrical charge across the mitochondrial membrane. 7. A model is proposed for the mechanism of the electrical field-induced conformational changes in the cytochrome aa3 and ATP synthetase complexes, and the significance of these changes in the mechanism and control of mitochondrial energy conservation is discussed.

On the lack of ATP-induced midpoint potential shift for cytochrome b-566 in plant mitochondria.

Recently proposed mechanisms of site II energy transduction that assign a key role to cytochrome b-566 are based on the finding that the apparent midpoint potential of b-566 in animal mitochondria increases by more than 250 mV upon addition of ATP [Chance et al. (1970) Proc. Nat. Acad. Sci. USA 66, 1175-1182]. However, since it has never been shown that the redox mediators used in the midpoint potential measurements equilibrate directly with b-566, the observed midpoint potential shift could merely reflect reversed electron transport. In mung bean mitochondria, the apparent midpoint potential of b-566 is known to be unaffected by addition of ATP [Dutton and Storey (1971) Plant Physiol. 47, 282-288]. In the present work, mung bean b-566 is shown to undergo an ATP-induced reduction similar to that observed for b-566 in animal mitochondria. However, in mung bean mitochondria the reduction is found to be rapidly relaxed by addition of redox mediator (phenazine methosulfate, PMS) and concomitantly PMS causes a marked, antimycinsensitive stimulation of ATPase activity. These results suggest that the ATP-induced reduction in mung bean mitochondria is due to reversed electron transport and that PMS can effectively short-circuit reversed electron transport in this system, bringing it close to equilibrium. Moreover, since mung bean and animal b-566 are identical in all other respects tested, the results support the idea that the apparent midpoint potential shift in animal mitochondria is also merely due to reversed electron transport, and that the mediators are now not effective enough to bring the system to equilibrium.