Principles of Biochemistry, Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Electron transport, oxidative phosphorylation, and the regulation of ATP synthesis
The enzyme catalyzing ATP synthesis was isolated and reconstituted

Let us now examine the ATP-synthesizing enzyme system embedded in The inner mitochondrial membrane. This enzyme complex, known as ATP synthase or the F₀F₁-ATPase, consists of two main components: F₀ and F₁ (where F stands for "factor"). The F₁ component is shaped somewhat like a round doorknob facing the mitochondrial matrix, or a mushroom cap (hence these structures are also referred to as mushroom-like projections; Figs. 17-2 and 17-15). The "cap," more commonly called the HEAD, is attached via a stalk to the F₀ component, which is embedded within and spans the inner membrane. (The index "o" is not a zero, but the letter "o", indicating that this part of the ATP synthase molecule binds the toxic antibiotic oligomycin, a potent inhibitor of this enzyme and, consequently, of Oxidative Phosphorylation as well.)

F₁ was first isolated in purified form from the inner mitochondrial membrane by Efraim Racker and his colleagues. In isolation, the F₁ component lacks The ability to synthesize ATP from ADP and phosphate, but it can cleave ATP into ADP and phosphate, which is why it is also called F₁-ATPase. If F₁ is gently extracted from inverted membrane vesicles obtained by disrupting the inner mitochondrial membrane (Fig. 17-15), the respiratory chains in these vesicles remain intact and capable of electron transport. However, vesicles depleted of F₁ (the absence of F₁ heads is confirmed by Electron Cell/15.html">Microscopy) are no longer able to synthesize ATP. If isolated F₁ is added back to such vesicles under appropriate conditions, the normal Structure OF THE inner mitochondrial membrane (an essential feature of which is the presence of F₁ heads) is restored, along with the energy coupling between Electron Transport and ATP synthesis (Fig. 17-15). Such membrane reconstitution experiments, first pioneered by Racker, marked the beginning of extensive and fruitful investigations into membrane Structure and function.

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Fig. 17-15. Disruption of the inner mitochondrial membrane by ultrasonication, preparation of membrane vesicles lacking oxidative phosphorylation capability, and reconstitution of structures capable of carrying out this process. Ultrasonication disrupts the cristae of the inner mitochondrial membrane. The edges of the membrane fragments then seal to form closed membrane vesicles in which the heads of the mushroom-like projections, or F₁ heads, face outward rather than inward. Treatment of these inverted vesicles with urea or Trypsin causes the F₁ heads to detach. Vesicles treated in this manner, which still contain the F₀ components, retain the capacity for electron transport but can no longer carry out phosphorylation. If F₁ molecules are subsequently added to these head-depleted vesicles, the molecules reassociate with the F₀ units preserved in the vesicle membrane. In such reconstituted vesicles, both electron transport and oxidative phosphorylation will occur once again.

Fig. 17-16. Structure of the F₀F₁-ATPase (ATP synthase). A. F₀F₁-ATPase was first discovered in the form of mushroom-like projections on the inner surface of the mitochondrial membrane (seen here in an electron micrograph). B. A model of the F₀F₁-ATPase showing the tentative arrangement of its subunits. C. Crystals of the F₁ component of the complex from rat Liver Mitochondria. D. An electron micrograph showing two molecules of F₀F₁-ATPase isolated from rat liver mitochondria.

Later, the F₁ component was isolated in pure crystalline form (Fig. 17-16). Its molecular weight is approximately 380,000. The F₁ molecule is composed of nine subunits of five different types, grouped together and containing Multiple binding sites for ATP and ADP. Researchers also succeeded in obtaining highly purified preparations of

F₀F₁-ATPase. High-resolution electron microscopic examination of the intact enzyme molecule revealed that it consists of an F₁ head, a stalk, and a base that typically spans the entire thickness of the inner mitochondrial membrane (Fig. 17-16). The F₀F₁-ATPase was named an ATPase because, in isolation, it catalyzes the Hydrolysis of ATP to ADP and P_i. However, in intact mitochondria, its primary biological function is not the breakdown, but the synthesis of ATP from ADP and P_i; therefore, it is more accurately designated as ATP synthase.