Divalent metals in beef heart mitochondrial adenosine triphosphatase. Demonstration of the metals in membrane-bound enzyme and studies of the interconversion of the "1-Mg" and "2-Mg" forms of the enzyme.

Tight divalent metal binding sites in the beef heart mitochondrial adenosine triphosphatase were studied using the procedure of reconstitution of soluble F1 with F1-depleted membranes (SU particles). Soluble F1 has been shown previously to contain two tight-binding site for Mg. Both of these sites were present on membrane-bound enzyme. Co and Mn, substituted at the second of the two Mg-binding sites on soluble F1, became incorporated with F1 into membrane-bound enzyme. Use of radioactive Co and Mn showed that they behaved differently during short bursts of succinate oxidation or ATP hydrolysis. Co remained stably bound, whereas Mn was released to the extent of 55-80%. The results extend previous work to show that the membrane proton-ATPase is an Mg-metalloenzyme containing a structural Mg site and a second Mg site possibly involved in catalysis. The conversion of 2-Mg F1 to 1-Mg F1 during purification and storage is shown to be due to use of ammonium sulfate precipitation, and the dependence of reuptake of Mg (1-Mg F1 leads to 2-Mg F1) on nucleotides is described.