Changes in conformation of the -subunit of the bovine heart mitochondrial F-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the -subunit are evident at high ligand concentration added to both the nucleotide-depleted F (Nd-F) and the F preparation containing the three tightly bound nucleotides (F(2,1)). Since the effects observed are similar in both the F forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the -subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F(3,0)). Phosphorescence lifetime measurements on this F form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the -subunit. This implies that the binding to the vacant noncatalytic site of F(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F and F(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F(2,1) in the absence of Mg provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the -subunit is discussed.

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