CONFORMATIONAL-CHANGES OF THE MITOCHONDRIAL F-1-ATPASE EPSILON-SUBUNIT INDUCED BY NUCLEOTIDE-BINDING AS OBSERVED BY PHOSPHORESCENCE SPECTROSCOPY

Changes in conformation of the E-subunit of the bovine heart mitochond rial F-1-ATPase complex as a result of nucleotide binding have been de monstrated from the phosphorescence emission of tryptophan. The triple t state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the p rotein structure surrounding the chromophore, nucleoside diphosphate a cts in an opposite manner, enhancing the rigidity of this region of th e macromolecule. Such changes in dynamic structure of the epsilon-subu nit are evident at high ligand concentration added to both the nucleot ide depleted F-1 (Nd-F-1) and the F-1 preparation containing the three tightly bound nucleotides (F-1(2,1)). Since the effects observed are similar in both the F-1 forms, the binding to the low affinity sites m ust be responsible for the conformational changes induced in the epsil on-subunit. This is partially supported by the observation that the Tr p lifetime is not significantly affected by adding an equimolar concen tration of adenine nucleotide to Nd-F-1. The effects on protein struct ure of nucleotide binding to either catalytic or noncatalytic sites ha ve been distinguished by studying the phosphorescence emission of the F-1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F-1(3,0)). Phosphorescence lifetime meas urements on this F-1 form demonstrate that the binding of Mg-NTP to ca talytic sites induces a slight enhancement of the rigidity of the epsi lon-subunit. This implies that the binding to the vacant noncatalytic site of F-1(2,1) must exert the opposite and larger effect of enhancin g the flexibility of the protein structure observed in both Nd-F-1 and F-1(2,1). The observation that enhanced flexibility of the protein oc curs upon addition of adenine nucleotides to F-1(2,1) in the absence o f Mg2+ provides direct support for this suggestion. The connection bet ween changes in structure and the possible functional role of the epsi lon-subunit is discussed.