Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Abstract

Changes in conformation of the ⑀-subunit of the bovine heart mitochondrial F 1 -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 2؉ 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 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 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 1 . 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 1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F 1 (3,0)). Phosphorescence lifetime measurements on this F 1 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 1 (2,1) must exert the opposite and larger effect of enhancing 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 occurs upon addition of adenine nucleotides to F 1 (2,1) in the absence of Mg 2؉ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the ⑀-subunit is discussed.The ATPase (ATP synthase) is the enzyme responsible for ATP synthesis during oxidative phosphorylation in all energytransducing membranes. It is composed of two main parts: F 0

Characterization of the binding of Fe(III) to F1ATPase from bovine heart mitochondria

“…This result shows that removal of ATP from the loose non-catalytic site induces the loss of the 2nd Fe(III) equivalent, indicating that the conformational changes produced by the detachment of ATP from the loose non-catalytic site, or by the binding of ATP to such site, modulate the binding of Fe(III) to the second site. In line with this result, is the recent finding that binding of ATP or ADP to such site is most responsible for the different conformations assumed by F~ATPase in the presence of saturating ATP or ADP [19].…”

“…Since it has been reported that phosphate, under conditions comparable to those of Fig. 3, does not produce conformational changes transmitted over long distances [5,19], the spectral changes of the Fe(III)-protein adducts induced by phosphate are attributed to a direct interaction of the anion with the metal ions, implying that one of the two iron-binding sites is located in the proximity of a catalytic site. Nothing may be said concerning the location of the second iron centre.…”

“…To avoid interference with the endogenous iron, the interaction of FIATPase with Fe(III) has been carried out on an enzyme in which all the iron has been depleted by EDTA [9]. In addition, to avoid the conformational changes produced by the ATP and ADP binding to nucleotide sites [4,19], both the unbound and loosely bound nucleotides have been removed from the enzyme. FIATPase sample so obtained is named Iron-Loose Nucleotide-Depleted FIATPase (I-LN-DF~) and contains 2.9 + 0.2 mol of nucleotides per mol of protein, which are bound, according to [3], in the two tight non-catalytic sites and in the tight catalytic site.…”

The Atp Synthase—a Splendid Molecular Machine

“…Nucleotide binding changed signals from His-179 and His-200, but not from His-119, although substitution of His-119, located at a long loop opposite the catalytic site, suppressed ATPase activity and thermostability. In an unusual approach it was shown that changes in conformation of the ε-subunit of MF 1 can be observed by the phosphorescence emission of Trp (200). An interesting cross-reconstitution of F 1 from MF 1 or EcF 1 with F 0 -containing but F 1 -stripped membranes of E. coli or heart mitochondria was achieved (201).…”

Modification of the mitochondrial F1-ATPase ∊ subunit, enhancement of the ATPase activity of the IF1–F1 complex and IF1-binding dependence of the conformation of the ∊ subunit

“…The ε subunit of the bovine mitochondrial F " complex is a peptide of 50 amino acid residues [3] that has been cloned, sequenced and studied in several laboratories ; however, its functional role is far from clear. It has only been reported that its conformation is sensitive to different nucleotides binding to F " [14] and that its suppression in a yeast mutant promotes an uncoupling between the two sectors F ! and F " [15].…”