Fluorescence resonance energy transfer between coumarin-derived mitochondrial F1-ATPase γ subunit and pyrenylmaleimide-labelled fragments of IF1 and c subunit

We introduced a reporting group into a critical position of the mitochondrial F1-ATPase in order to gain structural information about enzyme—ligand complexes. Incubation of 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) with bovine heart mitochondrial F1-ATPase pretreated with 1mM sodium arsenite modified the only cysteine residue in the γ subunit (γ-Cys78), resulting in an enzyme—CPM fluorescent complex (CPM—F1) with an ATPase activity similar to that of the native enzyme. Transferred fluorescence of F1-bound CPM occurred when different peptide fragments of naturally binding polypeptides carrying a pyrenylmaleimide (PM) moiety were bound to the enzyme. Fluorescence resonance energy transfer (RET) from PM bound to cysteine residues associated with Glu40, Lys47 and Lys58 of fragments of the inhibitor protein (IF1) with CPM—F1 occurred with an efficiency of approx. 20, 21 and 3% respectively. The distance at which the efficiency of energy transfer was 50%, R0, for the CPM and PM donor/acceptor pair was 4.1nm, indicating that the three IF1 fragments must be within 6.7nm of γ-Cys78. RET from the PM-bound hydrophilic fragment of c subunit (residues 37–42) of the F1F0-ATPase complex and CPM-bound γ-Cys78 occurred with an efficiency of approx. 30%, indicating a distance of 4.7nm between the two fluorophores. Based on previous observations and on the present RET measurements, the hydrophilic loop of c subunit was located at the base of the F1 foot, and the N-terminal region of IF1 was located on the surface of F1 in the lower part of the α3β3 hexamer ring.