Abstract
Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the single sulfhydryl residue of isolated oligomycin sensitivity-conferring protein (OSCP), which was then reconstituted with F1. Fluorescence and phosphorescence resonance energy transfer yields from the lone tryptophan residue of F1 present in the epsilon-polypeptide and the fluorescence labels attached to the F1 complex established that tryptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstituted OSCP-F1 complex, by 4.9 nm from its closest catalytic site and by more than 6.4 nm from the two other catalytic sites, including the lowest affinity ATP site. These separations together with the crystallographic coordinates of the F1 complex (Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F1 molecule in a unique asymmetrical position relative to the catalytic sites of the enzyme.