STRUCTURAL MAPPING OF THE EPSILON-SUBUNIT OF MITOCHONDRIAL H-ATPASE COMPLEX (F1)()

Phosphorescence and fluorescence energy transfer measurements have bee n used to locate the epsilon-subunit within the known structural frame of the mitochondrial soluble part of F-type H+-ATPase complex (F-1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the p robe hylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached t o the single sulfhydryl residue of isolated oligomycin sensitivity-con ferring protein (OSCP), which was then reconstituted with F-1. Fluores cence and phosphorescence resonance energy transfer yields from the lo ne tryptophan residue of F-1 present in the epsilon-polypeptide and th e fluorescence labels attached to the F-1 complex established that try ptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstitut ed OSCP-F-1 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 low est affinity ATP site. These separations together with the crystallogr aphic coordinates of the F-1 complex (Abrahams, J. P., A. G. W. Leslie , R. Lutter, and J. E. Walker. 1994. Structure at 2.8 Angstrom resolut ion of F-1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F-1 molecule in a unique asymmetrical position relative to the catalytic sites of the e nzyme.