Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase (original) (raw)

2000, Journal of Biological Chemistry

The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3 ␤ 3 hexamer stationary relative to the a subunit as the ␥⑀c 9-12 complex rotates. Despite their essential nature, the contacts between b and the ␣, ␤, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24-156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete F 1 F 0 was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to ␣ and ␤), and 109 and 110 (to ␣ only). Mass spectrometric analysis of peptide fragments derived from the b 24-156 A92C crosslink revealed that cross-linking took place within the region of ␣ between Ile-464 and Met-483. This result indicates that the b dimer interacts with the ␣ subunit near a non-catalytic ␣/␤ interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F 0 in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b 24-156 A92C and ␤ as well as b 24-156 I109C and ␣ are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex. ATP synthase, or F 1 F 0-ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9-12. The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and ␤ subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each ␤ subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the