Cross-linking of engineered subunit δ to (αβ)3 in chloroplast F-ATPase

Abstract

Ser → Cys mutations were introduced into subunit δ of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis. The engineered δ subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F1-ATPase (CF1) lacking the δ subunit (CF1(-δ)). By modification with eosin-5-maleimide, it was shown that residues 10, 57, 82, 160, and 166 were solvent-accessible in isolated CF1 and all but residue 166 also in membrane-bound CF0CF1. Modification of the engineered δ subunit with photolabile cross-linkers, binding of δ to CF1(-δ), and photolysis yielded the same SDS gel pattern of cross-link products in the presence or absence of ADP, phosphate, and ATP and both in soluble CF1 and in CF0CF1. By chemical hydrolysis of cross-linked CF1, it was shown that δS10C was cross-linked within the N-terminal 62 residues of subunit β. δS57C, δS82C, and δS166C were cross-linked within the N-terminal 192 residues of subunit α. Cross-linking affected neither ATP hydrolysis by soluble CF1 nor its ability to reassemble with CF0 and to structurally reconstitute ATP synthesis. Functional reconstitution, however, seemed to be impaired.