Cross-linking of engineered subunit delta to (alpha beta)(3) in chloroplast F-ATPase

Abstract

Ser --> Cys mutations were introduced into subunit delta of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis. The engineered delta subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F-1-ATPase (CF1) lacking the delta subunit (CF1(-delta)). 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 delta subunit with photolabile cross-linkers, binding of delta to CF1(-delta), 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 delta(S10C) was cross-linked within the N-terminal 62 residues of subunit beta. delta(S57C), delta(S82C), and delta(S166C) were cross-linked within the N-terminal 192 residues of subunit alpha. Crosslinking 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.