Cross‐Linking of Chloroplast F 0 F 1 ‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor

Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6 → Ser mutant of subunit ɛ of spinach chloroplast F 0 F 1 ATP synthase. Wild‐type and engineered ɛ were expressed in Escherichia coli , purified in the presence of urea, refolded and reassembled with spinach chloroplast F 1 lacking the ɛ subunit [F 1 (‐ɛ)]. Cys‐containing ɛ variants were modified with a sulfhydryl‐reactive photolabile cross‐linker. Photocross‐linking of ɛ to F 1 (‐ɛ) yielded the same SDS gel pattern of cross‐link products independent of the presence or absence of Mg 2+ ADP, phosphate and Mg 2+ ATP. ɛ (wild type) [Ser6, Cys28]ɛ and [Ser6,Cys41]ɛ were cross‐linked with subunit γ. With chloroplast F 0 F 1 the same cross‐link pattern was obtained, except for one extra cross‐link, probably between [Ser6, Cys28]ɛ and F 0 subunit III. [Ser6, Cys17]ɛ and [Ser6, Cys85]ɛ did not produce cross‐links. Cross‐linking of ɛ, [Ser6,Cys28]ɛ, [Ser6,Cys41]ɛ to γ in soluble chloroplast F 1 impaired the ability of ɛ to inhibit Ca 2+ ‐ATPase activity. The Mg 2+ ‐ATPase activity of soluble F 1 (measured in the presence of 30% MeOH) was not affected by cross‐linking ɛ with γ. Functional reconstitution of photophosphorylation in F 1 ‐depleted thylakoids was observed with F 1 in which γ was cross‐linked to [Ser6,Cys28]ɛ or [Ser6,Cys41]ɛ but not with wild‐type ɛ. In view of the intersubunit rotation of γ relative to (αβ) 3 , which is driven by ATP hydrolysis, γ and ɛ would seem to act concertedly as parts of the ‘rotor’ relative to the ‘stator’ (αβ) 3 .