Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6 → Ser mutant of subunit ɛ of spinach chloroplast F0F1 ATP synthase. Wild‐type and engineered ɛ were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F1 lacking the ɛ subunit [F1(‐ɛ)]. Cys‐containing ɛ variants were modified with a sulfhydryl‐reactive photolabile cross‐linker. Photocross‐linking of ɛ to F1(‐ɛ) yielded the same SDS gel pattern of cross‐link products independent of the presence or absence of Mg2+ ADP, phosphate and Mg2+ ATP. ɛ (wild type) [Ser6, Cys28]ɛ and [Ser6,Cys41]ɛ were cross‐linked with subunit γ. With chloroplast F0F1 the same cross‐link pattern was obtained, except for one extra cross‐link, probably between [Ser6, Cys28]ɛ and F0 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 F1 impaired the ability of ɛ to inhibit Ca2+‐ATPase activity. The Mg2+‐ATPase activity of soluble F1 (measured in the presence of 30% MeOH) was not affected by cross‐linking ɛ with γ. Functional reconstitution of photophosphorylation in F1‐depleted thylakoids was observed with F1 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.

Schulenberg, B., Wellmer, F., Lill, H., Junge, W., & Engelbrecht, S. (1997). Cross‐Linking of Chloroplast F0F1‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor. European Journal of Biochemistry, 249(1), 134–141. Portico.