The formation or the reduction of a disulfide bridge on the subunit of chloroplast ATP synthase affects the inhibitory effect of the ε subunit

Abstract

We have studied the change of the catalytic activity of chimetic complexes that were formed by chloroplast coupling factor 1 (CF1) -γ, α and β subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different γ subunits modified by oligonucleotide- directed mutagenesis techniques. For this purpose, three mutant γ subunits were produced: γΔ194-230, here 37 amino acids from Pro-194 to Ile- 230 are deleted, Γ(C199A), Cys-199 is changed to Ala, and γΔ200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimetic subunit complexes produced from each of these mutant CF1-γ subunits and a and β subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-γ. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between γ subunit and ε subunit was strongly influenced by the type of γ subunit itself. Although the ATPase activity of the chimeric complex that contained γΔ200-204 or γ(C199A) was inhibited by the addition of recombinant ε subunit from CF1 similarly to complexes containing the reduced wild-type subunit, the recombinant ε subunit did not inhibit the ATPase of the complex, which contained the oxidized form of γ subunit. Therefore the affinity of the E subunit to the γ subunit may be dependent on the state of the subunit or the ε subunit may bind to the oxidized form of γ subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains γΔ194-230 was not efficiently inhibited by ε subunit. These results show that the formation or reduction of the disulfide bond on the γ subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent ε subunit.