Molecular Approach to the Regulatory Mechanism of Chloroplast ATP Synthase

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We have studied the change of the catalytic activity of chimeric complexes which were formed by CF_1-gamma, and alpha and beta subunits of thermophilic bacterial F_1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced : gamma_<DELTA194-230>, here 37 amino acids from Pro-194 to Ile-230 are deleted, gamma_<C199A>, Cys-199 is changed to Ala, and gamma_<DELTA200-204>, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF_1-gamma subunits and alpha and beta subunits from thermophilic bacterial F_1 lost the sensitivity against thiol reagents when compared to the complex containing wild type CF_1-gamma. The pH optimum (pH 8.5 to pH 9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction o … More f the mutations did not change the main features of ATPase activity of the chimeric complex.However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex which contained gamma_<DELTA200-204> or gamma_<C199A> was inhibited by the addition of recombinant epsilon subunit from CF_1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex which contained the oxidized form of gamma subunit, suggesting the affinity of the epsilon subunit to the gamma subunit is dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex which contains gamma_<DELTA194-230> was not efficiently inhibited by epsilon subunit. These results show that the formation or the reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region which directly affects the interaction of this subunit with the adjacent epsilon subunit. Less