THE FORMATION OR THE REDUCTION OF A DISULFIDE BRIDGE ON THE GAMMA-SUBUNIT OF CHLOROPLAST ATP SYNTHASE AFFECTS THE INHIBITORY EFFECT OF THE EPSILON-SUBUNIT

We have studied the change of the catalytic activity of chimeric compl exes that were formed by chloroplast coupling factor 1 (CF1) -gamma, a lpha and beta subunits of thermophilic bacterial F-1 after formation o r reduction of the disulfide bridge of different gamma subunits modifi ed by oligonucleotide-directed mutagenesis techniques. For this purpos e, three mutant gamma subunits were produced: gamma(Delta 194-230), he re 37 amino acids from Pro-194 to Ile-230 are deleted, gamma(C199A), C ys-199 is changed to Ala, and gamma(Delta 200-204), amino acids from A sp-200 to Lys-204 are deleted. All of the chimeric subunit complexes p roduced from each of these mutant CF1-gamma subunits and alpha and bet a subunits from thermophilic bacterial F-1 lost the sensitivity agains t thiol reagents when compared with the complex containing wild-type C F1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methano l 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 gamma subunit and epsilon subunit was strongl y influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma(Delta 200-204) o r gamma(C199A) was inhibited by the addition of recombinant epsilon su bunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the AT Pase of the complex, which contained the oxidized form of gamma subuni t, Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subu nit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that con tains gamma Delta 194-230 was not efficiently inhibited by epsilon sub unit, These results show that the formation or reduction of the disulf ide bond on the gamma subunit may induce a conformational change in th e region that directly affects the interaction of this subunit with th e adjacent epsilon subunit.