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 complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 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, gammaC199A, 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 CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. 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 gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 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. Therefore the affinity of the epsilon subunit to the gamma subunit may be 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 that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.
Hisabori
T
T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan. thisabor@res.titech.ac.jp
Motohashi
K
K
Kroth
P
P
Strotmann
H
H
Amano
T
T
D000595:Amino Acid Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002736:Chloroplasts; D004220:Disulfides / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D004220:Disulfides; D011993:Recombinant Fusion Proteins; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D000432:Methanol