STUDY OF CONFORMATIONAL-CHANGES OF CHLOROPLAST COUPLING FACTOR BY ACID-BASE TITRATION

The state of ionization of ionizable groups in chloroplast coupling fa ctor CF1 was studied by acid-base titration. About 330 ionizable group s in CF1 bind protons in the pH range 3.0-9.0. About 180 ionizable gro ups in the native enzyme are solvent-inaccessible; these are exposed b y denaturation of the enzyme with sodium dodecyl sulfate. The titratio n curve is mainly determined by the carboxyl groups of the alpha- and beta-subunits of CF1. About half of the carboxyl groups have abnormall y high pK values, i.e., 5.5-5.7. Hysteresis in the acid-base titration curves for CF1 is found at pH 5.0-9.0. The hysteresis is not eliminat ed by decreasing the titration rate, and it is not related to pH-induc ed denaturation of the enzyme because the same hysteresis is seen if t he titration cycle is repeated. The hysteresis is due to pH-induced co nformational changes of the enzyme. ADP binding decreases the number o f groups which are titrated in pH 5.0-9.0 range. ATP and octyl glucosi de, which activate the ATPase reaction, inhibit the protonation of CF1 in the pH 7.0-9.0 range. Triton X-100 does not change the catalytic p roperties of CF1 but increases the protonation of 100 ionizable groups of the enzyme. These data suggest a relationship between conformation al changes of CF1 and the state of ionization of the enzyme.