ROTATIONAL-DYNAMICS OF CHLOROPLAST ATP SYNTHASE IN PHOSPHOLIPID-VESICLES

The rotational dynamics of the purified dicyclohexylcarbodiimide-sensitive H+-ATPase (DSA) reconstituted into phospholipid vesicles and of the DSA coreconstituted with the proton pump bacteriorhodopsin were examined by using the technique of time-resolved phosphorescence emission anisotropy. The phosphorescent probe erythrosin isothiocyanate was used to covalently label the 7-polypeptide of DSA before reconstitution. Rotational correlation times were measured under a variety of conditions. The rotational correlation time was independent of the viscosity of the external medium but increased significantly as the microviscosity of the membrane increased. This indicates the rotational correlation times are a measure of the enzyme motion within the membrane. The activation energy associated with the rotational correlation time is 8–10 kcal/mol. At 4 °C, the correlation time, typically ~ 100–180 µs, was unaffected by the addition of substrates and the presence of a membrane pH gradient. Therefore, molecular rotation of the DSA does not appear to play an important role in enzyme catalysis or ion pumping. © 1990, American Chemical Society. All rights reserved.