Rotational mobility and domain flexibility of membrane-bound bacterial coupling factor as detected with the triplet probe eosin-isothiocyanate

Abstract

The membrane-bound coupling factor (BF 1) of chromatophores from the photosynthetic bacerium Rhodopseudomonas sphaeroides was covalently labeled with the triplet probe eosin-isothiocyanate. The labeled enzyme was isolated and functionally reconstituted into depleted chromatophores from the same bacterium. ATPase and ATP synthase activities of the reconstituted vesicles were strongly dependent on the labeling conditions, decreasing at increasing load of eosin molecules per BF 1. When labeling was carried out in the dark and in the presence of ATP, one molecule of eosin isothiocyanate was bound per BF 1 and the activities catalyzed by the reconstituted and labeled enzyme were as high as in untreated chromatophores. Upon light energization of the chromatophore membrane, a large conformational change of BF 1 could be detected by using the triplet probe as a spectroscopic tool. The domain flexibility and rotational mobility of the reconstituted coupling enzyme were directly related to the enhancement of the ATPase activity induced by light. Both the light-stimulated ATPase activity and conformational changes could be prevented by addition of ADP or oligomycin and affected to the same extent by uncouplers and inhibitors of electron transport. Moreover, the detected conformational changes were reversible in time, appearing with a half-time of 10 ms upon illumination of the chromatophores, and disappearing with a half-time of 70 ms in the dark. The results obtained prove the feasibility of the spectroscopic technique in detecting conformational changes of the membrane-bound BF 1, similarly to what already has been observed for chloroplast coupling factor (Wagner, R. and Junge, W. (1980) FEBS Lett. 114, 327–333), and add to the possibility of characterizing, by this method, energy transduction at a molecular level.