CONTROL OF THE UNIDIRECTIONAL TOPOLOGICAL ORIENTATION OF A CROSS-LINKED COMPLEX COMPOSED OF THE BACTERIAL PHOTOSYNTHETIC REACTION-CENTER AND HORSE HEART CYTOCHROME-C RECONSTITUTED INTO PROTEOLIPOSOMES

Control of the unidirectional topological orientation was achieved for a cross-linked complex composed of the bacterial photosynthetic react ion center and horse heart cytochrome c (RC/cyt c) reconstituted into proteoliposomes. Using the method of Ueno et al, [Ueno et al, (1995) M ater, Sci, Eng, C3, 1-6], we prepared RC/cyt c by conjugating cyt c to the H-subunit of RC of Rhodobacter sphaeroides R-26 using a bifunctio nal cross-linking reagent, N-succinimidyl 3-(2-pyridyldithio)propionat e (SPDP), as previously reported, The freeze-thaw method was used to i ncorporate RC/cyt c into liposomes that contained dipalmitoyl-L-alpha- phosphatidylcholine and dipalmitoyl-L-alpha-phosphatidylglycerol (1:9) , The topological orientation of RC/cyt c in the proteoliposomes was d etermined using three methods: (i) release of the cyt c moiety from th e proteoliposomes by cleaving the disulfide bond in the linker residue , (ii) electron transfer from free cyt c outside the proteoliposomes t o the RC moiety, and (iii) photo-induced membrane potential of RC- and RC/cyt c-reconstituted proteoliposomes. The results indicated that ab out 90% of the RC/cyt c in proteoliposomes was oriented with the H-sub unit exposed on the outside of the liposomes, whereas only about 60% o f the RC in proteoliposomes had this orientation. Thus, we successfull y controlled the unidirectional topological orientation of the RC moie ty in liposomes using the RC/cyt c complex.