PURIFICATION AND CHARACTERIZATION OF RHODOCYCLUS-GELATINOSUS PHOTOCHEMICAL-REACTION CENTER

Photoactive Rhodocyclus gelatinosus reaction centers were isolated from photoreceptor units by temperature induced phase separation in the presence of octyl beta-D-thioglucopyranoside/decyltetraoxyethylene mixture (Agalidis, I. and Reiss-Husson, F. (1991) Biochem. Biophys. Res. Commun. 177, 1107-1112) and further purified by ion exchange chromatography. They consisted of three polypeptides L, M, H, with apparent molecular weights of 24, 28 and 34 kDa, respectively. The tetraheme cytochrome c associated to the reaction center (RC) in vivo was removed during purification. Spectral properties of the reaction center indicated a pigment composition similar to that of Rhodobacter sphaeroides. The reaction center contained only the primary quinone (Q(A)) which was identified with menaquinone MK8 (Agalidis, I. et al. (1991) Z. Naturforsch. 46c, 99-105). The absorption spectrum of Q(A)- was typical of a menasemiquinone exhibiting a major peak at 412 nm. Charge recombination time for P+ Q(A)- was 35 ms. Q(A) site could be reconstituted with ubiquinones UQ0, UQ6, UQ9 and UQ10, with P+ Q(B)- charge recombination decay within 1-2.3 s. Multiflash-induced binary oscillations of Q(B)- in the presence of UQ10 demonstrated that the secondary acceptor functioned as a two-electron gate. In contrast, MK8 did not reconstitute Q(B) activity as its midpoint redox potential was probably two low relative to that of Q(A).