ISOLATION AND CHARACTERIZATION OF A 2-SUBUNIT CYTOCHROME B-C(1) SUBCOMPLEX FROM RHODOBACTER-CAPSULATUS AND RECONSTITUTION OF ITS UBIHYDROQUINONE OXIDATION (Q(O)) SITE WITH PURIFIED FE-S PROTEIN SUBUNIT

The presence of a two-subunit cytochrome (cyt) b-c(1) subcomplex in ch romatophore membranes of Rhodobacter capsulatus mutants lacking the Ri eske iron-sulfur (Fe-S) protein has been described previously [Davidso n, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in larg e quantities, and its properties were characterized. As expected, it c ontained stoichiometric amounts of cyt b and cyt c(1) subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to th ose of a wild-type bc(1) complex, except that those of its cyt b(L) he me were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (E-m7) values o f cytochromes b(H), bL, and c(1) were very similar to those of a wild- type bc(1) complex. The purified b-c(1) subcomplex had a nonfunctional ubihydroquinone (UQH(2)) oxidation (Q(o)) site, but it contained an i ntact ubiquinone (UQ) reductase (Q(i)) site as judged by its ability t o bind the Q(i) inhibitor antimycin A, and by the presence of antimyci n A sensitive Q(i) semiquinone. Interestingly, its Q(o) site could be readily reconstituted by addition of purified Fe-S protein subunit. Re activated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR g(x) signal comparable to that observed with a bc(1) complex when the Q(o) site is partially occupied with UQ/UQH(2). However, a mutant derivative of the Fe-S prot ein subunit lacking its first 43 amino acid residues was unable to rea ctivate the purified b-c(1) subcomplex although it could bind to its Q (o) site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain o f the Fe-S protein subunit is necessary for UQH(2) oxidation even thou gh its carboxyl-terminal domain is sufficient to provide wild-type-lik e interactions with stigmatellin at the Q(o) site of the bc(1) complex .