INTERACTIONS BETWEEN THE CYTOCHROME-B, CYTOCHROME C(1), AND FE-S PROTEIN SUBUNITS AT THE UBIHYDROQUINONE OXIDATION SITE OF THE BC(1) COMPLEX OF RHODOBACTER-CAPSULATUS

Ubihydroquinone:cytochrome (cyt) c oxidoreductase (bc(1) complex and i ts plant counterpart b(6)f complex) is a vital component of energy-tra nsducing systems in most organisms from bacteria to eukaryotes. In the facultative phototrophic (Ps) bacterium Rhodobacter capsulatus, it is constituted by the cyt b, cyt c(1), and Rieske Fe-S protein subunits and is essential for Ps growth. Of these subunits, cyt b has two nontr ansmembrane helices, cd1 and cd2, which are critical for its structure and function. In particular, substitution of threonine (T) at positio n 163 on cd1 with phenylalanine (F) or proline (P) leads to the absenc e of the bc(1) complex. Here, Ps(+) revertants of B:T163F were obtaine d, and their detailed characterizations indicated that position 163 is important for the assembly of the bc(1) complex by mediating subunit interactions at the Q(o) site. The loss of the hydroxyl group at posit ion 163 of cyt b was compensated for by the gain of either a hydroxyl group at position 182 of cyt b or 46 of the Fe-S protein or a sulfhydr yl group at position 46 of cyt c(1). Examination of the mitochondrial bc(1) complex crystal structure [Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I,, Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature 392, 677-684] revealed that the counterp arts of B:G182 (i.e., G167) and F:A46 (i.e., A70) are located close to B:T163 (i.e., T148), whereas the C:R46 (i.e., R28) is remarkably far from it. The revertants contained substoichiometric amounts of the Fe- S protein subunit and exhibited steady-state and single-turnover, elec tron transfer activities lower than that of a wild-type bc(1) complex. Interestingly, their membrane supernatants contained a smaller form o f this subunit with physicochemical properties identical to those of i ts membrane-bound form. Determination of the amino-terminal amino acid sequence of this soluble Fe-S protein revealed that it was derived fr om the wild-type protein by proteolytic cleavage at V44. This work rev ealed for the first time that position 163 of cyt b is important both for proper subunit interactions at the Q(o) site and for inactivation of the bc(1) complex by proteolytic cleavage of its Fe-S protein subun it at a region apparently responsible for its mobility during Q(o) sit e catalysis.