Interactions between the cytochrome b, cytochrome c1, and Fe-S protein subunits at the ubihydroquinone oxidation site of the bc1 complex of Rhodobacter capsulatus

Abstract

Ubihydroquinone:cytochrome (cyt) c oxidoreductase (bc1 complex and its plant counterpart b6f complex) is a vital component of energy-transducing systems in most organisms from bacteria to eukaryotes. In the facultative phototrophic (Ps) bacterium Rhodobacter capsulatus, it is constituted by the cyt b, cyt c1, and Rieske Fe-S protein subunits and is essential for Ps growth. Of these subunits, cyt b has two nontransmembrane helices, cd1 and cd2, which are critical for its structure and function. In particular, substitution of threonine (T) at position 163 on cd1 with phenylalanine (F) or proline (P) leads to the absence of the bc1 complex. Here, Ps+ revertants of B:T163F were obtained, and their detailed characterizations indicated that position 163 is important for the assembly of the bc1 complex by mediating subunit interactions at the Qo site. The loss of the hydroxyl group at position 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 sulfhydryl group at position 46 of cyt c1. Examination of the mitochondrial bc1 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 counterparts 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, electron transfer activities lower than that of a wild-type bc1 complex. Interestingly, their membrane supernatants contained a smaller form of this subunit with physicochemical properties identical to those of its membrane-bound form. Determination of the amino-terminal amino acid sequence of this soluble Fe-S protein revealed that it was derived from the wild-type protein by proteolytic cleavage at V44. This work revealed for the first time that position 163 of cyt b is important both for proper subunit interactions at the Qo site and for inactivation of the bc1 complex by proteolytic cleavage of its Fe-S protein subunit at a region apparently responsible for its mobility during Qo site catalysis.