The three-dimensional structure of the mitochondrial bc1 complex reveals that the extrinsic domain of the Fe - S subunit, which carries the redox-active [2Fe2S] cluster, is attached to its transmembrane anchor domain by a short flexible hinge sequence (amino acids D43 to S49 in Rhodobacter capsulatus). In various structures, this extrinsic domain is located in different positions, and the conformation of the hinge region is different. In addition, proteolysis of this region has been observed previously in a bc1 complex mutant of R. capsulatus [Saribas, A. S., Valkova-Valchanova, M. B., Tokito, M., Zhang, Z., Berry E. A., and Daldal, F. (1998) Biochemistry 37, 8105-8114]. Thus, possible correlations between proteolysis, conformation of the hinge region, and position of the extrinsic domain of the Fe - S subunit within the bc1 complex were sought. In this work, we show that thermolysin, or an endogenous activity present in R. capsulatus, cleaves the hinge region of the Fe - S subunit between its amino acid residues A46-M47 or D43-V44, respectively, to yield a protease resistant fragment with a M(r) of approximately 18 kDa. The cleavage was affected significantly by ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) site inhibitors and by specific mutations located in the bc1 complex. In particular, using either purified or detergent dispersed chromatophore-embedded R. capsulatus bc1 complex, we demonstrated that while stigmatellin blocked the cleavage, myxothiazol hardly affected it, and antimycin A greatly enhanced it. Moreover, mutations in various regions of the Fe - S subunit and cyt b subunit changed drastically proteolysis patterns, indicating that the structure of the hinge region of the Fe - S subunit was modified in these mutants. The overall findings establish that protease accessibility of the Fe - S subunit of the bc1 complex is a useful biochemical assay for probing the conformation of its hinge region and for monitoring indirectly the position of its extrinsic [2Fe2S] cluster domain within the Q(o) pocket.
ASJC Scopus subject areas