Proteolytic cleavage of the Fe-S subunit hinge region of Rhodobacter capsulatus bc(1) complex: Effects of inhibitors and mutations

The three-dimensional structure of the mitochondrial bc(1) complex reveals that the extrinsic domain of the Fe-S subunit, which carries the redox-acti ve [2Fe2S] cluster, is attached to its transmembrane anchor domain by a sho rt flexible hinge sequence (amino acids D43 to S49 in Rhodobacter capsulatu s). In various structures, this extrinsic domain is located in different po sitions, and the conformation of the hinge region is different. In addition , proteolysis of this region has been observed previously in a bc(1) comple x mutant of R. capsulatus [Saribas, A. S., Valkova-Valchanova, M. B., Tokit o, 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 wit hin the bc(1) complex were sought. In this work, we show that thermolysin, or an endogenous activity present in R. capsulatus, cleaves the hinge regio n of the Fe-S subunit between its amino acid residues A46-M47 or D43-V44, r espectively, to yield a protease resistant fragment with a M, of approximat ely 18 kDa. The cleavage was affected significantly by ubihydroquinone oxid ation (Q(o)) and ubiquinone reduction (Q(i)) site inhibitors and by specifi c mutations located in the bcr complex. In particular, using either purifie d or detergent dispersed chromatophore-embedded R. capsulatus bc(1) complex , we demonstrated that while stigmatellin blocked the cleavage, myxothiazol hardly affected it, and antimycin A greatly enhanced it. Moreover, mutatio ns in various regions of the Fe-S subunit and cyt b subunit changed drastic ally proteolysis patterns, indicating that the structure of the hinge regio n of the Fe-S subunit was modified in these mutants. The overall findings e stablish that protease accessibility of the Fe-S subunit of the bc(1) compl ex 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.