IMMUNOELECTRON MICROSCOPY AND EPITOPE MAPPING WITH MONOCLONAL-ANTIBODIES SUGGEST THE EXISTENCE OF AN ADDITIONAL N-TERMINAL TRANSMEMBRANE HELIX IN THE CYTOCHROME-B SUBUNIT OF BACTERIAL UBIQUINOL-CYTOCHROME-C OXIDOREDUCTASES

The topology of the ubiquinol:cytochrome-c oxidoreductase (cytochrome bc(1) complex) from Paracoccus denitrificans was investigated by immun oelectron microscopy with sequence-specific murine monoclonal antibodi es. Epitope mapping with synthetic peptides and enzymic proteolytic cl eavage of the cytochrome be, complex were employed to localize precise ly the respective antibody epitopes on the subunits of this membrane p rotein complex. Localization of defined epitopes on the cytochrome be, complex by immunoelectron microscopy clearly demonstrates that the N- terminus of the cytochrome b subunit is exposed to the periplasmic spa ce. This finding is in agreement with a nine-transmembrane-helices top ology model (I-IX) as predicted before for cytochrome b. However, due to other published evidence we favour the existence of an additional t ransmembrane helix (helix 0) complementing a more recently published e ight-helices model (A-C,cd, D-H), at least for prokaryotes.