ACTIVATION OF A MATRIX PROCESSING PEPTIDASE FROM THE CRYSTALLINE CYTOCHROME BC(1) COMPLEX OF BOVINE HEART-MITOCHONDRIA

No mitochondrial processing peptidase (MPP) activity is detected in cr ystalline bovine heart mitochondrial cytochrome be, complex, which pos sesses full electron transfer activity. However, when the complex is t reated with increasing concentrations of Triton X-100 at 37 degrees C, the electron transfer activity decreases, whereas peptidase activity increases. Maximum MPP activity is obtained when the electron transfer activity in the complex is completely inactivated with 1.5 mar of Tri ton X-100, This result supports our suggestion that the lack of MPP ac tivity in the mammalian cytochrome be, complex is because of binding o f an inhibitor polypeptide to the active site of MPP located at the in terface of core subunits I and II. This suggestion is based on the thr ee-dimensional structural information for the be, complex and the sequ ence homology between subunits of MPP and the core subunits of the bee f complex. Triton X-100, at concentrations that disrupt the structural integrity of the be, complex as indicated by the loss of its electron transfer activity, weakens the binding of inhibitor polypeptide to th e active site of MPP in core subunits, thus activating MPP, The Triton X-100-activated MPP is pH-, buffer system-, ionic strength-, and temp erature-dependent. Maximum activity is observed with an assay mixture containing 15 mM Tris-HCl buffer at neutral pH (6.5-8.5) and at 37 deg rees C, Activated MPP is completely inhibited by metal ion chelators s uch as EDTA and o-phenanthroline and partially inhibited by myxothiazo l (58%), ferricyanide (28%), and dithiothreitol (81%). The metal ion c helator-inhibited activity can be partially restored by the addition o f divalent cations such as Zn2+ (68%), Mg2+ (44%), Mn2+ (54%), Co2+ (6 2%), and Fe2+ (92%), indicating that metal ion is required for MPP act ivity. The cleavage site specificity of activated MPP depends more on the length of amino acid sequence from the mature protein portion and less on the presequence portion, when a synthetic peptide composed of NH2-terminal residues of a mature protein and the COOH-terminal residu es of its presequence is used as a substrate.