Abstract
Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity.
Highlights
Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with presequences for targeting into mitochondria [1]
Three types of processing peptidases are involved in removal of the presequence from precursors: mitochondrial processing peptidase (MPP)1 [2], mitochondrial intermediate peptidase [3], and inner membrane protease I [4, 5]
Based on the three-dimensional structure of this complex [29, 30], the lack of MPP activity in the crystalline bovine complex was thought to be due to the binding of an inhibitor polypeptide to the active site of MPP, which is located at the interface of core I and core II [28]
Summary
Indicated by the loss of its electron transfer activity, weakens the binding of inhibitor polypeptide (subunit IX) to the active site of MPP in core subunits and restores MPP activity [28]. The cleavage site specificity of activated MPP/bovine bc depends more on the length of the amino acid sequence in the mature protein portion than on that in the presequence portion, when a synthetic peptide composed of N-terminal residues of mature protein and C-terminal residues of its presequence is used as a substrate [28]. This finding is inconsistent with the present popular speculation that substrate recognition by MPP requires only structural elements in the presequence [35,36,37,38,39,40,41]. The structural importance of Tyr of core I in reconstitution is investigated
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