Domains important for the activity of the heterodimeric mitochondrial processing peptidase (MPP) were investigated, by inserting one alanine residue at ten positions along the polypeptide chain of the beta-subunit (beta-MPP). An alanine residue inserted after Glu70, Ser114, Lys215 and Ser314 respectively, abolished the cleavage activity of MPP. When the alpha-subunit (alpha-MPP) was co-expressed with N-terminal hexa-histidine tagged beta-MPP, alpha-MPP was co-eluted from a nickel-derivatized affinity resin, with a 1:1 stochiometry, both with wild-type beta-MPP and with the mutants with alanine inserted after Ser114 and Ser314. The mutants with alanine inserted after Glu70 and Lys215 did not associate with alpha-MPP. The mutagenesis studies indicate that: (1) the whole HXXEHX76H region of beta-MPP is important for the proper conformation of the active site of MPP and may also be in contact with alpha-MPP; (2) the non-conserved central region surrounding Lys215 is involved in the interaction with alpha-MPP; and (3) the carboxy-terminal region of beta-MPP surrounding Ser314 is also of importance for the catalysis. Cross-linking studies indicated that purified alpha-MPP bound a precursor protein in the absence of any beta-MPP. Furthermore, the interaction of MPP and its subunits with a peptide substrate, as analyzed by surface plasmon resonance, showed that alpha-MPP bound a peptide substrate as efficiently as MPP. The data suggest that the alpha-subunit is responsible for the binding of mitochondrial presequences prior their presentation to the catalytic site of MPP.