Abstract All three tryptophan residues in α-subunit of mitochondrial processing peptidase (MPP) were subsequently substituted. While substitutions of Trp 223 led to misfolded non-functional protein, mutations of Trp 147 and/or Trp 481 did not affect the enzyme processing activity. Thus, fluorescence properties of the mutants with fewer tryptophans were used for observation of both α-MPP domain translocation and visualization of conformational changes in the interdomain linker evoked by substrate. We found that in the presence of substrate the C-terminal penultimate Trp 481 was approaching Trp 223, which is localized at the border of N-terminal domain and interdomain linker. Also, excision of the α-MPP C-terminal 30 amino acid residues (ΔC30) led to a complete loss of protein function. Even shorter deletions of the α-MPP C-terminus destabilized the protein slightly (ΔC2) or dramatically (ΔC17). It suggests that the extreme C-terminus of α-MPP provides mechanical support to the C-terminal domain during its extensive conformational change accompanying the substrate recognition process.