Mitochondrial processing peptidase (MPP) consists of alpha- and beta-subunits (alpha-MPP and beta-MPP). beta-MPP has a putative metal-binding sequence (HXXEH). To determine whether the sequence of beta-MPP is essential for the enzymatic activity, we individually mutated the histidines and glutamic acid to arginines and glutamine, respectively. The wild-type and mutated beta-MPPs were co-expressed with alpha-MPP in Escherichia coli. All three mutants had completely lost the activity, whereas the lost activity was recovered on the addition of wild-type beta-MPP. The activity of the wild-type enzyme was reduced by the mutant beta-MPPs. We conclude from these observations that the HXXEH region is involved in the formation of the active site and that beta-MPP is the catalytic subunit of MPP.