Catalytic mechanism of mitochondrial processing peptidase: fluorescence studies

Abstract

Processing of nuclear-encoded precursor proteins by mitochondrial processing peptidase (MPP) is an essential step for their sorting and function in mitochondria. We report spectroscopic studies on the catalytic mechanism of Neurospora crassa MPP. It is a complex enzyme consisting of two different subunits termed alpha-and beta-MPP. Following changes in the protein intrinsic fluorescence we register and characterize a complex formation between (i) the alpha- and the beta-subunit of MPP, (ii) the two subunits and a precursor protein, and (iii) the two subunits and some metal ions. The presequence of the precursor protein was absolutely necessary for its binding to MPP subunits. Mn2+ ions in concentrations enhancing the processing activity did not influence the substrate binding, whereas EDTA in concentrations inhibiting the enzyme completely abolished the binding of the substrate to the MPP subunits. Both MPP subunits bind metal ions such as Mn2+, Mg2+, and Zn2+. beta-MPP interacts stronger with these ions but alpha-MPP-Mn2+ conjugates seem to be important for the processing activity.