Published: October 11, 2011 r2011 American Chemical Society 17824 dx.doi.org/10.1021/ja207065v | J. Am. Chem. Soc. 2011, 133, 17824–17831 ARTICLE pubs.acs.org/JACS A Proposal for Mitochondrial Processing Peptidase Catalytic Mechanism Orazio Amata, Tiziana Marino, Nino Russo, and Marirosa Toscano* Dipartimento di Chimica and Centro di Calcolo ad Alte Prestazioni per Elaborazioni Parallele e Distribuite-Centro d'Eccellenza MURST, Universita' della Calabria, I-87030 Arcavacata di Rende (CS), Italy b S Supporting Information ’ INTRODUCTION The process of growth and division of preexisting mitochon- dria that give rise to new mitochondria requires a constant supply of proteins. Most of these proteins destined for the mitochon- drial matrix or for the intermediate and inner membrane spaces are primarily synthesized as larger precursor polypeptides whose N-terminal extensions, which serve as the leader sequences, are proteolytically removed by specific processing peptidase en- zymes during or after the precursors have been imported into mitochondria. 1 Mitochondrial Processing Peptidase (MPP; EC 3.4.24.64) is a metallopeptidase that cleaves off most of the N-terminal presequences from precursor proteins at single sites, 2,3 while the inner membrane peptidase (IMP) and mitochondrial inter- mediate peptidase (MIP) process only some of precursor proteins. 4 Elimination of peptide chains presequences is required for proper protein folding: otherwise enzymes are less active and less stable thermodynamically. 4,5 The MPP, therefore, plays a crucial role in the life, hydrolyzing virtually all mitochondrial proteins from the cytoplasm. Many human diseases are related to lack of mitochondrial proteins encoded by the nucleus of the cell. 4À6 An alteration of the MPP feature leads to an accumulation of peptides precursors in the matrix that results in a slowing of cell growth and death. 7À9 The MPP is a heterodimer enzyme that was first identified in the mitochondria of Neurospora crassa, 10 yeast Saccharomyces cerevisiae, 11 rat liver, 12 and a few plants. 13,14 Afterward, its pres- ence was ascertained in many other organisms. 7,8,15À18 It consists of α- and β-subunits whose localization is different dependently on the considered organism. In plants, they are part of a bc1 respiratory chain complex. In yeast, one of the subunits is in the matrix while the other continues to be part of the above-mentioned bc1 complex. Finally, in animals and mushrooms they are present in the matrix. 7,8,10À18 Both α- and β-subunits, which a crystallographic study 19 has demonstrated to have nearly identical architecture, are essential for MPP processing activity. In fact, they act cooperatively to remove the presequences from the precursor proteins. In parti- cular, α-MPP participates in the substrate recognition through many binding sites negatively charged and present in its C-term- inal domain 19À24 and a highly conserved region rich in glycine and histidine residues, 4,19,25,26 and β-MPP is the catalytic subunit. 27 The β-subunit also contains many conserved amino acid residues that are important for substrate recognition. 3,28,29 Over- all, then, the two catalytic subunits form a cavity of negative charge. 12,14,30 In addition, the walls of the cavity also contain β- sheet structures that make up a scaffold for the binding of substrate via hydrogen bonds. 14,29 This fits well with the fact that the pres- equences have several residues both basic (positively charged) and hydroxylated, but very rarely acidic (negatively charged). The electrostatic interactions alone, however, do not explain the specificity of the catalytic action of MPP: it is estimated that, in mammals, the MPP processes to a single and clearly defined site over 1000 presequences which have little in common in terms of length (varying from 8 to about 69 amino acids) and primary struc- ture. The MPP, therefore, must recognize structural elements. 18,20 In particular, the presence of distal basic residues and of one proximal to the cleavage site, connected by a region of variable length but rather flexible, 20,28,29,31À34 seems to be very important. Many preproteins show, for the cleavage site, a pattern consisting of the sequence RxVΦΨ, where V indicates the bond to be hydrolyzed, R is an arginine residue, and Φ and Ψ are a hydrophobic hindered and a hydrophilic residue, respectively. 12,31À33 In some cases the arginine residue is located three amino acids Received: July 28, 2011 ABSTRACT: The reaction mechanism of the Mitochondrial Processing Peptidase enzyme (MPP) was investigated by using hybrid density functional theory. This enzyme removes the NH 2 -terminal targeting signals of nuclear-encoded mitochon- drial protein precursors in the mitochondrial matrix. The catalytic process was studied using a model for the active site consisting of 161 atoms locating all the stationary points on the potential energy curve and determining the main energetic, structural, and electronic features that drive the catalysis. Despite the differences between the B3LYP and MPWB1K descriptions, it is possible hypothesize that the rate-limiting step of the reaction is most likely the nucleophilic attack of zinc-bound hydroxide to a carbonyl carbon of the substrate. The results allowed assignment of the proper roles to some active site residues in this mechanism.