Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redoxdriven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 3 Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8͞1.9-Å resolution in the fully oxidized͞ reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redoxcoupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.

X-ray structures of bovine heart cytochrome c oxidase have suggested that the enzyme, which reduces O 2 in a process coupled with a proton pumping process, contains a proton pumping pathway (H-pathway) composed of a hydrogen bond network and a water channel located in tandem across the enzyme. The hydrogen bond network includes the peptide bond between Tyr-440 and Ser-441, which could facilitate unidirectional proton transfer. Replacement of a possible proton-ejecting aspartate (Asp-51) at one end of the H-pathway with asparagine, using a stable bovine gene expression system, abolishes the proton pumping activity without influencing the O 2 reduction function. Blockage of either the water channel by a double mutation (Val386Leu and Met390Trp) or proton transfer through the peptide by a Ser441Pro mutation was found to abolish the proton pumping activity without impairment of the O 2 reduction activity. These results significantly strengthen the proposal that H-pathway is involved in proton pumping. mutagenesis ͉ mitochondrial import ͉ HeLa cell ͉ peptide bond ͉ keto-enol tautomerism

The mitochondrial respiratory chain is essential for oxidative phosphorylation and comprises multiple complexes, including cytochrome c oxidase, assembled in macromolecular supercomplexes. Little is known about factors that contribute to supercomplex organization. Here we identify COX7RP as a factor that promotes supercomplex assembly. Cox7rp-knockout mice exhibit decreased muscular activity and heat production failure in the cold due to reduced COX activity. In contrast, COX7RP-transgenic mice exhibit increased exercise performance with increased cytochrome c oxidase activity. Two-dimensional blue native electrophoresis reveals that COX7RP is a key molecule that promotes assembly of the III 2 /IV n supercomplex with complex I. Our study identified COX7RP as a protein that functions in I/III 2 /IV n supercomplex assembly and is required for full activity of mitochondrial respiration.

X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.

We have recently demonstrated that synthetic peptides modeled on the extension peptide of malate dehydrogenase can be a good substrate of mitochondrial processing peptidase and that arginine residues present at positions ؊2 or ؊3 and distant from the cleavage point were important for recognition by the enzyme (Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719 -24722). We further investigated the elements required for substrates of the protease. To analyze the reaction by a more rapid yet quantitative method, we have developed intramolecularly quenched fluorescent substrates. Using the fluorogenic substrates we demonstrated that at least one of the proline and glycine between the distal and proximal arginine residues was also important while other connecting sequences were dispensable. In addition, the protease showed considerable preference for aromatic and, to a lesser extent, hydrophobic amino acids in the P 1 -position. These results together with the previous data suggest that the proximal and distal arginine residues, proline and/or glycine between them, and P 1 amino acid could be critical determinants for the specific cleavage of the substrates by the protease.Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and transported into their correct mitochondrial component. The majority of them carry N-terminal extension peptides that target the protein molecules to the organelle. Mitochondrial processing peptidase (MPP) 1 is localized in the mitochondrial matrix and is responsible for proteolytic cleavage of the extension peptides after or during the transportation (1-3). The enzyme is a metalloprotease and forms a heterodimer consisting of structurally related ␣-and ␤-subunits (4 -11). We have recently demonstrated that the ␤-subunit is a catalytic one (12). MPP acts exclusively on the precursor forms of mitochondrial proteins, whose extension peptides are heterogeneous in sequence (13-15). The absence of apparent sequence homologies raises a question about how MPP specifically recognizes the extension peptides and cleaves them at a single site. Attempts have been made to solve the question, mainly by use of in vitro translated and radiolabeled mutants of mitochondrial precursors. By detecting mature forms from the in vitro translated precursors on SDS-polyacrylamide gel electrophoresis followed by fluorography, the processing activity has so far been evaluated. Those studies pointed out the importance of basic amino acids near the C terminus and neutral amino acids in the middle portion of the extension peptides (16 -20). The method, however, is time-consuming, and the obtained data are not quantitative. Thus the conventional method is unsuitable for kinetic analysis of the reaction. To overcome the limitation of the assay method, we developed a new method (21), which employed as the substrate synthetic peptides that were modeled on the extension peptide of rat malate dehydrogenase. This method enabled us to reveal the importance of argini...

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.

Mitochondrial processing peptidase, a metalloendopeptidase consisting of ␣-and ␤-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat ␤-subunit, which has a putative metal-binding motif, H 56 XXEH 60 , were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the ␣-subunit. Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with Nterminal extension peptides for targeting into mitochondria (1-3). During or after import of the precursors into mitochondria, the extension peptides are proteolytically removed by three types of processing peptidases. Mitochondrial processing peptidase (MPP 1 ; EC 3.4.24.64) (4 -8) generally cleaves off a large part of the extension peptide, including the mitochondrial matrix-targeting sequence as the initial step of the processing. Many precursors are converted into the mature forms by the one-step processing. The second enzyme is mitochondrial intermediate peptidase, which catalyzes second-step cleavage in the two-step processing of some precursor proteins (9, 10). These precursors are first cleaved by MPP, and then the residual octapeptides are removed by the mitochondrial intermediate peptidase. The last enzyme is inner membrane protease I, which processes the signal sequence for inner membrane and intermembrane space (11). The last two peptidases sequentially act after cleavage of the matrix targeting sequences by MPP. Thus, MPP plays an important role in proteolytical processing of precursors in mitochondria. MPP has been purified from mitochondria of Neurospora crassa (12), yeast Saccharomyces cerevisiae (13), rat liver (14), and a few plants (15,16). The enzymes, except for those of the plant, are soluble proteins in the mitochondrial matrix and consist of ␣ and ␤ subunits. The subunits from yeast and rat liver form a stable heterodimer, whereas Neurospora subunits could be separated from each other by gel filtration. Processing activity of the enzymes is sensitive to metal chelators, and the lost activity is restored by divalent metal ions (4 -7). MPP specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off the extension peptides at single sites (14,17,18). Contrary to the strict substrate specificity of the enzyme, amino acid sequences of the extension peptides are wide in length and poor in similarity (1). Many experimental observations have indicated that basic amino acid residues in the extension peptides were required for the effective processing by . A consensus sequence of the processing signals has...

Mitochondrial processing peptidase is a heterodimer consisting of ␣-mitochondrial processing peptidase (␣-MPP) and ␤-MPP. We investigated the role of ␣-MPP in substrate recognition using a recombinant yeast MPP. Disruption of amino acid residues between 10 and 129 of the ␣-MPP did not essentially impair binding activity with ␤-MPP and processing activity, whereas truncation of the C-terminal 41 amino acids led to a significant loss of binding and processing activity. Several acidic amino acids in the region conserved among the enzymes from various species were mutated to asparagine or glutamine, and effects on processing of the precursors were analyzed. Glu 353 is required for processing of malate dehydrogenase, aspartate aminotransferase, and adrenodoxin precursors. Glu 377 and Asp 378 are needed only for the processing of aspartate aminotransferase and adrenodoxin precursors, both of which have a longer extension peptide than the others studied. However, processing of the yeast ␣-MPP precursor, which has a short extension peptide of nine amino acids, was not affected by these mutations. Thus, effects of substitution of acidic amino acids on the processing differed with the precursor protein and depended on length of the extension peptides. ␣-MPP may function as a substrate-recognizing subunit by interacting mainly with basic amino acids at a region distal to the cleavage site in precursors with a longer extension peptide.Most mitochondrial proteins encoded in the nucleus are synthesized as precursor proteins with extension peptides at the N termini and are targeted to the mitochondria. After import of the precursors into the mitochondria, the extension peptides are proteolytically cleaved off by a matrix-located metallopeptidase, mitochondrial processing peptidase (MPP) 1 (1-3). Purification of MPP from various species demonstrated that this peptidase is a heterodimer consisting of ␣-and ␤-subunits (4 -6). Sequence analysis (7-14) revealed that subunits of MPP have significant sequence homology with a family of endopeptidases, the pitrilysin family, which includes Escherichia coli pitrilysin (also called protease III), the insulin-degrading enzymes from mammals and insects, and the N-arginine dibasic convertase from the rat (15). All members, except the ␣-MPPs, have a metal binding motif, His-Xaa-Xaa-Glu-His. The ␣-and ␤-subunits of the MPP are also homologous to core 2 and core 1 proteins, respectively, of the mitochondrial ubiquinol-cytochrome c oxidoreductase (bc 1 complex), a component of the respiratory chain (16). Plant MPP was purified as subunits of the bc 1 complex located in an inner membrane (16 -18). In Neurospora crassa, a core I protein of the bc 1 complex is identical with ␤-MPP in the matrix (19).Sequence analysis of extension peptides of mitochondrial protein precursors revealed that the peptides vary in length and sequence and are rich in positively charged amino acids among hydrophobic amino acids (20 -22). Studies using radiolabeled precursors and synthetic peptides as a substrate demonstra...