Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc 1 complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc 1 complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc 1 complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr 57 of core I is essential for reconstitutive activity.Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with presequences for targeting into mitochondria (1). These presequences are proteolytically removed during or after import of the precursors into mitochondria. Three types of processing peptidases are involved in removal of the presequence from precursors: mitochondrial processing peptidase (MPP) 1 (2), mitochondrial intermediate peptidase (3), and inner membrane protease I (4, 5).MPP cleaves all or part of the presequence as the initial processing step. Many proteins are mature after a one-step cleavage by MPP. Mitochondrial intermediate peptidase catalyzes a second-step cleavage in the two-step processing of some precursor proteins. The inner membrane protease I cleaves intermediate forms of proteins routed to the intermembrane space. The last two peptidases act sequentially after cleavage of the matrix targeting sequences by MPP. Thus, MPP plays an important role in the proteolytic processing of precursor proteins in the mitochondria.MPP is located in the matrix of fungal and mammalian mitochondria and in the inner membrane of plant mitochondria (2). Matrix-localized MPP has been studied extensively and purified to homogeneity from Neurospora crassa (6), Saccharomyces cerevisiae (7), and rat liver (8, 9). Purified, matrix-localized MPP contains two nonidentical subunits, ␣-MPP and ␤-MPP, each with molecular mass of around 50 kDa. The cDNAs encoding ␣-and ␤-MPP from these three sources have been cloned, sequenced (6, 7, 9 -13), and overexpressed in Escherichia coli cells (13-15). MPP is classified in the pitrilysin family (16) of zinc metalloproteases because of the presence of an inverted zinc binding motif, HXXEH 76 H, in the ␤-MPP (17). Processing activity requires both subunits because recombinant...

Recombinant subunit IV mutants which identify the regions essential for restoration of bc 1 activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized.

Previous studies indicate that the three-subunit cytochrome bc(1) core complex of Rhodobacter sphaeroides contains a fraction of the electron transfer activity of the wild-type enzyme. Addition of subunit IV to the core complex increases electron transfer activity to the same level as that of the wild-type complex. This activity increase may result from subunit IV preventing electron leakage, from the low potential electron transfer chain, and reaction with molecular oxygen, producing superoxide anion. This suggestion is based on the following observations: (1) the extent of cytochrome b reduction in the three-subunit core complex, by ubiquinol, in the presence of antimycin A, never reaches the same level as that in the wild-type complex; (2) the core complex produces 4 times as much superoxide anion as does the wild-type complex; and (3) when the core complex is reconstituted with subunit IVs having varying reconstitutive activities, the activity increase in reconstituted complexes correlates with superoxide production decrease and extent of cytochrome b reduction increase.

A region of subunit IV comprising residues 77-85 is identified as essential for interaction with the core complex to restore the bc(1) activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81-84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77-80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc(1) complex.

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.