No abstract

The electron carriers of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) are contained predominately in two extramembranous subcomplexes, a flavoprotein (FP) and an iron-sulfur protein (IP). FP contains three subunits with molecular masses of 51, 24, and 9 kDa. The 51-kDa subunit carries the NADH binding site and contains FMN and a tetranuclear iron-sulfur cluster. The 24-kDa subunit contains a binuclear iron-sulfur cluster. IP contains seven subunits with molecular masses of 75, 49, 30, 18, 15, 13, and 11 kDa. It contains a tetranuclear and very likely a binuclear iron-sulfur cluster in the 75-kDa subunit. FP and IP make contact through the 51- and the 75-kDa subunits. The remainder of complex I (hydrophobic protein (HP), 31 subunits) is largely membrane-intercalated and contains two iron-sulfur clusters apparently in a 23-kDa subunit and possibly another in a 20-kDa subunit. In this study, the stoichiometries of the FP and IP subunits in complex I were determined by radioimmunoassay. Per mole of complex I, there are 2 mol of the 15-kDa subunit and 1 mol each of the FP and the four largest IP subunits. The stoichiometries of the 13- and the 11-kDa subunits could not be determined separately, because they comigrate upon gel electrophoresis. In addition, the effect of substrates (NADH, NADPH, NAD, and NADH plus potassium ferricyanide to rapidly oxidize NADH via FP) on the cross-linking patterns of FP and IP subunits was investigated, using three different cross-linking reagents of different molecular lengths.(ABSTRACT TRUNCATED AT 250 WORDS)

Coenzyme Q (Q) functions in the electron transport chain of both prokaryotes and eukaryotes. The biosynthesis of Q requires a number of steps involving at least eight Coq polypeptides. Coq5p is required for the C-methyltransferase step in Q biosynthesis. In this study we demonstrate that Coq5p is peripherally associated with the inner mitochondrial membrane on the matrix side. Phenotypic characterization of a collection of coq5 mutant yeast strains indicates that while each of the coq5 mutant strains are rescued by the Saccharomyces cerevisiae COQ5 gene, only the coq5-2 and coq5-5 mutants are rescued by expression of Escherichia coli ubiE, a homolog of COQ5. The coq5-2 and coq5-5 mutants contain mutations within or adjacent to conserved methyltransferase motifs that would be expected to disrupt the catalysis of C-methylation. The steady state levels of the Coq5-2 and Coq5-5 mutant polypeptides are not decreased relative to wild type Coq5p. Two other polypeptides required for Q biosynthesis, Coq3p and Coq4p, are detected in the wild type parent and in the coq5-2 and coq5-5 mutants, but are not detected in the coq5-null mutant, or in the coq5-4 or coq5-3 mutants. The effect of the coq5-4 mutation is similar to a null, since it results in a stop codon at position 93. However, the coq5-3 mutation (G304D) is located just four amino acids away from the C terminus. While C-methyltransferase activity is detectable in mitochondria isolated from this mutant, the steady state level of Coq5p is dramatically decreased. These studies show that at least two functions can be attributed to Coq5p; first, it is required to catalyze the C-methyltransferase step in Q biosynthesis and second, it is involved in stabilizing the Coq3 and Coq4 polypeptides required for Q biosynthesis.Ubiquinone, or coenzyme Q (Q), 1 is a polyprenylated benzoquinone lipid that is a critical component of the electron transport pathways of both eukaryotes and prokaryotes (1). Q n consists of a hydrophobic isoprenoid tail and a quinone head group. The tail length (n) varies depending on the organism studied; Saccharomyces cerevisiae contains Q 6 , Escherichia coli contains Q 8 , and humans contain Q 10 . The tail anchors Q in the membrane, while the head group is responsible for the redox chemistry, undergoing reversible redox cycling between the quinone (Q), semiquinone, and hydroquinone (QH 2 ) forms. In eukaryotes Q is primarily associated with the inner mitochondrial membrane and is best known for its role in respiratory metabolism as a member of the electron transport chain shuttling electrons from Complex I (NADH:Q oxidoreductase) and Complex II (succinate:Q oxidoreductase) to Complex III (the cytochrome bc 1 complex) (2). QH 2 also acts as a lipid-soluble antioxidant, capable of scavenging lipid peroxyl radicals directly, or indirectly, by reducing ␣-tocopheroxyl radicals (3, 4). In the plasma membrane, Q participates in a trans-plasma membrane electron transport chain, in which intracellular NADH is oxidized and extracellular ascorbate free radicals ...

The well characterized subunits of the bovine ATP synthase complex are the ␣, ␤, ␥, ␦, and ⑀ subunits of the catalytic sector, F 1 ; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin sensitivityconferring protein), F 6 , and A6L, which are present in the membrane sector, F 0 , and the 45-Å-long stalk that connects F 1 to F 0 . It has been shown recently that bovine ATP synthase preparations also contain three small polypeptides, designated e, f, and g, with respective molecular masses of 8.2, 10.2, and 11.3 kDa. To ascertain their involvement as bona fide subunits of the ATP synthase and to investigate their membrane topography and proximity to the above ATP synthase subunits, polyclonal antipeptide antibodies were raised in the rabbit to the COOH-terminal amino acid residues 57-70 of e, 75-86 of f, and 91-102 of g. It was shown that (i) e, f, and g could be immunoprecipitated with anti-OSCP IgG from a fraction of bovine submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH 2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (iii) the COOH termini of all three polypeptides are exposed on the cytosolic side of the inner membrane; and (iv) f cross-links to A6L and to g, and e crosslinks to g and appears to form an e-e dimer. Thus, the bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.

Factor B is a subunit of the mammalian ATP synthase complex, whose existence has been controversial. This paper describes the molecular and functional properties of a recombinant human factor B, which when added to bovine submitochondrial particles depleted of their factor B restores the energy coupling activity of the ATP synthase complexes. The mature human factor B has 175 amino acids and a molecular mass of 20,341 Da. The preparation is water-soluble, monomeric, and is inactivated by monothiol-and especially dithiol-modifying reagents, probably reacting at its cysteine residues Cys-92 and Cys-94. A likely factor B gene composed of 5 exons has been identified on chromosome 14q21.3, and the functional role of factor B in the mammalian ATP synthase complex has been discussed.It is generally considered that the mammalian mitochondrial ATP synthase complex is composed of 16 unlike subunits (1-3). These subunits are ␣, ␤, ␥, ␦, and ⑀ in the catalytic F 1 domain; OSCP, a, b, c, d, e, f, g, F 6 and A6L in F O and stator; and the ATPase inhibitor protein, IF 1 , which binds reversibly to F 1 to inhibit ATP hydrolysis. In 1967, Sanadi and co-workers (4) showed that submitochondrial particles prepared by sonication from bovine mitochondria suspended in 0.25 M sucrose and 0.6 mM EDTA and adjusted to pH 9.0 with ammonium hydroxide lost considerable activity for respiration-driven ATP synthesis and ATP hydrolysis-driven electron transfer from succinate to NAD. Addition to the ammonia-EDTA-treated particles (AE-SMP) 1 of a partially purified soluble protein extracted from mitochondrial acetone powders partially restored these activities (4, 5). Sucrose density gradient centrifugation suggested a molecular mass of 32 kDa for the active peak of the soluble preparation, which was designated factor B (4, 5). By using bovine mitochondrial acetone powder extracts, we isolated a pure and monodisperse protein, which restored ATP synthasecoupled activities to AE-SMP (6). Its molecular mass as estimated from sedimentation equilibrium and gel filtration experiments was 11-12 kDa, and it was immunoprecipitated by Sanadi's anti-factor B antiserum in an Ouchterlony double diffusion experiment (7). Sanadi and co-workers (5, 8) revised the molecular mass of their preparation to 29.2 kDa and suggested that it is a dimer of monomer molecular mass of 14.6 kDa. They also obtained preparations of relative molecular mass of 13-15 and 47 kDa, which exhibited factor B-like activity (5, 9, 10), and in 1990 (11) they published the sequence of the 55 amino-terminal amino acids of a factor B preparation with a relative molecular mass of 22 kDa.The existence of factor B as a component of the ATP synthase complex remained controversial, however. Although Sanadi (5, 12) claimed that the ATP synthase complex prepared in his laboratory contained factor B, a thorough analysis of the polypeptide composition of an ATPase complex prepared in Walker's laboratory demonstrated the existence of the 16 unlike polypeptides mentioned above but no factor B (1, 2). T...