Complete amino acid sequence of the cytochrome subunit and amino-terminal sequence of the flavin subunit of flavocytochrome c (sulfide dehydrogenase) from Chlorobium thiosulfatophilum

The complete amino acid sequence of the 86-residue heme subunit of flavocytochrome c (sulfide dehydrogenase) from the green phototrophic bacterium Chlorobium thiosulfatophilum strain Tassajara has been determined as follows: APEQSKSIPRGEILSLSCAGCHGTDGKSESIIPTIYGRSAEYIESALLDFKSGA- RPSTVMGRHAKGYSDEEIHQIAEYFGSLSTMNN. The subunit has a single heme-binding site near the N terminus, consisting of a pair of cysteine residues at positions 18 and 21. The out-of-plane ligands are apparently contributed by histidine 22 and methionine 60. The molecular weight including heme is 10,014. The heme subunit is apparently homologous to small cytochromes c by virtue of the location of the heme-binding site and its extraplanar ligands. However, the amino acid sequence is closer to Paracoccus sp. cytochrome c554(548) (37%) than it is to the heme subunit from Pseudomonas putida p-cresol methylhydroxylase flavocytochrome c (20%). The flavocytochrome c heme subunit is only 14% similar to the small cytochrome c555 also found in Chlorobium. Secondary structure predictions suggest N- and C-terminal helices as expected, but the midsection of the protein probably folds somewhat differently from the small cytochromes of known three-dimensional structure such as Pseudomonas cytochrome c551. Analyses of the residues near the exposed heme edges of the cytochrome subunits of P. putida and C. thiosulfatophilum flavocytochromes c (assuming homology to proteins of known structure) indicate that charged residues are not conserved, suggesting that electrostatic interactions are not involved in the association of the heme and flavin subunits. The N-terminal sequence of the flavoprotein subunit of flavocytochrome has also been determined. It shows no similarity to the comparable region of the p-cresol methylhydroxylase flavoprotein subunit from P. putida. The flavin-binding hexapeptide, isolated and sequenced earlier (Kenney, W. C., McIntire, W., and Yamanaka, T. (1977) Biochim. Biophys. Acta 483, 467-474), is situated at positions 40-46.     

The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822).     

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.     

Sequence of the gene encoding flavocytochrome c from Shewanella putrefaciens: a tetraheme flavoenzyme that is a soluble fumarate reductase related to the membrane-bound enzymes from other bacteria.

Flavocytochrome c from the Gram-negative, food-spoiling bacterium Shewanella putrefaciens is a soluble, periplasmic fumarate reductase. We have isolated the gene encoding flavocytochrome c and determined the complete DNA sequence. The predicted amino acid sequence indicates that flavocytochrome c is synthesized with an N-terminal secretory signal sequence of 25 amino acid residues. The mature protein contains 571 amino acid residues and consists of an N-terminal cytochrome domain, of about 117 residues, with four heme attachment sites typical of c-type cytochromes and a C-terminal flavoprotein domain of about 454 residues that is clearly related to the flavoprotein subunits of fumarate reductases and succinate dehydrogenases from bacterial and other sources. A second reading frame that may be cotranscribed with the flavocytochrome c gene exhibits some similarity with the 13-kDa membrane anchor subunit of Escherichia coli fumarate reductase. The sequence of the flavoprotein domain demonstrates an even closer relationship with the product of the yeast OSM1 gene, mutations in which result in sensitivity to high osmolarity. These findings are discussed in relation to the function of flavocytochrome c.     

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.     

Preparation of subunits of flavocytochromes c derived from Chlorobium limicola f. thiosulfatophilum and Chromatium vinosum.

The subunits of Chlorobium limicala f. thiosulfatophilum cytochrome c-553 and of Chromatium vinosum cytochrome c-552 have been obtained. Chlorobium cytochrome c-553 is split into the cytochrome and flavoprotein subunits by treatment with trichloroacetic acid; after the cytochrome is precipitated by 1–2% trichloroacetic acid, the cytochrome subunit is extractable with buffer, while the flavoprotein subunit is not dissolved. The subunits of Chromatium cytochrome c-552 can not be obtained by the trichloroacetic acid-treatment. The flavoprotein subunit of the Chromatium cytochrome is obtained by isoelectric focusing in the presence of 6 M urea and 1% mercaptoethanol, while the cytochrome subunit is prepared by gel filtration in the presence of 6 M urea with Sephacryl S-200. Molecular weights of the cytochrome and flavoprotein subunits from the Chlorobium cytochrome are 11,000 and 47,000, respectively, while those of the two subunits from the Chromatium cytochrome are 21,000 and 46,000, respectively. The molecule of each flavocytochrome c is composed of one molecule of each of the cytochrome and flavoprotein subunits.     

Amino acid sequence of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis

The complete nucleotide sequence of the gene encoding the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequence are presented. The nucleotide sequence of the gene reveals the existence of a typical bacterial signal peptide of 20 amino acid residues which is not found in the mature cytochrome subunit. The gene encoding the cytochrome subunit is preceded by the gene encoding the M subunit. Both genes overlap by 1 bp. The mature cytochrome subunit consists of 336 amino acid residues; 73% of its amino acid sequence was confirmed by protein sequencing work. The mol. wt of the cytochrome subunit including the covalently bound fatty acids and the bound heme groups is 40 500. The internal sequence homology is low, despite the symmetric structure of the cytochrome subunit previously shown by X-ray crystallographic analysis of the intact photosynthetic reaction centre. Sequence homologies to other cytochromes were not found.     

Orientation of succinate dehydrogenase and cytochrome b558 in the Bacillus subtilis cytoplasmic membrane.

The orientation of the three subunits of the membrane-bound succinate dehydrogenase (SDH)-cytochrome b558 complex in Bacillus subtilis was studied in protoplasts ("right side out") and isolated membranes (random orientation), using immunoadsorption and surface labeling with [35S]diazobenzenesulfonate. Anti-SDH antibodies were adsorbed by isolated membranes but not by protoplasts. The SDH Mr 65,000 flavoprotein subunit was labeled with [35S]diazobenzenesulfonate in isolated membranes but not in protoplasts. The flavoprotein subunit is thus located on the cytoplasmic side of the membrane. The location of the SDH Mr 28,000 iron-protein subunit was not definitely established, but most probably the iron-protein subunit also is located on the cytoplasmic side of the membrane. Antibodies were not obtained to the hydrophobic cytochrome b558. The cytochrome was strongly labeled with [35S]diazobenzenesulfonate in protoplasts, and labeling was also obtained with isolated membranes. Cytochrome b558 is thus exposed on the outside of the membrane. In B. subtilis SDH binds specifically to cytochrome b558, which suggests that the cytochrome is exposed also on the cytoplasmic side of the membrane. The results obtained suggest that the B. subtilis SDH is exclusively located on the cytoplasmic side of the membrane where it is bound to cytochrome b558, which spans the membrane.     

Cloning and sequencing of the cDNA for a 13th different subunit (IHQ) of beef heart cytochrome c oxidase

The mammalian cytochrome c oxidase is a complex of 13 different subunits. We present the full amino acid sequence of the one remaining uncharacterized subunit, subunit IHQ in the nomenclature used here, VIIb in the numbering system of Kadenbach and colleagues (e.g. Kadenbach, B., and Merle, P. (1981) FEBS Lett. 135, 1-11). A partial protein sequence was obtained from the purified subunit isolated by gel filtration procedures. This information was used to synthesize an oligonucleotide probe which was then used to isolate a cDNA clone encoding the subunit. This cDNA for subunit IHQ is 480 base pairs long and encodes a polypeptide which is either 83 or 88 amino acids long, including an N-terminal leader sequence of either 27 or 32 residues. The molecular weight of the mature subunit IHQ is 6350 based on the amino acid sequence deduced from the gene. The leader sequence is typical of other mitochondrial target sequences in having several positively charged residues but no negatively charged side chains.     

The amino acid sequence of cytochrome c oxidase subunit VI from Saccharomyces cerevisiae

The complete amino acid sequence of the nuclearly coded cytochrome c oxidase subunit VI was determined for a genetically defined haploid strain of Saccharomyces cerevisiae. The subunit contains 108 amino acids, has Mr = 12,627, is acidic (net charge of -9.7 at pH 7) and is quite polar (polarity index, 50.9%). Distribution of charges within the polypeptide chain is highly non-random. The NH2- and COOH-terminal regions are predominantly acidic whereas an apolar and a basic region are found in the interior, Subunit VI shows between 28 and 40% sequence homology (depending on the method of alignment) with subunit V of bovine cytochrome c oxidase; since the yeast subunit VI lacks methionine and contains only a single histidine residue very close to the NH2 terminus, it is unlikely that either of the two subunits carries heme alpha in the native enzyme.     

Characterization of the 25-kilodalton subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: sequence similarity to the 24-kilodalton subunit of the flavoprotein fraction of mammalian complex I

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. Structural genes encoding the subunits of this enzyme complex constitute at least one gene cluster [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428]. The 25-kDa subunit (NQO2), which has been isolated from sodium dodecyl sulfate-polyacrylamide gels, is a polypeptide of this enzyme complex. The partial N-terminal amino acid sequence and amino acid composition of the NQO2 subunit have been determined. On the basis of the amino acid sequence, the NQO2 gene was found to be located 1.7 kilobase pairs upstream of the gene for NADH-binding subunit (NQO1). The complete nucleotide sequence of the NQO2 gene was determined. It is composed of 717 base pairs and codes for 239 amino acid residues with a calculated molecular weight of 26,122. The NQO2 subunit is homologous to the Mr 24,000 subunit of the mammalian mitochondrial NADH-ubiquinone oxidoreductase which bears an electron paramagnetic resonance-visible binuclear iron-sulfur cluster (probably cluster N1b). Comparison of the predicted amino acid sequence of the Paracoccus NQO2 subunit with those of its mammalian counterparts suggests putative binding sites for the iron-sulfur cluster. In addition, nucleotide sequencing shows the presence of two unidentified reading frames between the NQO1 and NQO2 genes. These are designated URF1 and URF2 and are composed of 261 and 642 base pairs, respectively. The possible function of the protein coded for the URF2 is discussed.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

Isolation and partial amino acid sequence of three subunit species of porcine spleen ferritin: evidence of multiple H subunits

A partial amino acid sequence for three different subunits of the iron storage protein, ferritin, has been determined. Ferritin (Mr approximately 480,000) was isolated from porcine spleen and dissociated into its component subunits (Mr approximately 20,000). The subunits, in turn, were separated into three fractions by reversed-phase HPLC. The fractions appeared to be of equal size by sedimentation velocity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and size-exclusion chromatography in 6 M guanidinium chloride. All three fractions were shown to be monomeric and to have no covalently attached carbohydrate (J. F. Collawn et al. (1984) Arch. Biochem. Biophys. 233, 260-266). Determination of the amino acid sequence of the C-terminal 70-80 residues from each of the fractions demonstrated three different sequences. Comparison with human liver H and L subunit sequences indicates that two of the porcine ferritin subunits are H-type subunits and one is an L-type subunit. Application of the Chou-Fasman algorithm on the three partial sequences suggests that these respective regions from each of the three subunits would probably adopt the same conformation.     

Covalent structure of the flavoprotein subunit of the flavocytochrome c: sulfide dehydrogenase from the purple phototrophic bacterium Chromatium vinosum.

The amino acid sequence of the flavoprotein subunit of Chromatium vinosum flavocytochrome c-sulfide dehydrogenase (FCSD) was determined by automated Edman degradation and mass spectrometry in conjunction with the three-dimensional structure determination (Chen Z et al., 1994, Science 266:430-432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass, including FAD, of 43,568 Da, compared with a mass of 43,652 +/- 44 Da measured by LDMS. There are six cysteine residues, among which Cys 42 provides the site of covalent attachment of the FAD. Cys 161 and Cys 337 form a disulfide bond adjacent to the FAD. The flavoprotein subunit of FCSD is most closely related to glutathione reductase (GR) in three-dimensional structure and, like that protein, contains three domains. However, approximately 20 insertions and deletions are necessary for alignment and the overall identity in sequence is not significantly greater than for random comparisons. The first domain binds FAD in both proteins. Domain 2 of GR is the site of NADP binding, but has an unknown role in FCSD. We postulate that it is the binding site for a cofactor involved in oxidation of reduced sulfur compounds. Domains 1 and 2 of FCSD, as of GR, are homologous to one another and represent an ancient gene doubling. The third domain provides the dimerization interface for GR, but is the site of binding of the cytochrome subunit in FCSD. The four functional entities, predicted to be near the FAD from earlier studies of the kinetics of sulfite adduct formation and decay, have now been identified from the three-dimensional structure and the sequence as Cys 161/Cys 337 disulfide, Trp 391, Glu 167, and the positive end of a helix dipole.     

The most conserved nuclear-encoded polypeptide of cytochrome c oxidase is the putative zinc-binding subunit: primary structure of subunit V from the slime mold Dictyostelium discoideum.

A full-length 515 base pairs cDNA for cytochrome c oxidase subunit V of D. discoideum was isolated from a lambda gt11 expression library. The encoded polypeptide, whose identity was confirmed by partial protein sequencing, is 119 amino acids long (Mr = 13,352) and does not contain a cleavable presequence. The protein, which is homologous to human subunit Vb and yeast subunit IV, exhibits the highest degree of sequence conservation found among nuclear-encoded subunits of cytochrome c oxidase from distantly related organisms. All the invariant residues are clustered in two regions of the C-terminus which include the putative amino acids involved in the coordination of the Zn ion tightly associated to eukaryotic oxidase.