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.     

Gene cluster of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans: characterization of four structural gene products.

In previous reports from our laboratory, the three structural genes (NQO1, NQO2, and NQO3) of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans were characterized [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428; (1991) Biochemistry 30, 8678-8684; (1992) Arch. Biochem. Biophys. 296, 40-48]. In this report, the four structural genes NQO4, NQO5, NQO6, and NQO7 of the same Paracoccus denitrificans oxidoreductase were cloned and sequenced. On the basis of sequence homology and immunological cross-reactivity, these genes encode counterparts of the 49-, 30-, and 20-kDa polypeptides and the mitochondrial DNA ND3 polypeptides of bovine mitochondrial complex I. These seven structural genes were found to be located in the same gene cluster. The order of the seven structural genes of the Paracoccus NADH-quinone oxidoreductase in the gene cluster is NQO7, NQO6, NQO5, NQO4, NQO2, NQO1, and NQO3. Upstream of the NQO7 gene, an open reading frame encoding a predicted polypeptide homologous to the UV repair enzyme A of Escherichia coli and Micrococcus lysodeikticus was detected. The 5'-terminus of the gene cluster carrying the Paracoccus NADH-quinone oxidoreductase was studied, and the possible promoter region is discussed. The NQO4 and NQO5 genes appear to code for the M(r) 48,000 and 21,000 polypeptides of the isolated Paracoccus NADH dehydrogenase complex [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311] on the basis of amino acid analyses and N-terminal protein sequence analyses. The antisera to the bovine complex I 49- and 30-kDa polypeptides cross-reacted with the Paracoccus 48- and 21-kDa subunits, respectively.     

The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: gene cloning and deduced primary structure

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. The NADH-binding subunit (Mr = 50,000) of this enzyme complex was identified by direct photoaffinity labeling with [32P]NADH [Yagi, T., & Dinh, T.M. (1990) Biochemistry 29, 5515-5520]. Primers were synthesized on the basis of the N-terminal amino acid sequence of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the alpha subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex I were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.     

Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. When the Paracoccus NADH dehydrogenase complex was irradiated by UV light in the presence of [adenylate-32P]NAD, radioactivity was incorporated exclusively into one of three polypeptides of Mr approximately 50,000. Similar results were obtained when [adenylate-32P]NADH was used. The labeling of the Mr 50,000 polypeptide was diminished when UV irradiation of the enzyme with [adenylate-32P]NAD was performed in the presence of NADH, but not in the presence of NADP(H). The labeled polypeptide was isolated by preparative sodium dodecyl sulfate gel electrophoresis and was shown to cross-react with antiserum to the NADH-binding subunit (Mr = 51,000) of bovine NADH-ubiquinone oxidoreductase. Its amino acid composition was also very similar to that of the bovine NADH-binding subunit. These chemical and immunological results indicate that the Mr 50,000 polypeptide is an NADH-binding subunit of the Paracoccus NADH dehydrogenase complex.     

Characteristics of the aerobic respiratory chains of the microaerophiles Campylobacter jejuni and Helicobacter pylori

The respiratory chain enzymes of microaerophilic bacteria should play a major role in their adaptation to growth at low oxygen tensions. The genes encoding the putative NADH:quinone reductases (NDH-1), the ubiquinol:cytochrome c oxidoreductases (bc1 complex) and the terminal oxidases of the microaerophiles Campylobacter jejuni and Helicobacter pylori were analysed to identify structural elements that may be required for their unique energy metabolism. The gene clusters encoding NDH-1 in both C. jejuni and H. pylori lacked nuoE and nuoF, and in their place were genes encoding two unknown proteins. The NuoG subunit in these microaerophilic bacteria appeared to have an additional Fe-S cluster that is not present in NDH-1 from other organisms; but C. jejuni and H. pylori differed from each other in a cysteine-rich segment in this subunit, which is present in some but not all NDH-1. Both organisms lacked genes orthologous to those encoding NDH-2. The subunits of the bc1 complex of both bacteria were similar, and the Rieske Fe-S and cytochrome b subunits had significant similarity to those of Paracoccus denitrificans and Rhodobacter capsulatus, well-studied bacterial bc1 complexes. The composition of the terminal oxidases of C. jejuni and H. pylori was different; both bacteria had cytochrome cbb3 oxidases, but C. jejuni also contained a bd-type quinol oxidase. The primary structures of the major subunits of the cbb3-type (terminal) oxidase of C. jejuni and H. pylori indicated that they form a separate group within the cbb3 protein family. The implications of the results for the function of the enzymes and their adaptation to microaerophilic growth are discussed.     

The amino acid sequences of two 13 kDa polypeptides and partial amino acid sequence of 30 kDa polypeptide of complex I from bovine heart mitochondria: possible location of iron-sulfur clusters

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated system in the respiratory chain. It consists of many subunits, some of which hold iron-sulfur clusters, but structural information is still limited. The amino acid sequences of two 13 kDa polypeptides, 13 kDa-A and 13 kDa-B polypeptides, of iron-sulfur protein fraction (IP) of bovine heart mitochondrial complex I were determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion. The 13 kDa-A polypeptide was composed of 96 amino acids with a molecular weight of 10,536. The 13 kDa-B polypeptide consisted of 114 amino acids and had an acetylated amino terminus. The molecular weight of this protein was calculated to be 13,130 including the acetyl group. These proteins had no obvious sequence similarity to other known proteins. The partial amino acid sequence of 30 kDa-B polypeptide of IP was also determined to reveal a characteristic arrangement of cysteine residues that could be involved in iron-sulfur cluster formation.     

Mitochondrial NADH-ubiquinone reductase: complementary DNA sequences of import precursors of the bovine and human 24-kDa subunit

The 24-kDa subunit of mitochondrial NADH-ubiquinone reductase (complex I) is an iron-sulfur protein that is present in the flavoprotein or NADH dehydrogenase II subcomplex. It is a nuclear gene product and is imported into the organelle. A group of human patients with mitochondrial myopathy have been shown to have reduced levels of subunits of complex I in skeletal muscle mitochondria, and in one patient the 24-kDa subunit appears to be absent (Schapira et al., 1988). To investigate the genetic basis of this type of myopathy, cDNA clones have been isolated from a bovine library derived from heart and liver mRNA by hybridization with two mixtures of 48 synthetic oligonucleotides 17 bases in length that were designed on the basis of known protein sequences. The recombinant DNA sequence has been determined, and it encodes a precursor of the mature 24-kDa protein. The N terminus of the mature protein is preceded by a presequence of 32 amino acids that has properties that are characteristic of mitochondrial import sequences. The sequence of the mature protein deduced from the cDNA contains a segment of nine amino acids that was not determined in an earlier partial protein sequence analysis. The bovine clone has been employed as a hybridization probe to identify cDNA clones of the human homologue of the 24-kDa protein. Its DNA sequence has also been determined, and it codes for a protein that is closely related to the bovine protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR signals assigned to Fe/S cluster N1c of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) derive from cluster N1a

The proton-pumping NADH:ubiquinone oxidoreductase, which is also called respiratory complex I, transfers electrons from NADH to ubiquinone via one flavin mononucleotide (FMN) and up to nine iron-sulfur clusters. A structural minimal form of complex I consisting of 14 different subunits called NuoA to NuoN (or Nqo1 to Nqo14) is found in bacteria. The isolated Escherichia coli complex I can be split into a NADH dehydrogenase fragment, a connecting fragment, and a membrane fragment. The soluble NADH dehydrogenase fragment represents the electron input part of the complex and consists of the subunits NuoE, F, and G. The FMN and four iron-sulfur clusters have been detected in this fragment by means of EPR spectroscopy. One of the EPR signals, called N1c, has spectral properties, which are not found in preparations of the complex from other organisms. Therefore, it is attributed to an additional binding motif on NuoG, which is present only in a few bacteria including E. coli. Here, we show by means of EPR spectroscopic analysis of the NADH dehydrogenase fragment containing site-directed mutations on NuoG that the EPR signals in question derived from cluster N1a on NuoE. The mutations in NuoG disturbed the assembly of the overproduced NADH dehydrogenase fragment indicating that a yet undetected cluster might be bound to the additional motif. Thus, there is no third binuclear iron-sulfur "N1c" in the E. coli complex I but an additional tetranuclear cluster that may be coined N7.     

Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.     

Exploring the membrane domain of the reduced nicotinamide adenine dinucleotide-quinone oxidoreductase of Paracoccus denitrificans: characterization of the NQO7 subunit

The proton-translocating reduced nicotinamide adenine dinucleotide- (NADH-) quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 different subunits (NQO1-14). In addition, this enzyme complex houses one flavin mononucleotide (FMN) and 7-8 iron-sulfur clusters as cofactors. The expression and partial characterization of the NQO7 subunit, one of the seven subunits that constitute the hydrophobic sector of the enzyme complex, have been performed and are reported here. Expression of the NQO7 subunit was achieved by use of the glutathione-S-transferase (GST) fusion system together with Escherichia coli strains BLR(DE3)pLysS and BL21(DE3)pLysS. The GST-fused NQO7 subunit was expressed in the membrane fraction of the host cells and was extracted from the membranes by nonionic detergents (Triton X-100, dodecyl maltoside). The extracted polypeptide was purified by glutathione affinity column chromatography and characterized. The isolated GST-fused NQO7 subunit (but not the GST alone) was determined to interact with phospholipid vesicles and suppress the membrane fluidity. Antibodies against both the N- and C-terminal regions of the deduced primary structure of the NQO7 subunit reacted with a single band (15 kDa) of the Paracoccus membranes. By use of immunochemical and cysteine residue modification techniques, the topology of the Paracoccus NQO7 subunit in the membranes has been examined. The data suggest that the Paracoccus NQO7 subunit contains three transmembrane segments and that its N- and C-terminal regions are directed toward the cytoplasmic and periplasmic phases of the membrane, respectively. The proposed topology of the GST-fused NQO7 subunit expressed in E. coli membranes is consistent with that of the NQO7 subunit in the Paracoccus membranes.     

The 30-kilodalton subunit of bovine mitochondrial complex I is homologous to a protein coded in chloroplast DNA

In cattle, 7 of the 30 or more subunits of the respiratory enzyme NADH:ubiquinone reductase (complex I) are encoded in mitochondrial DNA, and potential genes (open reading frames, orfs) for related proteins are found in the chloroplast genomes of Marchantia polymorpha and Nicotiana tabacum. Homologues of the nuclear-coded 49- and 23-kDa subunits are also coded in chloroplast DNA, and these orfs are clustered with four of the homologues of the mammalian mitochondrial genes. These findings have been taken to indicate that chloroplasts contain a relative of complex I. The present work provides further support. The 30-kDa subunit of the bovine enzyme is a component of the iron-sulfur protein fraction. Partial protein sequences have been determined, and synthetic oligonucleotide mixtures based on them have been employed as hybridization probes to identify cognate cDNA clones from a bovine library. Their sequences encode the mitochondrial import precursor of the 30-kDa subunit. The mature protein of 228 amino acids contains a segment of 57 amino acids which is closely related to parts of proteins encoded in orfs 169 and 158 in the chloroplast genomes of M. polymorpha and N. tabacum. Moreover, the chloroplast orfs are found near homologues of the mammalian mitochondrial genes for subunit ND3. Therefore, the plant chloroplast genomes have at least two separate clusters of potential genes encoding homologues of subunits of mitochondrial complex I. The bovine 30-kDa subunit has no extensive sequences of hydrophobic amino acids that could be folded into membrane-spanning alpha-helices, and although it contains two cysteine residues, there is no clear evidence in the sequence that it is an iron-sulfur protein.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human NADH dehydrogenase

The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of the human respiratory chain NADH dehydrogenase has been investigated to test the possibility that any of them is synthesized in mitochondria. For this purpose, antibodies specific for individual subunits of the bovine enzyme, which cross-reacted with the homologous human subunits in immunoblot assays, were tested against HeLa cell mitochondrial proteins labeled in vivo with [35S]methionine in the absence or presence of inhibitors of mitochondrial or cytoplasmic protein synthesis. The results clearly indicated that all the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human complex I are synthesized in the cytosol and are, therefore, encoded in nuclear genes.     

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.     

H(+)-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans. Studies on topology and stoichiometry of the peripheral subunits.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane. In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P. denitrificans NDH-1 in the membranes were investigated using immunological techniques. Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes. Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex. Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic N-terminal stretch plays an important role in such an association. In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed. In addition, determination of stoichiometry of the peripheral subunits of the P. denitrificans NDH-1 was completed by radioimmunological methods. All the peripheral subunits are present as one molecule each in the enzyme complex. These results estimated the total number of cofactors in the P. denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2x[2Fe-2S] and 6x[4Fe-4S], provided that the NQO6 subunit bears one [4Fe-4S] cluster.     

The NADH-binding subunit of respiratory chain complex I is nuclear-encoded in plants and identified only in mitochondria

In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato ( Solanum tuberosum ). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers.
Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a ΔΨ-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific crossreactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.     

The genes of the Paracoccus denitrificans bc1 complex. Nucleotide sequence and homologies between bacterial and mitochondrial subunits

The genes for the three subunits of the cytochrome bc1 complex from the bacterium Paracoccus denitrificans were identified by screening a gene library constructed in pBR 322 for expression using a cytochrome c1-specific antibody. These three genes coding for the FeS subunit, cytochrome b, and cytochrome c1 were located on contiguous sites on the genome in a presumed operon arrangement. The DNA-deduced amino acid sequence shows that all three subunits are homologous to corresponding polypeptides of the mitochondrial cytochrome bc1 complex. Cytochrome c1 of Paracoccus is much larger than its mitochondrial counterpart due to an extra 150 amino acids of unique, highly acidic composition; in addition, it is most likely synthesized as a precursor polypeptide.     

Gene cluster of Rhodothermus marinus high-potential iron-sulfur Protein: oxygen oxidoreductase, a caa(3)-type oxidase belonging to the superfamily of heme-copper oxidases

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.     

Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid/iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila.

The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma). The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced. The cdhD gene is upstream of and separated by 3 bp from cdhE. Both genes are preceded by apparent ribosome-binding sites. Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD. The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum. The CdhE sequence had a four-cysteine motif with the potential to bind a 4Fe-4S cluster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy. A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli. The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration. The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom.