Nucleotide sequence of the gene coding for cytochrome oxidase subunit I from the thermophilic bacterium PS3

The gene coding for cytochrome oxidase subunit I (COI) was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminal of the COI protein was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequence of COI protein is composed of 536 amino acid residues and its molecular mass is 59,510. The protein is clearly homologous to the corresponding subunit in the mitochondrial cytochrome oxidase and similarly appears to have 12 trans-membrane segments. The proposed ligands to two hemes (cytochrome aa3) and a copper atom (CuB) in this protein (Holm et al. (1987) EMBO J. 6, 2819-2823) are conserved in the sequence.     

Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.     

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.     

Determination of the ligands of the low spin heme of the cytochrome o ubiquinol oxidase complex using site-directed mutagenesis.

The cytochrome o complex of Escherichia coli is a ubiquinol oxidase which is the predominant respiratory terminal oxidase when the bacteria are grown under high oxygen tension. The amino acid sequences of three of the subunits of this quinol oxidase reveal a substantial relationship to the aa3-type cytochrome c oxidases. The two cytochrome components (b563.5 and o) and the single copper (CuB) present in the E. coli quinol oxidase appear to be equivalent to cytochrome a, cytochrome a3, and CuB of the aa3-type cytochrome c oxidases, respectively. These three prosthetic groups are all located within subunit I of the oxidase. Sequence alignments indicate only six totally conserved histidine residues among all known sequences of subunit I of the cytochrome c oxidases of various species plus the E. coli quinol oxidase. Site-directed mutagenesis has been used to change each of these totally conserved histidines with the presumption that two of these six must ligate to the low spin cytochrome center of the E. coli oxidase. The presence of the low spin cytochrome b563.5 component of the oxidase can be evaluated both by visible absorbance properties and by its EPR spectrum. The results unambiguously indicate that His-106 and His-421 are the ligands of the six-coordinate low spin cytochrome b563.5. Although the data are not definitive in making additional metal ligation assignments of the remaining four totally conserved histidines, a reasonable model is suggested for the structure of the catalytic core of the cytochrome o complex and, by extrapolation, of cytochrome c oxidase.     

Molecular cloning of the cytochrome aa3 gene from the archaeon (Archaebacterium) Halobacterium halobium.

A novel aa3-type cytochrome oxidase from the extremely halophilic archaeon, Halobacterium halobium, differs significantly from those of other prokaryotic and eukaryotic cytochrome oxidases (Fujiwara, T., Fukumori, Y., and Yamanaka, T. (1989) J. Biochem. 105, 287-292). In the present study, we cloned and sequenced the gene which encodes the cytochrome aa3 by using the polymerase chain reaction methods. The deduced amino acid sequence of subunit I of H. halobium cytochrome aa3 was more similar to that of subunit I of the eukaryotic cytochrome (44%, maize mitochondria) than that of the cytochrome from other bacteria (36%, Paracoccus denitrificans). The consensus sequence in putative metal binding residues is well-conserved also in H. halobium cytochrome aa3.     

The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli. This enzyme catalyzes the oxidation of ubiquinol-8 to ubiquinone-8 within the cytoplasmic membrane and the concomitant reduction of O2 to H2O. The hydropathy profiles of the deduced amino acid sequences suggest that all five of the gene products of the cyo operon contain multiple membrane-spanning helical segments. The goal of this work was to obtain experimental evidence for the topology of the five gene products in the cytoplasmic membrane by using the technique of gene fusions. A number of random gene fusions were generated in vitro encoding hybrid proteins in which the amino-terminal portion was provided by the subunit of interest and the carboxyl-terminal portion by one of two sensor proteins, alkaline phosphatase lacking its signal sequence or beta-galactosidase. Results obtained are self-consistent, and topological models are proposed for all of the five gene products encoded by the cyo operon. Based on the sequence similarities with subunits of the aa3-type cytochrome c oxidases, the experimental evidence obtained here can be used to infer topological models for the mitochondrial encoded subunits of the eukaryotic cytochrome c oxidases.     

Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.     

A novel double heme substitution produces a functional bo3 variant of the quinol oxidase aa3 of Bacillus cereus. Purification and paratial characterization

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3. The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg-1 protein. The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa3 oxidase. The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 microM, a Vmax of 21 micromol of O2 min-1mg-1, and a calculated turnover of 55 s-1. The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 microM, respectively. Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.     

Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides.

It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R. sphaeroides. This correlates with a second wild-type R. sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase. Subunit I of the heme-copper oxidase superfamily contains the binuclear center. Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species. The polymerase chain reaction was used to show that the chromosome of R. sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome aa3 in Haloferax volcanii

A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN(-) complex spectrum that indicates the presence of heme a and heme a(3). This cytochrome aa(3) consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa(3), providing physiological evidence for electron transfer from cytochrome c to cytochrome aa(3) in archaea.     

The Bacillus subtilis cytochrome-c oxidase. Variations on a conserved protein theme.

The structural genes of cytochrome-c oxidase in Bacillus subtilis have been isolated and sequenced. Five genes, ctaB-F, are closely spaced. ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively, ctaB, which may encode an assembly factor, is separated and upstream from the others. In comparison to its mitochondrial counterparts, subunit I has an extended C-terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N-terminus. The C-terminal extension in subunit II is a covalent cytochrome-c domain, previously characterized only in the thermophilic oxidases. Subunit IVB, a small hydrophobic protein, is a novel subunit. These predictions suggest that the B. subtilis cytochrome-c oxidase is structurally more related to the four-subunit Escherichia coli cytochrome-bo complex than, for instance, to the Paracoccus denitrificans enzyme. Cytochrome aa3, which was previously isolated from B. subtilis [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103] is not encoded by the ctaC-F genes; thus, there seems to be two different cytochrome-aa3-type oxidases in this Gram-positive bacterium.     

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.     

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species.

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.     

The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast.

Yeast cytochrome c oxidase contains three large subunits made in mitochondria and at least six smaller subunits made in the cytoplasm. There is evidence that the catalytic centers (heme a and copper) are associated with the mitochondrially-made subunits, but the role of the cytoplasmically-made subunits has remained open. Using a gene interruption technique, we have now constructed a Saccharomyces cerevisiae mutant which lacks the largest of the cytoplasmically-made subunits (subunit IV). This mutant is devoid of cyanide-sensitive respiration, the absorption spectrum of cytochrome aa3 and cytochrome c oxidase activity. It still contains the other cytochrome c oxidase subunits but these are not assembled into a stable complex. Active cytochrome c oxidase was restored to the mutant by introducing a plasmid-borne wild-type subunit IV gene; no restoration was seen with a gene carrying an internal deletion corresponding to amino acid residues 28-66 of the mature subunit. Subunit IV is thus necessary for proper assembly of cytochrome c oxidase.     

黄河裸裂尻鱼细胞色素C氧化酶Ⅰ、Ⅱ和Ⅲ亚基基因的克隆及序列特征分析

采用RT-PCR技术克隆获得了黄河裸裂尻鱼(Schizopygopsis pylzovi)CO Ⅰ、Ⅱ、Ⅲ基因的编码序列,并对此进行了初步分析.结果表明,黄河裸裂尻鱼CO I基因全长为1 551 bp,开放阅读框(ORF)由基因全长组成,编码516个氨基酸;COⅡ基因全长为691 bp,开放阅读框为690 bp,编码2...     

Soluble CuA domain of cyanobacterial cytochrome c oxidase

The genomes of several cyanobacteria show the existence of gene clusters encoding subunits I, II, and III of aa(3)-type cytochrome c oxidase. The enzyme occurs on both plasma and thylakoid membranes of these oxygenic phototrophic prokaryotes. Here we report the expression and purification of a truncated subunit II copper A (Cu(A)) domain (i.e. the electron entry and donor binding site) of cytochrome c oxidase from the cyanobacterium Synechocystis PCC 6803 in high yield. The water-soluble purple redox-active bimetallic center displays a relatively low standard reduction potential of 216 mV. Its absorption spectrum at pH 7 is similar to that of other soluble fragments from aa(3)-type oxidases, but the insensitivity of both absorbance and circular dichroism spectra to pH suggests that it is less exposed to the aqueous milieu compared with other Cu(A) domains. Oxidation of horse heart cytochrome c by the bimetallic center follows monophasic kinetics. At pH 7 and low ionic strength the bimolecular rate constant is (2.1 +/- 0.3) x 10(4) m-1 s(-1), and the rates decrease upon the increase of ionic strength. Sequence alignment and modeling of cyanobacterial Cu(A) domains show several peculiarities such as: (i) a large insertion located between the second transmembrane region and the putative hydrophobic cytochrome c docking site, (ii) the lack of acidic residues shown to be important in the interaction between cytochrome c and Paracoccus Cu(A) domain, and (iii) an extended C terminus similar to Escherichia coli ubiquinol oxidase.     

Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151-158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a more widely distributed type including the B. stearothermophilus enzyme, suggesting that the latter type is evolutionarily older.     

Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase.

Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikstr?m, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605 cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B. subtilis.     

The cytochrome C oxidase genes in blue-green algae and characteristics of the deduced protein sequence for subunit II of the thermophilic cyanobacterium Synechococcus vulcanus.

Blue-green algae (cyanobacteria) contain both primitive photosynthetic and respiratory systems in their membranes. The controversial genes coding for an alpha alpha 3-type cytochrome oxidase in cyanobacteria were examined. The DNA probe coding for the most conserved part of subunit I hybridized with DNA fragments from four cyanobacterial species. We have cloned the genes coding for subunits I and II from the genomic library of the thermophilic cyanobacterium Synechococcus vulcanus and determined the nucleotide sequence of the subunit II gene. The deduced protein sequence (327 amino acid residues) indicates that there are two hydrophobic segments near the N-terminus and a hydrophilic intermembrane domain containing ligands for CuA (the ESR-active Copper) similar to other subunit IIs. The S. vulcanus subunit II does not contain the cytochrome c moiety that is present in bacilli and thermophiles.