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N Sone

Publications and source records attributed to N Sone.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cytochrome bd-type quinol oxidase in a mutant of Bacillus stearothermophilus deficient in caa3-type cytochrome c oxidase.

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as a terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated B. stearothermophilus mutants defective in the caa3-type oxidase activity. One mutant contained little cytochrome a and had low cytochrome c oxidase activity. However, growth and the respiratory activity of membranes in the presence of NADH were close to normal, suggesting that the mutant contains an alternative electron transfer pathway. A novel oxidase was isolated from the membrane fraction of the mutant. The enzyme is a cytochrome bd-type quinol oxidase composed of two subunits of 52 and 40 kDa, whose N-terminal regions show sequence similarity to polypeptides of the bd-type oxidase from Escherichia coli and Azotobacter vinelandii. This is the first report of a bd-type terminal oxidase purified from a Gram-positive bacterium.

Amino Acid Sequence↗
PubMed1996

Bacillus stearothermophilus qcr operon encoding rieske FeS protein, cytochrome b6, and a novel-type cytochrome c1 of quinol-cytochrome c reductase.

The gcr of Bacillus stearothermophilus K1041 encoding three subunits of the quinol-cytochrome c oxidoreductase (cytochrome reductase, b6c1 complex) was cloned and sequenced. The gene (qcrA) for a Rieske FeS protein of 19,144 Da with 169 amino acid residues, and the gene (qcrC) for cytochrome c1 of 27,342 Da with 250 amino acid residues were found at adjacent upstream and downstream sides of the previously reported qcrB (petB) for cytochrome b6 of subunit 25,425 Da with 224 residues (Sone, N., Sawa, G., Sone, T., and Noguchi, S. (1995) J. Biol. Chem. 270, 10612-10617). The three structural genes for thermophilic Bacillus cytochrome reductase form a transcriptional unit. In the deduced amino acid sequence for the FeS protein, the domain including four cysteines and two histidines binding the 2Fe-2S cluster was conserved. Its N-terminal part more closely resembled the cyanobacteria-plastid type than the proteobacteria-mitochondria type when their sequences were compared. The amino acid sequence of cytochrome c1 was not similar to either type; the thermophilic Bacillus cytochrome c1 is composed of an N-terminal part corresponding to subunit IV with three membrane-spanning segments, and a C-terminal part of cytochrome c reminiscent of cytochrome c-551 of thermophilic Bacillus. The subunit IV in the enzyme of cyanobacteria and plastids is the counterpart of C-terminal part of cytochrome b of proteobacteria and mitochondria. These characteristics indicate that Bacillus cytochrome b6c1 complex is unique.

Amino Acid Sequence↗
PubMed1996

Nucleotide and amino acid sequences for cytochrome caa3-type oxidase of Bacillus stearothermophilus K1041 and non-Michaelis-type kinetics with cytochrome c.

A pseudo-sigmoidal cytochrome c-dependence curve of oxidase activity was observed with cytochrome oxidase from the Bacillus stearothermophilus strain K1041, while the other thermophilic Bacillus PS3 which has been extensively studied possessed normal Michaelis-Menten type kinetics. The genes coding for four subunits of cytochrome caa3-type oxidase and for heme O synthase were isolated from a genomic DNA library of K1041 by using a PS3 DNA fragment containing the highly-conserved region of the largest subunit as a probe, and sequenced. Most residues in subunits I (COI/caaB product), III (COIII/caaC product), and IV (COIV/caaD product) of K1041 were highly conserved when compared with those of PS3. However, the sequence of K1041 subunit II (COII/caaA product) was distinctly different from that of the PS3 subunit II. These Bacillus COIIs have an additional sequence for cytochrome c after the CuA binding protein portion with two transmembrane segments which is homologous to the mitochondrial counterpart, and represents the site of electron ingress. Several charged residues in the vicinity of cytochrome c moiety are replaced by oppositely charged residues. It is likely that these amino acid replacements in subunit II are the cause of the abnormal sigmoidal saturation curve for extrinsic cytochromes c of the K1041 enzyme.

Amino Acid Sequence↗
PubMed1996

A flash-photolysis study of the reactions of a caa3-type cytochrome oxidase with dioxygen and carbon monoxide.

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase from Bacillus PS3 in the presence of O2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochrome c subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 microseconds, 30 microseconds, 100 microseconds, and I ms at 445 nm. The initial, 10-microsecond absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving heme a and CuA are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochrome c, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O2 were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from heme a3 to a in PS3 oxidase compared to the bovine enzyme.

Animals↗
PubMed1996

Intra- and inter-complex cross-linking of subunits in the quinol oxidase super-complex from thermophilic Bacillus PS3.

Gram-positive thermophilic Bacilli contain quinol-cytochrome c reductase and cytochrome c oxidase as two major respiratory complexes of the electron transfer chain, and these enzymes can be extracted with mild detergents as an associated quinol oxidase super-complex. The reductase is composed of three subunits; cytochrome b6, cytochrome c1, and FeS protein, whereas cytochrome c oxidase consists of four subunits numbered 1 through 4. In order to clarify the interactions between the subunits, the super-complex isolated from Bacillus PS3 was cross-linked with three bifunctional cross-linkers; disuccinimidyl tartrate, 3,3'-dithiobis(succinimidylpropionate), and ethylene glycolbis(sulfosuccinimidylsuccinate). The most prominent cross-linking was observed for the combination of subunit 1 plus 2 in cytochrome c oxidase, and for that of cytochrome b6 plus cytochrome c1 in the reductase. In addition to these intra-complex cross-linkings, inter-complex linking was observed for the combination of cytochrome b6 plus subunit 1 with ethylene glycolbis(sulfosuccinimidylsuccinate), and for the combinations of cytochrome b6 plus subunit 1 and cytochrome b6 plus subunit 2 with 3,3'-dithiobis(succinimidylpropionate). Incubation in the presence of Triton X-100, which was confirmed to cleave the two enzyme complexes, selectively reduced the inter-complex cross-linking, suggesting that the chemical cross-linking reflect the spatial arrangement of subunits in the super-complex.

Bacillus↗
PubMed1996

A cb-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori.

A Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). When ascorbate was used as reductant, the Vmax and apparent Km values were 612 nmol electron min-1 (mg protein)-1 and 14 microM for yeast, and 419 nmol electron min-1 (mg protein)-1 and 19 microM for equine cytochrome c, respectively. For TMPD oxidation, the Vmax and Km values were 640 nmol electron min-1 (mg protein)-1 and 182 microM, respectively. These oxidase activities showed a high affinity for oxygen. Inhibition of both cytochrome-c and TMPD oxidase activities by 50% was caused by about 4 microM cyanide and about 0.5 mM azide. Redox difference spectra of the membrane solubilized with Triton X-100 showed b- or c-type cytochromes but not aa3-type cytochromes. c-type and a part of some b-type cytochromes were reduced with ascorbate plus TMPD. A CO difference spectrum revealed that protohaem, but not an aa3-type cytochrome, may be interacting with CO/oxygen. Only protohaem was detected in the haem fraction extracted from the membrane. Three polypeptides (60, 38 and 29 kDa) were found to be bearing haem c after SDS-PAGE of the membrane. From these results, it was suggested that the cbb3-type cytochrome-c oxidase, having a haem-copper binuclear centre like the cytochrome aa3-type oxidase, but differing in a few other properties, functions as a terminal oxidase in the respiratory chain of H. pylori.

Animals↗
PubMed1996

Identification of peptide fragments chemically cross-linked in cytochrome c oxidase from thermophilic Bacillus PS3.

In order to study steric arrangement of subunits in cytochrome c oxidase isolated from thermophilic Bacillus PS3, we developed a simple procedure including chemical cross-linking, two consecutive runs of electrophoresis, proteolytic digestion, and peptide sequencing for simultaneous identification of two cross-linked fragments. By this procedure, the cytochrome c domain of subunit 2 was found cross-linked to C-terminal region of subunit 1 including two hydrophobic transmembrane segments, suggesting that these two regions were located close each other. The present simple procedure might be applicable to proteins whose crystal structures are not revealed.

Amino Acid Sequence↗
PubMed1995

Thermophilic bacilli have split cytochrome b genes for cytochrome b6 and subunit IV. First cloning of cytochrome b from a gram-positive bacterium (Bacillus stearothermophilus).

The genes of Bacillus stearothermophilus K1041 encoding cytochrome b(6) (Bacillus cytochrome b is referred to as cytochrome b(6) for its resemblance to plastid b6) and subunit IV of the quinol:cytochrome c oxidoreductase (bc1 complex) were cloned and sequenced. For preparation of the probe for cloning, polymerase chain reaction was carried out using oligonucleotide mixtures targeting for N-terminal regions of cytochrome bc and subunit IV of the thermophilic Bacillus PS3. The deduced amino acid sequences contained 224 residues of 25,425 daltons for cytochrome b(6) and 173 residues of 19,371 daltons for subunit IV, and both open reading frames were separated by 67 base pairs. Cytochrome b and subunit IV contained 4 and 3 hydrophobic transmembrane segments, respectively, indicating that the fourth segment of subunit IV (eighth segment of cytochrome b) is lacking. Four histidine residues supposed to ligand two protohemes were conserved, but the two His in the fourth segment were separated by 14 amino acid residues like cytochrome b6, not like mitochondrial cytochrome b. The residues that might have conferred the two quinol-binding sites were mostly conserved, but especially the third His residue in the fourth segment of mitochondrial cytochrome b was replaced by Arg in Bacillus cytochrome b6 as in cytochrome b6. These characteristics and quantitative comparison of the protein sequences indicate that this Bacillus sequence is unique and meanwhile rather close to the cyanobacteria-plastids type than the purple bacteria-mitochondria type.

Amino Acid Sequence↗
PubMed1995

Kinetics of platelets in dogs with thrombocytopenia induced by antiglycoprotein IIb/IIIa receptor monoclonal antibody.

To experimentally assess the kinetics of platelets in thrombocytopenia, we constructed a canine model using 111In-oxine labeled autologous platelets and an intact antiplatelet monoclonal antibody (MAb) NNKY2-11 (IgG2a). With the infusion of radiolabeled autologous platelets into dogs, the peripheral platelet count and blood radioactivity level were examined, and the radioactivity in the liver, spleen and heart was determined with scintigraphic analysis. Thereafter, i.v. injection of 100 micrograms/kg of NNKY2-11 had no effect on platelet counts or the biodistribution of radiolabeled platelets. However, 200 and 300 micrograms/kg of MAb reduced the platelets, and the radioactivity of the liver and spleen augmented clearly after injection of MAb. Platelet radioactivity in serum, which had decreased after MAb infusion, did not recover, even when peripheral platelet counts returned to the normal levels, indicating that these new platelets might be derived from the platelet-storage pool or new thrombocytogenesis. This model of antiplatelet MAb induced thrombocytopenia seems to be useful for analyzing the kinetics of platelets in thrombocytopenia.

Animals↗
PubMed1995

Preparation and characterization of the hydrophilic CuA-cytochrome c domain of subunit II of cytochrome c oxidase from thermophilic bacillus PS3.

Cytochrome c oxidase of the thermophilic bacterium, PS3, was treated with trypsin. The hydrophilic domain of 26 kDa can be easily cleaved off from the hydrophobic anchor domain at the N-terminal region of subunit II, but remains attached to the rest of the enzyme upon gel-filtration in the presence of 0.2% lauroyl sarcosinate. The separation occurred in the presence of 5 M urea in addition to 0.2% lauroyl sarcosinate. After relatively prolonged proteolysis, that induced severe activity decay, and subunit I fragmentation, the 26 kDa fragment of subunit II can be easily isolated from the rest, suggesting that this fragment with cytochrome c and CuA interacts with subunit I. The separated fragment showed absorption spectra due to CuA and cytochrome c. Reconstitution of the cytochrome oxidase activity occurred on addition of the 26 kDa fragment to the proper gel-filtration chromatographic fraction.

Amino Acid Sequence↗
PubMed1994

Over-expression of membrane-bound cytochrome c-551 from thermophilic Bacillus PS3 in Bacillus stearothermophilus K1041.

Cytochrome c-551 is a lipoprotein of about 10500 Da, found in thermophilic Bacillus PS3 grown under air-limited conditions. An expression vector was constructed from a structural gene of PS3 cytochrome c-551, synthetic oligonucleotide as a promoter for Bacillus stearothermophilus and a shuttle vector for Escherichia coli and B. stearothermophilus. The transformed cells of B. stearothermophilus K1041 expressed cytochrome c-551 as much as 5 nmol/mg membrane protein. The effects of over-expression on the host cells are analyzed; a slightly slower growth rate and an increased synthesis of cytochrome oxidase (about twofold) occurred. Over-expressed (4-10-fold) cytochrome c-551 were purified and its properties were examined to know whether the protein is processed as in PS3 cells grown under air-limited conditions. The molecular mass determination and treatment with Rhizopus lipase suggested that the same processes, cleavage of signal peptidase, blocking of the N-terminal group and acylation of glycerol residue by two fatty acids, took place in the over-expression system. Fatty acylation seems useful for the cytochrome c to be effectively oxidized.

Acylation↗
PubMed1994

Membrane-bound Bacillus cytochromes c and their phylogenetic position among bacterial class I cytochromes c.

Gram-positive bacteria lack a periplasmic compartment and contain only membrane-bound cytochromes c. There are at least two types. One is found in subunit II of cytochrome oxidase, and the other is small cytochrome c which is also membrane-bound because of an unprocessed signal sequence or post-translational acylation at the N-terminal end of the protein. These Bacillus cytochromes c are compared with known class I cytochromes c, and a phylogenetic tree has been constructed by the neighbour-joining method.

Amino Acid Sequence↗
PubMed1994

Proton pumping activity and visible absorption and resonance Raman spectra of a cao-type cytochrome c oxidase isolated from the thermophilic bacterium Bacillus PS3.

Cytochrome c oxidase having heme O in addition to heme C and heme A (cytochrome cao) [Sone, N., & Fujiwara, Y. (1991) FEBS Lett. 288, 154-158] was isolated from a thermophilic bacterium, Bacillus PS3, grown under slightly air-limited conditions. Cytochrome cao could oxidize yeast cytochrome c and N,N,N',N'-tetramethyl-p-phenylenediamine twice as fast as cytochrome caa3, which this organism yielded under normal growing conditions. Cytochrome cao also pumped protons upon cytochrome c oxidation in a way similar to cytochrome caa3. Binding of cyanide to cytochrome cao caused spin-state conversion of heme O at the binuclear center and seriously inhibited its physiological activity. A low Ki value (0.4 microM) for cyanide was found to be mainly due to a small "off" constant. Resonance Raman spectra of cytochrome cao bore close resemblance to those of cytochrome caa3 in both oxidized and reduced states, although the formyl stretching (vCH=O) band was absent. The Fe-histidine stretching (vFe-His) and Fe-CO stretching (vFe-CO) frequencies of cytochrome cao were very close to those seen for cytochrome caa3, but were distinct from those of hemoglobin and peroxidases, suggesting that the protein structure in the vicinity of heme O resembles that of the heme a3 moiety of cytochrome caa3.

Bacillus↗
PubMed1993

The genes in the thermophilic cyanobacterium Synechococcus vulcanus encoding cytochrome-c oxidase.

It is still controversial whether cyanobacteria (blue-green algae) contain an aa3-type cytochrome-c oxidase. We have approached this problem using DNA analysis. Using a DNA probe coding for the most conserved part of subunit I of the Bacillus enzymes, structural genes for the oxidase of a thermophilic cyanobacterium Synechococcus vulcanus were cloned and sequenced. We found genes for subunits II, I, III and IV of this order like those of the Bacillus enzymes, and a terminator structure after the gene for subunit IV. The deduced protein sequences for the subunits II, I and III showed consensus amino-acid residues atevery important portion, suggesting that these genes are operating. However, the S. vulcanus oxidase lacked a cytochrome-c-moiety fused to subunit II, the 13th and 14th hydrophobic segments of subunit I which are lacking in the Paracoccus enzyme, and the 1st and 2nd ones of subunit III which are lacking in the Bacillus enzyme, were not found. A gene homologous to ctaB gene, which locates at the 5'-upstream region of the gene for subunit II and co-transcribed in Bacillus subtilis, was not found. Comparison of protein sequences showed that S. vulcanus cytochrome oxidase is closer to Bacillus cytochrome oxidases than the mitochondrial and Paracoccus enzymes, or quinol oxidases from B. subtilis and Escherichia coli.

Amino Acid Sequence↗
PubMed1993

Cytochrome c-551 of the thermophilic bacterium PS3, DNA sequence and analysis of the mature cytochrome.

The structural gene for cytochrome c-551 was isolated from genomic DNA of the thermophilic bacterium PS3. The amino acid sequence of cytochrome c-551 as deduced from the DNA sequence consists of 111 amino acid residues and contains one heme c-binding site (-CASCH-) located approximately in the middle of the polypeptide. The N-terminus of isolated cytochrome c-551 was blocked, but treatment with Rhizopus lipase and molecular weight measurement of the mature and lipase-treated forms by ion spray mass spectroscopy suggest that the mature c-551 may have 93 or 94 amino acid residues with a diacylated glycerol-cysteine at the N-terminal region. The first 17 or 18 amino acid residues in the N-terminal region of the nascent polypeptide, rich in hydrophobic and basic amino acid residues, may be a signal peptide to translocate the major portion of cytochrome c-551 to the extracellular surface and to be processed. Similarity of amino acid sequence of this protein is discussed in relation to other c-type cytochromes of bacilli as well as bacterial small cytochromes c such as Pseudomonas aeruginosa cytochrome c-551 and cytochrome c6 of cyanobacteria.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Haem O2 can replace haem A in the active site of cytochrome c oxidase from thermophilic bacterium PS3.

Thermophilic bacterium PS3 cultured under slightly air-limited conditions showed a mitochondrion-like cytochrome pattern similar to that in vigorously aerated cells, but an o-type cytochrome replaced cytochrome a3 as the CO-binding centre. Cytochrome cao-type oxidase was purified from the cell membranes by almost the same procedure as used for cytochrome caa3. The turnover number of cytochrome cao was higher than that of cytochrome caa3, but the Km's of the two enzymes for cytochrome c and O2 were almost the same. Gel electrophoresis in the presence of sodium dodecyl sulfate gave bands of four subunits at the identical positions both for cytochrome cao and cytochrome caa3. Cytochrome cao contained a novel kind of haem in addition to haems C and A. This novel haem is likely to be haem O, very recently found as the chromophore of the cytochrome bo complex in Escherichia coli. These data suggest that cytochrome cao is an alternative form of cytochrome c oxidase (cytochrome caa3), in which the cytochrome a3 centre of the enzyme is replaced with cytochrome o.

Binding Sites↗