Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated ...several mutants from B. stearothermophilus defective in the cao3-type oxidase activity Sakamoto, J. et al. (1996) FEMS Microbiol. Lett. 143, 151–158. A novel oxidase was isolated from membrane preparations of one of the mutants, K17. The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme O, heme A, and Cu in a ratio of 1:0.7:0.2:3. CO difference spectra indicate that the high-spin heme is mainly heme O. These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A. The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus. The turnover rate of the activity (Vmax = 190 s−1) and its affinity for cytochrome c-551 (Km =0.15 μM) were much higher than those for yeast and equine heart cytochromes c. The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a K1 value of 19 μM. The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase. Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the heme-copper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.
Evidence for the presence of a quinol oxidase super-complex composed of a cytochrome bc1 complex and cytochrome oxidase in the respiratory chain of a Gram-positive thermophilic bacterium PS3 is ...reported. On incubation with an octyl glucoside-solubilized fraction of the total membranes of PS3 anti-serum against PS3 cytochrome oxidase gave an immunoprecipitate that showed both quinol-cytochrome c reductase and cytochrome c oxidase activities. When the cholate-deoxycholate and LiCl-treated membranes of PS3 were solubilized and subjected to ion-exchange chromatography in the presence of octaethyleneglycol dodecyl ether, most of the A-, B-, and C-type cytochromes were copurified as a peak having both quinol-cytochrome c reductase and cytochrome oxidase activities. The immunoprecipitate and quinol oxidase preparation contained hemes a, b, and c in a ratio of about 2:2:3, indicating the presence of one-to-one complex of cytochrome oxidase containing 2 hemes a and one heme c, and a bc1 complex containing 2 hemes b and 2 hemes c. Gel electrophoresis in the presence of dodecyl sulfate showed that the immunoprecipitate and quinol oxidase preparation were composed of seven subunits; those of 51 (56-kDa), 38, and 22 kDa for cytochrome oxidase and those of 29, 23, 21, and 14 kDa for the bc1 complex. The 38-, 29-, and 21 kDa components possessed covalently bound heme c. The apparent molecular mass of the super complex was estimated to be as 380 kDa by gel filtration.
Cytochrome c-551, the electron donor of SoxB-type cytochrome c oxidase in thermo-philic bacilli, can be over-expressed in Bacillus thermodernitrificans cells by tranforma-tion with pSTEc551. Several ...mutant cytochromes c-561 were prepared by site-directed mutagenesis to this expression plasmid. Among them, several Lys residues were changed to Ala/Ser, and we found that these mutant cytochromes retained their activity as substrates, although their Km values were 0.04–0.12 μM, depending on the site replaced. In contrast, the C19A mutant cytochrome, which was produced in Brevibacil-lus choshinesis as a secretion protein, lost its activity as a substrate, suggesting that the fatty acyl-glyceryl residue covalently bound to the cysteine residue of the wild-type c-551 plays a very important role in the activity. The importance of the hydrophobic fatty acid residue for the binding of cytochrome c-551 to the oxidase was also shown by the loss of substrate activity in deacylated cytochrome c-551. These results show the importance of the hydrophobic interaction between this cytochrome and SoxB-type oxi-dase, despite the fact that the importance of an electrostatic interaction between cyto-chrome c and mitochondrial cytochrome aa3 oxidase has already been established.
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(sulfosuccinimi-dylsuccinate). 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 c 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 crosslinking reflect the spatial arrangement of subunits in the super-complex.
A quinol-cytochrome c oxidoreductase (cytochrome bc1 complex) has been purified from plasma membranes of a thermophilic Bacillus, PS3, by ion-exchange chromatography in the presence of Triton X-100. ...The purified enzyme shows absorption bands at 561-562 nm and 553 nm at room temperature, and 560, 551, and 547 nm at 80 K upon reduction, and gives an ESR signal similar to that of a Rieske-type iron sulfur center. Its contents of protohemes, heme c, and non-heme iron are about 23, 10, and 21 nmol/mg of protein, respectively. The enzyme consists of four polypeptides with molecular masses of 29, 23, 21, and 14 kDa judging from their electrophoretic mobilities in the presence of sodium lauryl sulfate. Since the staining intensities of the respective bands are almost proportional to their molecular masses, the monomer complex (87 kDa) of the subunits probably consists of a cytochrome b having two protohemes, a cytochrome c1 and an Fe2-S2-type iron sulfur center. The 29 and 21 kDa subunits were identified as cytochromes c1 and b, respectively, and the 23-kDa subunit is probably an iron-sulfur protein, since the 14-kDa polypeptide can be removed with 3 M urea without reducing the content of non-heme iron. Several characteristics of the subunits and chromophores indicate that the PS3 enzyme is rather similar to cytochrome b6f (a bc1 complex equivalent) of chloroplasts and Cyanobacteria. The PS3 complex catalyzes reduction of cytochrome c with various quinol compounds in the presence of P-lipids and menaquinone. The turnover number at pH 6.8 was about 5 s-1 at 40 degrees C and 50 s-1 at 60 degrees C. The enzyme is heat-stable up to 65 degrees C.
(AlN)
1/(GaN)
n1
m
/(AlN)
n2
-based quantum wells (QWs) constructed by periodically introducing several atomic layers of AlN into (AlN)
1/(GaN)
n1
short-period superlattices have a great potential ...for the application to mid-infrared quantum-cascade lasers. Effective electron injection from the first to second subbands in the (AlN)
1/(GaN)
n1
short-period superlattice is expected through the inserted (AlN)
n2
layer which has a large polarization field. The (AlN)
1/(GaN)
n1
m
/(AlN)
n2
QWs were prepared by hot wall epitaxy, and the structure was characterized by transmission electron microscopy and X-ray diffraction. The quality of (AlN)
1/(GaN)
n1
m
/(AlN)
n2
QW depends significantly on the quality of GaN buffer layer, and high-quality QW structures were prepared on GaN films grown on Al
2O
3 (
0
0
0
1
).
One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are ...exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively.
Helicobacter pylori is a microaerophilic Gram-negative spiral bacterium residing in human stomach. A cb-type cytochrome c oxidase that terminates the respiratory chain was purified to near ...homogeneity by solubilizing the membranes with Triton X-100 and applying anion exchange, Cu-chelating, and gel filtration chromatographies. Redox- and CO-difference spectra and pyridine ferrohaemochrome analysis showed the enzyme to contain three haems C, one low-spin protohaem, and one high-spin protohaem that probably forms a dioxygen-reducing bimetalic center with a copper atom. The enzyme actively oxidizes soluble cytochrome c from this bacterium (TNmax of about 250 s-1) with aof 0.9 μM. Yeast cytochrome c and N,N,N,N-tetramethyl p-phenylenediamine (TMPD) are also oxidized at similar maximal velocities with larger Km's. Oxygen pulse experiments on resting cells in the presence of ascorbate plus TMPD or L-lactate indicated that this sole terminal oxidase pumps H+, although the H+ pumping activity by proteoliposomes reconstituted from the enzyme and P-lipids was low. Two main bands with haem C at 58 and 26 kDa were observed upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and succeeding protein and haem staining. Sequencing of the operon encoding the subunits of the enzyme revealed the presence of ccoNOQP. N-terminal analysis of the 58 kDa band showed 15 or 13 amino acids coinciding with the amino acid sequences deduced from the DNA of ccoN and ccoO. CcoN, the largest subunit bearing two protohaems and copper, and ccoO, a mono-haem cytochrome subunit form a protein complex with an apparent molecular mass of 58 kDa, even in the presence of sodium dodecyl sulfate. The 26 kDa band is tentatively assumed to be ccoP with two haems C.
Helicobacter pylori, a microaerophilic Gram-negative spiral bacterium residing in the human stomach, contains a small size soluble cytochorme c. This cytochrome c was purified from the soluble ...fraction of H.pylori by conventional chromatographies involving octyl-cellulose and CM-Toyopearl. Its reduced form gave an alpha absorption band at 553 nm, and thus the cytochrome was named H.pylori cytochrome c-553. The cytochrome, giving a band below 10,000 Da upon SDS-PAGE, was determined to have a mass of 8,998 by time of flight mass spectroscopy. Its N-terminal peptide sequence was TDVKALAKS—, indicating that the nascent polypeptide was cleaved to produce a signal peptide of 19 amino acid residues and a mature protein composed of 77 amino acid residues. The cb-type cytochrome c oxidase oxidized ferrocytochrome c-553 of this bacterium actively (Vmax of about 250 s-1) with a small Km (0.9 μM). Analysis of the effect of the salt concentration on the oxidase activity indicated that oxidation of cytochrome c-553 is highly inhibited under high ionic conditions. The amino acid sequence of H.pylori cytochorme c-553 showed the closest similarity to that the Desulfovibrio vulgaris cytochrome C-553, and these sequences showed a weak relationship to that of the cytochrome c8- group among class I cytochromes c.
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