Purified dicyclohexylcarbodiimide-sensitive ATPase (TF0· F1) from thermophilic bacterium PS3 is composed of a water soluble part with ATP hydrolytic activity (TF1) and a water insoluble moiety (TF0). ...All of the five subunits (α , β , γ , δ , and ε ) of TF1were isolated. TF1was reconstituted from the five subunits, which catalyzed an ATP-32Piexchange and an ATP-driven enhancement of fluorescence of 1-anilinona-phthalene-8-sulfonate, when adsorbed on proteoliposome inlaid with TF0(TF0-vesicles). Subunit ε and/or δ became firmly bound to TF0-vesicles and there was no preferential sequence in the binding. Both subunits were required for binding of the remaining subunits of TF1to TF0-vesicles, but they did not modify the high H+-permeability of TF0-vesicles. The addition of γ subunit together with ε and δ subunits caused a marked decrease of H+-permeability of TF0-vesicles, similar to that induced by TF1. We conclude tentatively that the ε and δ subunits connect TF0and the other subunits forming a part of a proton pathway, γ is a gate of proton flow coupled to ATP hydrolysis (or synthesis), and α and β subunits contain the active site for energy transformation. A possible model of subunit structure of TF1is proposed.
Structural genes encoding quinol-cytochrome
c reductase (QcR) were cloned and sequenced from nocardia-form actinomycete
Rhodococcus rhodochrous.
QcrC and
qcrA encode diheme cytochrome
cc and the ...Rieske Fe–S protein, respectively, while the
qcrB product is a diheme cytochrome
b. These amino acid sequences are similar to those of
Corynebacterium and
Mycobacterium, the members of high G+C content firmicutes. The presence of diheme cytochrome
cc subunit as a sole
c-type cytochrome in these organisms suggests the direct elecron transfer to cytochrome
c oxidase. The N-terminal half of the Rieske Fe–S proteins of these bacteria has a unique structure with three transmembrane helices, while the C-terminal half sequence is conserved. A phylogenetic tree using the latter region showed that high G+C firmicutes form a clear clade with
Thermus, but not with low G+C firmicutes.
Gram-positive thermophilic
Bacillus species contain cytochrome
caa
3-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
caa
3-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 α-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 50 195 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 38 992 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.
1. A stable ATPase complex with sensitivity to dicyclohexylcarbodiimide (TFo-F1) was purified from the membranes of the thermophilic aerobic bacterium PS3, by ion exchange chromatography in the ...presence of Triton X-100. 2. The ATPase of TFo-F1 was maximal at 70 degrees at pH 8.6 and was stable after monomerization in 4 M urea and 0.5% Triton X-100 at 25 degrees. The activity was dependent on Mg2+, Co2+, or Mn2+, and it became insensitive to dicyclohexylcarbodiimide when Ca2+ or Cd2+ was added instead. 3. TFo-F1 required P-lipids of this bacterium contained branched fatty acyl groups but no unsaturated groups and were stable against oxidation and heat. 4. Studies by electron microscopy, gel electrophoresis, and use of anti-ATPase antibody and 3Hacetyl-ATPase indicated that the TFo-F1 complex was composed of an ATPase moiety (TF1, five different subunits) and a hydrophobic moiety (TFo, three different subunits. TFo conferred TF1 with sensitivity to dicyclohexylcarbodiimide. 5. Vesicles catalyzing 32Pi-ATP exchange and ATP-driven enhancement of fluorescence of anilinonaphthalene sulfonate were reconstituted by dialyzing pure TFo-F1 and P-lipids together, and were active even at 50-75 degrees. The vesicles reconstituted from TFo-F1 and bacterial P-lipids were more stable than those reconstituted from TFo-F1 and soybean P-lipids.
Vesicles were reconstituted from a purified dicyclohexyl-carbodiimide-sensitive ATPase complex (TF0-F1) and phospholipids of a thermophilic bacterium PS3. These vesicles synthesized ATP from ADP and ...Pi with energy from an electrochemical proton gradient (delta-micronH+) formed by a pH gradient and an electrical potential across their membranes. Maximal ATP synthesis was achieved by incubating the vesicles in malonate at pH 5.5 with valinomycin, and then rapidly transferring them to a solution of pH 8.4 and 150 mM K+. Under these conditons ATP synthesis continued at a decreasing rate for 30 s at 40 degrees. Appreciable formation of ATP (40 to 150 nmol/mg of TF0-F1) occurred at an initial delta-micronH+ above 205 mV and moderate formation at an initial value above 180 mV. ATP hydrolysis by the vesicles produced a delta-micronH+, and the additions of 32Pi and hexokinase to them resulted in 32Pi esterification. Analysis of the time courses of 32Pi esterification and decays of the pH difference and membrane potential, followed using 9-aminoacridine and 8-anilinonaphthalene-1-sulfonate, respectively, as probes, showed a relationship between delta-micronH+ and the rate of ATP synthesis. These results demonstrate that purified TF0-F1 is itself a reversible H+-translocating ATPase of oxidative phosphorylation.
1. The membrane-integrated portion (TF0) of the proton translocating ATPase complex (TF0-F1) of the thermophilic bacterium PS3 was highly purified. Its proton-conducting activity was investigated in ...vesicles reconstituted from TF0 and phospholipids (TF0 vesicles). 2. The rate of proton conduction through TF0 was proportional to the membrane potential imposed (6H+ uptake/s/TF0 molecule with 103 mV at pH 8.0). The pH profile of the rate revealed that a proton, not a hydroxy ion, was the true substrate conducted and that there was a monoprotic proton binding site in TF0 (pKa = 6.8). The temperature coefficient of proton conductance of TF0 showed a considerable variation depending on the phospholipids of the vesicles with respective transition temperatures. 3. Passive proton conduction through TF0 was inhibited stoichiometrically by addition of either the soluble ATPase portion (TF1) of TF0-F1, or an energy transfer inhibitor dicyclohexylcarbodiimide or an antibody against TF0. 4. The proton conductance of TF0 was concluded to represent its intrinsic activity in the original TF0-F1 complex.
A convenient and reliable method to measure passive H+-translocating activity (H+ conductivity) was developed; vesicles reconstituted from the membrane moiety (F0) of H+-ATPase (F0 . F1) and soybean ...phospholipids were loaded with KCl by a freeze-thaw-sonication procedure and the rate of H+ uptake caused by the K+ diffusion potential upon addition of valinomycin was followed with a pH meter. Of the methods tested, a dialysis method using cholate plus deoxycholate gave the best results for reconstitution. Using this method, H+ conductivity of the membrane moiety of H+-ATPase from a thermophilic bacterium PS3 (TF0) was analyzed. Dependence of H+ conductivity of TF0 on H+ concentration fitted a Michaelis-Menten equation showing a Vmax of 31.3 microgram ion/min . mg of TF0 and a Km of 0.095 microgram ion/liter. Upon modification of a tyrosyl residue of TF0 with iodine, the Km value shifted to 0.71 microgram ion/liter, while the Vmax remained constant. These results were interpreted as indicating that a single tyrosyl residue in N,N'-dicyclohexylcarbodiimide-binding proteolipid of TF0 plays an important role as an H+ donor in the the rate-limiting step of H+ permeation through TF0. TF1, the catalytic moiety of H+-ATPase from the thermophilic bacterium PS3, blocked H+ conduction through TF0. A 1:1 stoichiometry of TF1 and TF0 was found in ATP-dependent membrane potential generation as well as H+ conduction.
sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of archaeal lipids. 4- sup(3)HNADH that had sup(3)H at the R side was produced from 4- ...sup(3)HNAD and glucose with glucose dehydrogenase (a pro-S type enzyme). The sup(3)H of this 4- sup(3)HNADH was transferred to dihydroxyacetonephosphate during the sn-glycerol-1-phosphate dehydrogenase reaction. On the contrary, in a similar reaction using alcohol dehydrogenase (a pro-R type enzyme), sup(3)H was not incorporated into glycerophosphate. These results confirmed a prediction of the tertiary structure of sn-glycerol-1-phosphate dehydrogenase by homology modeling.
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.
We isolated a K17q8 mutant from K17 mutant cells of Bacillus stearothermophilus which contain SoxB-type cytochrome bo(3) as well as cytochrome bd but not SoxM-type cytochrome caa(3), which is the ...main terminal oxidase in B. stearothermophilus K1041. The respiration of K17q8 was highly sensitive to as little as 10 microM cyanide, indicating that the main terminal oxidase is cytochrome bo(3). The aerobic growth yield of K17q8 was lower than that of wild-type K1041, but higher than that of parental K17. The H(+)/O ratio of K17q8 was about 5, i.e. a little lower than the 6.1-6.5 of K1041, but higher than the 2.9-3.1 of K17 Sone et al. (1999) J. Biosci. Bioeng. 87, 495-499. Analyses of membrane fragments indicated that K17q8 contains about 0.2 nmol cytochrome bo(3) per mg membrane protein, and scarcely any subunits of cytochromes caa(3) and bd. From the membrane fraction of K17q8, cytochrome bo(3) was purified and shown to be composed of two subunits with apparent molecular masses of 56 and 19 kDa. The enzyme contained protoheme IX and heme O, as the main low-spin heme and high-spin heme. Analysis of the substrate specificity indicated that the high-affinity site is very specific to cytochrome c-551, a cytochrome c which is a membrane-bound lipoprotein of thermophilic Bacillus. The I(50) of purified cytochrome bo(3) was determined to be 4 microM, indicating that cytochrome bo(3) among the three terminal oxidases in B. stearothermophilus was most susceptible to cyanide. The respiration of K17q8 was mostly inhibited by the addition of cyanide at this concentration.
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