Energy-yielding properties of SoxB-type cytochrome bo(3) terminal oxidase: analyses involving Bacillus stearothermophilus K1041 and its mutant strains.
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.
Sone
N
N
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu Iizuka, Fukuoka 820-8502, Japan. sone@bse.kyutech.ac.jp
Koyanagi
S
S
Sakamoto
J
J
D001426:Bacterial Proteins / Q000737:chemistry; D003486:Cyanides / Q000494:pharmacology; D003573:Cytochrome b Group; D003574:Cytochrome c Group / Q000737:chemistry*; D003580:Cytochromes / Q000737:chemistry*; D004734:Energy Metabolism; D029968:Escherichia coli Proteins; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006912:Hydroxyquinolines / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003486:Cyanides; D003573:Cytochrome b Group; D003574:Cytochrome c Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D006912:Hydroxyquinolines; C083850:cytochrome bo3, E coli; D009243:NAD; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D010088:Oxidoreductases; C064500:flavocytochrome c sulfide dehydrogenase