Sone N, Koyanagi S, Sakamoto J
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu Iizuka, Fukuoka 820-8502, Japan.
J Biochem. 2000 Apr;127(4):551-7. doi: 10.1093/oxfordjournals.jbchem.a022640.
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.
我们从嗜热脂肪芽孢杆菌的K17突变细胞中分离出一个K17q8突变体，该细胞含有SoxB型细胞色素bo(3)以及细胞色素bd，但不含有嗜热脂肪芽孢杆菌K1041中的主要末端氧化酶SoxM型细胞色素caa(3)。K17q8的呼吸作用对低至10微摩尔的氰化物高度敏感，这表明其主要末端氧化酶是细胞色素bo(3)。K17q8的好氧生长产量低于野生型K1041，但高于亲本K17。K17q8的H(+)/O比率约为5，即略低于K1041的6.1 - 6.5，但高于K17的2.9 - 3.1 [索尼等人（1999年）《生物科学与生物工程杂志》87卷，495 - 499页]。膜片段分析表明，K17q8每毫克膜蛋白含有约0.2纳摩尔细胞色素bo(3)，几乎不含细胞色素caa(3)和bd的亚基。从K17q8的膜部分纯化得到细胞色素bo(3)，并显示其由两个亚基组成，表观分子量分别为56和19 kDa。该酶含有原血红素IX和血红素O，分别作为主要的低自旋血红素和高自旋血红素。底物特异性分析表明，高亲和力位点对细胞色素c - 551非常特异，细胞色素c - 551是嗜热芽孢杆菌的一种膜结合脂蛋白细胞色素。纯化的细胞色素bo(3)的I(50)测定为4微摩尔，这表明嗜热脂肪芽孢杆菌的三种末端氧化酶中，细胞色素bo(3)对氰化物最敏感。在此浓度下添加氰化物可大部分抑制K17q8的呼吸作用。
