A high-affinity cbb(3)-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum
A high-affinity cbb(3)-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum
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DOI：
10.1128/jb.178.6.1532-1538.1996
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发表时间：
1996-03-01
影响因子：
3.2
通讯作者：
Hennecke, H
中科院分区：
文献类型：
--
作者：
Preisig, O;Zufferey, R;Hennecke, H
长期以来的一个假设是，内共生根瘤菌（类菌）通过使用一种特殊的呼吸电子传递链来应对豆科植物根瘤中浓度为 10 至 20 nM 的游离 O-2，该呼吸电子传递链以氧化酶终止，该氧化酶应该对 O-2 具有高亲和力。此前，我们认为微需氧和厌氧诱导的日本慢生根瘤菌的fixNOQP操纵子可能编码这种特殊的氧化酶，本文报道了这种末端氧化酶在厌氧生长的日本慢生根瘤菌野生型细胞膜上富集27倍后的生化特征，纯化的氧化酶具有TMPD (N,N,N',N'-四甲基对苯二胺)氧化酶活性以及细胞色素c氧化酶活性。其主要组成亚基的 N 端氨基酸测序证实了fixN、fixO 和fixP 基因产物的存在。 FixN 是一种高度疏水性的血红素 B 结合蛋白，FixO 和 FixP 是膜锚定的 c 型细胞色素（表观 M(r) 分别为 29,000 和 31,000），如它们在十二烷基硫酸钠-聚丙烯酰胺凝胶中的过氧化物酶活性所示。所有氧化酶特性均可诊断为血红素铜氧化酶 cbb(3) 型亚家族的成员。在从根瘤类菌体分离的膜中可通过免疫学方法检测到 FixP 蛋白，并且类菌体膜中总细胞色素 c 氧化酶活性的 85% 由 cbb(3) 型氧化酶贡献。纯化的酶和来自不同日本芽孢杆菌野生型和突变菌株的膜的 O-2 的 K-m 值通过分光光度法测定，使用氧化大豆豆血红蛋白作为唯一的 O-2 递送系统。膜中 cbb(3) 型氧化酶的 O-2 的导出 K-m 值为 7 nM，比需氧 aa(3) 型细胞色素 c 氧化酶测定的值低六到八倍。我们得出的结论是，cbb(3) 型氧化酶支持内共生类菌中的微有氧呼吸。
It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O-2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O-2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase, Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells, The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed the presence of the fixN, fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein, FixO and FixP are membrane-anchored c-type cytochromes (apparent M(r)s of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb(3)-type subfamily of the heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb(3)-type oxidase. The K-m values for O-2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O-2 delivery system. The derived K-m value for O-2 of the cbb(3)-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa(3)-type cytochrome c oxidase. We conclude that the cbb(3)-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.