Purification and characterization of an O2-utilizing cytochrome-c oxidase complex from Bradyrhizobium japonicum bacteroid membranes.
Purification and characterization of an O2-utilizing cytochrome-c oxidase complex from Bradyrhizobium japonicum bacteroid membranes.
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来自日本慢生根瘤菌类菌膜的利用 O2 的细胞色素 C 氧化酶复合物的纯化和表征。
DOI：
10.1016/0005-2728(93)90008-4
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Maier,RJ
中科院分区：
文献类型：
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作者：
Keefe,RG;Maier,RJ
从慢生根瘤菌的类菌体膜中纯化出一个由7-8个亚基组成的细胞色素-c(Cytc)氧化酶超复合体，其质量为358-425 kDa。至少有两个亚基具有c型血红素作为假体基团。其中一种含有c-血红素的成分在类菌膜中被检测到，但在自由细胞中没有检测到。该络合物还含有b-血红素，分光光度法显示b型和c型血红素蛋白都能与一氧化碳形成络合物。CO差分光谱显示在551.7 nm处有一个吸收极小值(谷值)，可能对应于先前描述的类菌膜中的Cytc-552。1 mM的奎纳克林(Atebrin)对细胞色素-Coxidase复合体的O2摄取无影响，但10 mM对O2的摄取抑制达90%。细胞色素bc1呼吸复合体的细胞色素带c1与来自B的这两种蛋白的抗体发生免疫反应，被鉴定为类细菌复合体的两种成分。日本产的。外源氧化的氧化酶复合体增加了马心细胞色素，伴随着氧气的摄取。在不添加细胞色素的情况下，也能氧化人工给电子体N，N，N‘，N’-四甲基-对苯二胺。10μM-NaCN可完全抑制摄氧活性，0.1μ-M-NaCN可完全抑制38%。该氧化酶络合物不能氧化具有单一异戊二烯单元侧链的泛喹酚同系物。在1.0 mM EDTA存在下，类菌膜的增溶导致细胞色素-Coxidase活性完全丧失。豆红蛋白脱氧数据表明，在游离氧浓度远低于1.0μM的情况下，该氧化酶复合体可以有效地发挥作用，尽管由于形成了氧化的豆红蛋白衍生物，因此未能成功地确定该酶对氧的特定亲和力。
A cytochrome-c(cytc) oxidase supercomplex consisting of 7–8 subunits and possessing a mass of 358–425 kDa was purified fromBradyrhizobium japonicumbacteroid membranes. At least two subunits possessc-type heme as a prosthetic group. One of thec-heme-containing components was detected in bacteroid membranes, but not in free-living cells. The complex also containsb-heme, and bothb-type andc-type heme proteins were spectrophotometrically shown to form complexes with carbon monoxide. A CO difference spectrum showed an absorption minimum (trough) at 551.7 nm, possibly corresponding to a previously described cytc-552 in bacteroid membranes. 1 mM quinacrine (Atebrin) had no effect on O2uptake by the cytochrome-coxidase complex, but 10 mM inhibited O2uptake by 90%. Cytochromesbandc1of the cytochromebc1respiratory complex were identified as two of the components of the bacteroid complex based upon immunoreaction with antibodies against these two proteins fromB. japonicum. The oxidase complex oxidized exogenously added horse heart ferrocytochromecconcomitant with the uptake of oxygen. It could also oxidize the artificial electron donorN,N,N′,N′-tetramethyl-p-phenylenediamine in the absence of added cytochromec. Oxygen uptake activity was completely inhibited by 10 μM NaCN and 38% by 0.1 μM NaCN. The oxidase complex was not able to oxidize a ubiquinol homolog possessing a single isoprenoid unit side chain. Solubilization of bacteroid membranes in the presence of 1.0 mM EDTA resulted in complete loss of cytochrome-coxidase activity. Leghemoglobin deoxygenation data indicated that the oxidase complex can efficiently function at free oxygen concentrations well below 1.0 μM, even though attempts to determine the oxidase's specific affinity for oxygen were unsuccessful due to the formation of oxidized leghemoglobin derivatives.