Keefe R G, Maier R J
Department of Biology, McCollum Pratt Institute, Johns Hopkins University, Baltimore, MD 21218.
Biochim Biophys Acta. 1993 Nov 2;1183(1):91-104. doi: 10.1016/0005-2728(93)90008-4.
A cytochrome-c (cyt c) oxidase supercomplex consisting of 7-8 subunits and possessing a mass of 358-425 kDa was purified from Bradyrhizobium japonicum bacteroid membranes. At least two subunits possess c-type heme as a prosthetic group. One of the c-heme-containing components was detected in bacteroid membranes, but not in free-living cells. The complex also contains b-heme, and both b-type and c-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 cyt c-552 in bacteroid membranes. 1 mM quinacrine (Atebrin) had no effect on O2 uptake by the cytochrome-c oxidase complex, but 10 mM inhibited O2 uptake by 90%. Cytochromes b and c1 of the cytochrome bc1 respiratory complex were identified as two of the components of the bacteroid complex based upon immunoreaction with antibodies against these two proteins from B. japonicum. The oxidase complex oxidized exogenously added horse heart ferrocytochrome c concomitant with the uptake of oxygen. It could also oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine in the absence of added cytochrome c. Oxygen uptake activity was completely inhibited by 10 microM NaCN and 38% by 0.1 microM 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-c oxidase activity. Leghemoglobin deoxygenation data indicated that the oxidase complex can efficiently function at free oxygen concentrations well below 1.0 microM, even though attempts to determine the oxidase's specific affinity oxygen were unsuccessful due to the formation of oxidized leghemoglobin derivatives.
从日本慢生根瘤菌类菌体膜中纯化出一种细胞色素c（cyt c）氧化酶超复合物，它由7 - 8个亚基组成，质量为358 - 425 kDa。至少有两个亚基含有c型血红素作为辅基。其中一个含c型血红素的组分在类菌体膜中被检测到，但在自由生活的细胞中未被检测到。该复合物还含有b型血红素，通过分光光度法显示b型和c型血红素蛋白都能与一氧化碳形成复合物。一氧化碳差示光谱在551.7 nm处显示出一个吸收最小值（波谷），可能对应于之前描述的类菌体膜中的细胞色素c - 552。1 mM喹吖因（阿的平）对细胞色素c氧化酶复合物的氧气摄取没有影响，但10 mM可抑制90%的氧气摄取。基于与来自日本慢生根瘤菌的针对这两种蛋白质的抗体的免疫反应，细胞色素bc1呼吸复合物的细胞色素b和c1被鉴定为类菌体复合物的两个组分。该氧化酶复合物在摄取氧气的同时氧化外源添加的马心亚铁细胞色素c。在没有添加细胞色素c的情况下，它也能氧化人工电子供体N，N，N'，N'-四甲基对苯二胺。10 μM NaCN可完全抑制氧气摄取活性，0.1 μM NaCN可抑制38%。该氧化酶复合物无法氧化具有单个异戊二烯单元侧链的泛醇同系物。在1.0 mM EDTA存在下对类菌体膜进行增溶导致细胞色素c氧化酶活性完全丧失。豆血红蛋白脱氧数据表明，即使由于氧化豆血红蛋白衍生物的形成而未能成功测定氧化酶对氧气的特异性亲和力，但该氧化酶复合物在远低于1.0 μM的游离氧浓度下仍能有效发挥作用。
