Gilmour R, Krulwich T A
Department of Biochemistry, Mount Sinai School of Medicine of CUNY, New York 10029, USA.
Biochim Biophys Acta. 1996 Aug 7;1276(1):57-63. doi: 10.1016/0005-2728(96)00028-x.
The presence of a cytochrome bo-type terminal oxidase in Bacillus firmus OF4 had been suggested from the effects of CO on the spectra of reduced membrane cytochromes (Hicks, D.B., Plass, R.J. and Quirk, P.G. (1991) J. Bacteriol. 173, 5010-5016). In that study the CO-binding b-type cytochrome was partially purified by anion exchange chromatography. No further purification was attempted but later HPLC analysis indicated the absence of significant heme O in the B. firmus OF4 membranes. The current work shows that the partially purified cytochrome b is actually composed of three different b-type cytochromes which can be separated and purified by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. Two of the cytochromes were CO-reactive but lacked the characteristic multisubunit composition of known terminal oxidases. Neither purified cytochrome catalyzed quinol or ferrocytochrome c oxidation. The more abundant CO-reactive b-type cytochrome (cytochrome b560) had an apparent molecular mass of 10 kDa, whereas the other, more minor component (cytochrome b558), was partially purified and showed two bands of 23 and 17 kDa on SDS-PAGE. The functions of the cytochromes b560 and b558 remain unknown but together they account for the spectrum originally attributed to cytochrome bo. The third, non-CO reactive, cytochrome b was associated with substantial succinate dehydrogenase activity and was purified as a three subunit succinate dehydrogenase complex with high specific activity (17.7 mumol/min/mg). Limited N-terminal sequence of each subunit demonstrated marked similarity to the complex from Bacillus subtilis. The cytochrome b of the alkaliphile enzyme was reduced about 50% by succinate compared to the level of reduction achieved by dithionite. The enzyme reacted with both napthoquinones and benzoquinones. The results presented indicate that Bacillus firmus OF4 contains a succinate dehydrogenase complex with very similar properties to the enzyme from Bacillus subtilis, but does not contain a cytochrome o-type terminal oxidase under the growth conditions studied.
从一氧化碳对还原态膜细胞色素光谱的影响推测，坚强芽孢杆菌OF4中存在细胞色素bo型末端氧化酶（希克斯，D.B.，普拉斯，R.J.和夸克，P.G.（1991年）《细菌学杂志》173，5010 - 5016）。在该研究中，通过阴离子交换色谱法对结合一氧化碳的b型细胞色素进行了部分纯化。未尝试进一步纯化，但后来的高效液相色谱分析表明坚强芽孢杆菌OF4膜中不存在大量的血红素O。目前的工作表明，部分纯化的细胞色素b实际上由三种不同的b型细胞色素组成，它们可以通过离子交换、羟基磷灰石和凝胶过滤色谱法的组合进行分离和纯化。其中两种细胞色素与一氧化碳反应，但缺乏已知末端氧化酶的特征性多亚基组成。两种纯化的细胞色素均未催化喹啉或亚铁细胞色素c的氧化。含量较多的与一氧化碳反应的b型细胞色素（细胞色素b560）的表观分子量为10 kDa，而另一种含量较少的成分（细胞色素b558）经部分纯化后，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上显示出23 kDa和17 kDa的两条带。细胞色素b560和b558的功能仍然未知，但它们共同构成了最初归因于细胞色素bo的光谱。第三种不与一氧化碳反应的细胞色素b与大量琥珀酸脱氢酶活性相关，并作为具有高比活性（17.7 μmol/分钟/毫克）的三聚体琥珀酸脱氢酶复合物被纯化。每个亚基的有限N端序列显示与枯草芽孢杆菌的复合物有显著相似性。与连二亚硫酸盐实现的还原水平相比，嗜碱酶的细胞色素b被琥珀酸还原约50%。该酶与萘醌和苯醌均发生反应。给出的结果表明，坚强芽孢杆菌OF4含有一种与枯草芽孢杆菌的酶性质非常相似的琥珀酸脱氢酶复合物，但在所研究的生长条件下不含有细胞色素o型末端氧化酶。
