Tan H M, Joannou C L, Cooper C E, Butler C S, Cammack R, Mason J R
Centre for the Study of Metals in Biology and Medicine, King's College London, United Kingdom.
J Bacteriol. 1994 May;176(9):2507-12. doi: 10.1128/jb.176.9.2507-2512.1994.
The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.
恶臭假单胞菌ML2的苯双加氧酶是一种多组分复合物，由一种黄素蛋白还原酶、一种铁氧还蛋白和一种末端铁硫蛋白（ISP）组成。分离得到的复合物的催化活性与细胞提取物中的蛋白质浓度呈非线性关系，体外活性的限制因素是铁氧还蛋白（BED）。使用125I标记的抗体分析了这三种组分的相对水平，结果表明ISP（BED）、铁氧还蛋白（BED）和还原酶（BED）的功能摩尔比分别为1:0.9:0.8。通过对全细胞铁氧还蛋白（BED）和ISP（BED）中2Fe-2S中心的定量电子顺磁共振光谱确定了铁氧还蛋白（BED）的浓度。这些结果表明，铁氧还蛋白（BED）组分是体外双加氧酶活性的限制因素。为了确定它在体内是否是限制因素，将过量表达铁氧还蛋白（BED）的质粒（pJRM606）导入恶臭假单胞菌ML2。在细胞提取物中测量的该菌株的苯双加氧酶活性比野生型高五倍，并且在细胞提取物中蛋白质浓度高于2mg/ml时活性与蛋白质浓度呈线性关系。蛋白质免疫印迹（免疫印迹）和电子顺磁共振光谱分析证实重组菌株中铁氧还蛋白（BED）蛋白和活性氧化还原中心的水平升高。然而，在这些细胞中，铁氧还蛋白（BED）水平的增加对全细胞苯氧化的总体速率没有影响。因此，我们得出结论，在细胞中发现的高细胞内浓度（0.48mM）下，铁氧还蛋白（BED）不是限制因素。
