DETECTION OF THE IN-VIVO INCORPORATION OF A METAL CLUSTER INTO A PROTEIN - THE FEMO COFACTOR IS INSERTED INTO THE FEFE PROTEIN OF THE ALTERNATIVE NITROGENASE OF RHODOBACTER-CAPSULATUS
DETECTION OF THE IN-VIVO INCORPORATION OF A METAL CLUSTER INTO A PROTEIN - THE FEMO COFACTOR IS INSERTED INTO THE FEFE PROTEIN OF THE ALTERNATIVE NITROGENASE OF RHODOBACTER-CAPSULATUS
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DOI：
10.1111/j.1432-1033.1993.tb18003.x
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发表时间：
1993-07-01
期刊：
影响因子：
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通讯作者：
KLIPP, W
中科院分区：
文献类型：
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作者：
GOLLAN, U;SCHNEIDER, K;KLIPP, W
光合细菌Rhodobacter capsulatus除了具有Mo固氮酶外，还具有第二种不依赖Mo的固氮系统，即“仅含铁”的固氮酶，该固氮酶被Mo强烈抑制。在nifHDK-细胞中引起交替固氮酶50%抑制的MoO 42-浓度为6 nM。如果MoO 42-被添加到已经表达了替代固氮酶的生长中的nifHDK-培养物中，则整个细胞从乙炔产生乙烷的过程被显著地刺激。尽管在实验持续期间（3天）C2 H4形成连续减少，但总C2 H6产量在前24小时内增加约两倍，而C2 H6的相对产率从2%增加到2%。（C2 H6/C2 H4 X 100），在MoO 42-（1 mM）存在下孵育72 h后达到最大值69%。培养基中MoO_（42-）浓度越高，培养时间越长，这种“Mo效应”越强。在ReO ~（4-）、WO ~（42-）或VO ~（43-）存在下，没有出现类似的效应。在不能合成FeMo辅因子的nifHDK-nifE-双突变体中没有观察到“Mo效应”，而在nifHDK-nifQ-突变体中则减少了。从这些提取物中纯化的组分1显示S = 3/2 EPR信号，其相对较弱但为FeMoco的特征。这些结果强烈支持了“Mo效应”是由替代性固氮酶的脱辅基蛋白和常规固氮酶的FeMo辅因子组成的杂合酶形成的结果。“Mo效应”不受影响添加氯霉素到培养物中。因此，“Mo效应”的发生似乎与从头蛋白质合成无关。对nifE-lacZ和nifN-lacZ融合基因的分析表明，FeMo辅因子合成所必需的两个基因在MoO_（42）缺乏的条件下也能表达，并对FeMoco掺入替代固氮酶组分1的可能原因进行了讨论。
The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. The MoO42- concentration causing 50% repression of the alternative nitrogenase in nifHDK- cells was 6 nM. If MoO42- was added to a growing nifHDK- culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H4 formation decreased continuously during the duration of the experiment (3 days), the total C2H6 Production increased about twofold within the first 24 h, whereas the relative yield of C2H6 increased from 2% (C2H6/C2H4 X 100) in the absence of MoO42-, to a maximal value of 69% in the presence of MoO42- (1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MoO42- concentration in the medium and the longer the incubation time. In the presence of ReO4-, WO42- or VO43-, a similar effect did not occur.The 'Mo effect' was not observed in a nifHDK-nifE- double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK-nifQ- mutant.Crude extracts from nifHDK- cells cultivated in the presence of MoO42-, also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase.The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis. The analysis of nifE-lacZ and nifN-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO42- deficiency.The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.