Gollan U, Schneider K, Müller A, Schüddekopf K, Klipp W
Fakultät für Chemie, Lehrstuhl für Anorganische Chemie I, Universität Bielefeld, Germany.
Eur J Biochem. 1993 Jul 1;215(1):25-35. doi: 10.1111/j.1432-1033.1993.tb18003.x.
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 MoO4(2-) concentration causing 50% repression of the alternative nitrogenase in nifHDK- cells was 6 nM. If MoO4(2-) 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 MoO4(2-), to a maximal value of 69% in the presence of MoO4(2-) (1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MoO4(2-) concentration in the medium and the longer the incubation time. In the presence of ReO4-, WO4(2-) or VO4(3-), 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 MoO4(2-), 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 MoO4(2-) deficiency. The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.
光合细菌荚膜红杆菌除了拥有钼固氮酶外，还有第二个不依赖钼的固氮系统，即一种“仅含铁”的固氮酶，它受到钼酸盐的强烈抑制。导致nifHDK⁻细胞中替代固氮酶50%抑制的MoO₄²⁻浓度为6 nM。如果将MoO₄²⁻添加到已经表达替代固氮酶的正在生长的nifHDK⁻培养物中，全细胞由乙炔产生乙烷的量会显著增加。尽管在实验期间（3天）乙烯的形成持续减少，但在最初的24小时内乙烷的总产量增加了约两倍，而乙烷的相对产量从无MoO₄²⁻时的2%（C₂H₆/C₂H₄×100）增加到孵育72小时后在存在MoO₄²⁻（1 mM）时的最大值69%。培养基中MoO₄²⁻浓度越高且孵育时间越长，这种“钼效应”似乎越强。在存在ReO₄⁻、WO₄²⁻或VO₄³⁻时，未出现类似效应。在无法合成铁钼辅因子的nifHDK⁻ nifE⁻双突变体中未观察到“钼效应”，而在nifHDK⁻ nifQ⁻突变体中该效应减弱。在存在MoO₄²⁻的情况下培养的nifHDK⁻细胞的粗提取物也显示出乙烷产量增加。从这些提取物中纯化的组分1显示出S = 3/2的EPR信号，该信号相对较弱，但具有铁钼辅因子的特征。这些结果有力地支持了“钼效应”是由替代固氮酶的脱辅基蛋白和传统固氮酶的铁钼辅因子组成的杂合酶形成的结果这一观点。向培养物中添加氯霉素不影响“钼效应”的出现。因此，“钼效应”的发生似乎与从头合成蛋白质无关。对nifE - lacZ和nifN - lacZ融合体的分析证明，铁钼辅因子合成所需的两个基因在MoO₄²⁻缺乏的条件下也会表达。文中讨论了铁钼辅因子掺入替代固氮酶组分1的可能解释。
