Hänzelmann P, Meyer O
Lehrstuhl für Mikrobiologie, Universität Bayreuth, Germany.
Eur J Biochem. 1998 Aug 1;255(3):755-65. doi: 10.1046/j.1432-1327.1998.2550755.x.
The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is expressed in a catalytically fully competent form during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO. We have adopted these conditions for studying the effect of molybdate (Mo) and tungstate (W) on the biosynthesis of CO dehydrogenase and its molybdopterin (MPT) cytosine-dinucleotide-(MCD)-type molybdenum cofactor. W was taken up by the Mo transport system and, therefore, interfered with Mo transport in an antagonistic way. Depletion of Mo from the growth medium as well as inclusion of excess W both resulted in the absence of intracellular Mo and led to the biosynthesis of CO dehydrogenase species of proper L2M2S2 subunit structure that carried the two 2Fe:2S type-I and type-II centers and two FAD molecules. EPR, ultraviolet/visible and CD spectroscopies established the full functionality of the cofactors. Due to the absence of the Mo-MCD cofactor, the enzyme species were catalytically inactive. Unexpectedly, the following cytidine nucleotides were present in inactive CO dehydrogenase: CDP, dCDP, CMP, dCMP, CTP or dCTP. The sum of cytidine nucleotides was two/mol enzyme. The binding specificities of inactive CO dehydrogenase for cytidine nucleotides (oxy > deoxy; diphosphate > monophosphate > triphosphate), and the absence of MPT suggest that, in active CO dehydrogenase, the cytidine diphosphate moiety of Mo-MCD provides the strongest interactions with the protein and determines the specificity for the type of nucleotide. In H. pseudoflava, the biosynthesis of MPT (identified as form A) was independent of Mo. Mo was, however, strictly required for the conversion of MPT to MCD (identified as form-A-CMP) as well as the insertion of Mo-MCD into CO dehydrogenase. These data support a model for the involvement of Mo in the biosynthesis of the Mo-MCD cofactor and of fully functional CO dehydrogenase in which the synthesis and insertion of Mo-MCD require Mo, and protein synthesis including integration of the FeS-centers and FAD are independent of Mo.
含钼铁硫黄素蛋白一氧化碳脱氢酶在好氧细菌嗜氢假黄单胞菌利用丙酮酸加一氧化碳进行异养生长期间以催化完全有活性的形式表达。我们采用这些条件来研究钼酸盐（Mo）和钨酸盐（W）对一氧化碳脱氢酶及其钼蝶呤（MPT）胞嘧啶二核苷酸 - （MCD）型钼辅因子生物合成的影响。W被钼转运系统摄取，因此以拮抗方式干扰钼的转运。从生长培养基中耗尽Mo以及加入过量W均导致细胞内无Mo，并导致具有适当L2M2S2亚基结构的一氧化碳脱氢酶物种的生物合成，该物种携带两个2Fe:2S I型和II型中心以及两个FAD分子。电子顺磁共振、紫外/可见光谱和圆二色光谱确定了辅因子的完全功能性。由于不存在Mo-MCD辅因子，这些酶物种无催化活性。出乎意料的是，无活性的一氧化碳脱氢酶中存在以下胞嘧啶核苷酸：CDP、dCDP、CMP、dCMP、CTP或dCTP。胞嘧啶核苷酸的总量为每摩尔酶两个。无活性的一氧化碳脱氢酶对胞嘧啶核苷酸的结合特异性（氧化型>脱氧型；二磷酸>单磷酸>三磷酸）以及不存在MPT表明，在活性一氧化碳脱氢酶中，Mo-MCD的胞嘧啶二磷酸部分与蛋白质提供最强的相互作用，并决定了对核苷酸类型的特异性。在嗜氢假黄单胞菌中，MPT（鉴定为A形式）的生物合成不依赖于Mo。然而，Mo是MPT转化为MCD（鉴定为A-CMP形式）以及将Mo-MCD插入一氧化碳脱氢酶所严格必需的。这些数据支持了一个模型，即Mo参与Mo-MCD辅因子的生物合成以及完全功能性一氧化碳脱氢酶的形成，其中Mo-MCD的合成和插入需要Mo，而包括FeS中心和FAD整合在内的蛋白质合成不依赖于Mo。
