Hartwig Stefanie, Pinske Constanze, Sawers R Gary
Institute for Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
Biochem Biophys Rep. 2015 Mar 30;1:62-67. doi: 10.1016/j.bbrep.2015.03.006. eCollection 2015 May.
synthesizes three selenocysteine-dependent formate dehydrogenases (Fdh) that also have a molybdenum cofactor. Fdh-H couples formate oxidation with proton reduction in the formate hydrogenlyase (FHL) complex. The activity of Fdh-H in solution can be measured with artificial redox dyes but, unlike Fdh-O and Fdh-N, it has never been observed by chromogenic activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE). Here, we demonstrate that Fdh-H activity is present in extracts of cells from stationary phase cultures and forms a single, fast-migrating species. The activity is oxygen labile during electrophoresis explaining why it has not been previously observed as a discreet activity band. The appearance of Fdh-H activity was dependent on an active selenocysteine incorporation system, but was independent of the [NiFe]-hydrogenases (Hyd), 1, 2 or 3. We also identified new active complexes of Fdh-N and Fdh-O during fermentative growth. The findings of this study indicate that Fdh-H does not form a strong complex with other Fdh or Hyd enzymes, which is in line with it being able to deliver electrons to more than one redox-active enzyme complex.
合成了三种依赖硒代半胱氨酸的甲酸脱氢酶（Fdh），这些酶也含有钼辅因子。Fdh-H在甲酸氢化酶（FHL）复合物中将甲酸氧化与质子还原偶联起来。溶液中Fdh-H的活性可用人工氧化还原染料测量，但与Fdh-O和Fdh-N不同，在非变性聚丙烯酰胺凝胶电泳（PAGE）后通过显色活性染色从未观察到它。在此，我们证明Fdh-H活性存在于稳定期培养细胞的提取物中，并形成单一的、快速迁移的条带。该活性在电泳过程中对氧气敏感，这解释了为什么以前没有观察到它作为一条离散的活性带。Fdh-H活性的出现依赖于一个活跃的硒代半胱氨酸掺入系统，但与[NiFe] - 氢化酶（Hyd）1、2或3无关。我们还在发酵生长过程中鉴定出了Fdh-N和Fdh-O的新活性复合物。本研究结果表明，Fdh-H不会与其他Fdh或Hyd酶形成强复合物，这与其能够将电子传递给不止一种氧化还原活性酶复合物是一致的。
