Department of Molecular Biology and Biochemistry, University of California, Irvine, California, USA.
Department of Chemistry, University of Basel, Basel, Switzerland.
mBio. 2018 Mar 13;9(2):e00310-18. doi: 10.1128/mBio.00310-18.
The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase. This is the first report on the generation and characterization of an M-cluster-containing V-nitrogenase hybrid. The "normalization" of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the "weighted" contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.
钼氮酶和钒氮酶是氮酶家族中的两个同源成员，主要区别在于它们的辅因子位点（M 或 V 簇）存在不同的杂金属（钼或钒）。然而，钒氮酶在环境条件下将 CO 还原为碳氢化合物的活性比钼氮酶高约 600 倍。在这里，我们在 中表达了一种含有 M 簇的杂化钒氮酶，并将其与天然的含有 V 簇的钒氮酶进行了比较，以评估蛋白质支架和辅因子种类对钼氮酶和钒氮酶对 CO 反应性的差异的影响。M 簇位于钒氮酶的 VFe 蛋白组分中，表现出与 V 簇相似的电子顺磁共振（EPR）特征，并显示出从 CO 还原形成碳氢化合物的活性增加了约 100 倍，这表明蛋白质环境对氮酶整体 CO 还原活性有重大影响。另一方面，在相同的蛋白质支架中，M 簇的活性仍比 V 簇低约 6 倍，并且它仍然无法从 CO 还原形成可检测量的甲烷，这说明了辅因子性质对这种氮酶催化反应的精细调节作用。这些结果为 CO 还原酶活性的两个主要决定因素提供了重要的见解，同时为进一步阐明氮酶 CO 反应性的基本催化要素建立了有用的框架。这是关于含有 M 簇的钒氮酶杂种的生成和表征的第一个报告。通过将蛋白质支架“标准化”为钒氮酶的蛋白质支架，可以直接比较 Mo 和 V 氮酶（M 和 V 簇）在 CO 还原中的辅因子种类，而 Mo 和 V 氮酶（MoFe 和 VFe 蛋白）的蛋白质支架之间的差异容纳相同的辅因子（M 簇）允许有效地评估蛋白质环境对氮酶 CO 反应性的影响。这项研究的结果提供了对蛋白质环境和辅因子性质对 CO 还原总活性的“加权”贡献的首次了解；更重要的是，它们为进一步研究导致氮酶 CO 还原活性的结构要素建立了一个有用的平台。
