Unique features of the nitrogenase VFe protein from Azotobacter vinelandii
Unique features of the nitrogenase VFe protein from Azotobacter vinelandii
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DOI：
10.1073/pnas.0904408106
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发表时间：
2009-06-09
影响因子：
11.1
通讯作者：
Ribbe, Markus W.
中科院分区：
文献类型：
--
作者：
Lee, Chi Chung;Hu, Yilin;Ribbe, Markus W.
固氮酶是一种重要的金属酶，催化生物转化氮(N-2)为氨(NH3)。钒(V)-固氮酶与传统的钼(Mo)-固氮酶非常相似，但它具有自己独特的性质，这可能会为固氮酶催化的一般机理提供有用的见解。到目前为止，对棕色固氮菌V-固氮酶的钒铁(VFe)蛋白的研究主要集中在该蛋白的两种不完全形式：α-β(2)和α(2)-β(2)，两者都含有少量的小三角洲亚基。虽然这些研究提供了关于V依赖固氮酶系统的重要信息，但它们受到蛋白质样本异质性的阻碍。在这里，我们报告了一种同源的，His标记的VFe蛋白的分离和鉴定。该VFe蛋白具有先前未曾察觉的α(2)β(2)β(4)异八聚体组成。此外，它包含一个P-簇，该P-簇在电子上以及可能在结构上不同于钼铁(MoFe)蛋白的P-簇。更重要的是，它在催化上不同于MoFe蛋白，特别是在H-2进化机制方面。对FEV辅因子和P簇相关信号的来源和相互作用进行了详细的EPR研究，为今后对VFe蛋白的动力学和结构分析奠定了基础。
Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N-2) to ammonia (NH3). The vanadium (V)-nitrogenase is very similar to the "conventional" molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of Azotobacter vinelandii V-nitrogenase has been focused on 2 incomplete forms of this protein: alpha beta(2) and alpha(2)beta(2), both of which contain the small delta-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from A. vinelandii. This VFe protein has a previously-unsuspected, alpha(2)beta(2)delta(4)-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein. More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H-2 evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor- and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein.