Truscott Steven, Lewis Randy S, Watt G D
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84604, United States of America.
Department of Chemical Engineering, Brigham Young University, Provo, UT 84602, United States of America.
Biophys Chem. 2021 Oct;277:106650. doi: 10.1016/j.bpc.2021.106650. Epub 2021 Jun 18.
The MoFe protein component of the nitrogenase enzyme complex is the substrate reducing site and contains two sets of symmetrically arrayed metallo centers called the P (FeS) and the FeMoco (MoFeS-C-homocitrate) centers. The ATP-binding Fe protein is the specific reductant for the MoFe protein. Both symmetrical halves of the MoFe protein are thought to function independently during nitrogenase catalysis. Forming [AlF] transition-state complexes between the MoFe protein and the Fe protein of Azotobacter vinelandii ranging from 0 to 2 Fe protein/MoFe protein produced a series of complexes whose specific activity decreases with increase in bound Fe protein/MoFe protein ratio. Reduction of 2H to H was inhibited in a linear manner with an x-intercept at 2.0 with increasing Fe protein binding, whereas acetylene reduction to ethylene decreased more rapidly with an x-intercept near 1.5. H reduction is a distinct process occurring independently at each half of the MoFe protein but acetylene reduction decreases more rapidly than H reduction with increasing Fe protein/MoFe protein ratio, suggesting that a response is transmitted between the two αβ halves of the MoFe protein for acetylene reduction as Fe protein is bound. A mechanistic model is derived to investigate this behavior. The model predicts that each site functions independently for 2H reduction to H. For acetylene reduction, the model predicts positive (synchronous) not negative cooperativity arising from acetylene binding to both sites before substrate reduction occurs. When this model is applied to inhibition by Cp2 and modified Av2 protein (L127∆) that form strong, non-dissociable complexes, positive cooperativity is absent and each site acts independently. The results suggest a new paradigm for the catalytic function of the MoFe protein during nitrogenase catalysis.
固氮酶复合物的钼铁蛋白组分是底物还原位点，包含两组对称排列的金属中心，称为P（铁硫）中心和铁钼辅因子（钼铁硫 - C - 高柠檬酸）中心。与ATP结合的铁蛋白是钼铁蛋白的特定还原剂。钼铁蛋白的两个对称半部分在固氮酶催化过程中被认为是独立发挥作用的。在维涅兰德固氮菌的钼铁蛋白和铁蛋白之间形成[AlF]过渡态复合物，铁蛋白与钼铁蛋白的比例范围为0至2个铁蛋白/钼铁蛋白，产生了一系列复合物，其比活性随着结合的铁蛋白/钼铁蛋白比例的增加而降低。随着铁蛋白结合增加，2H还原为H以线性方式受到抑制，x轴截距为2.0，而乙炔还原为乙烯的速率下降更快，x轴截距接近1.5。H还原是一个在钼铁蛋白的每个半部分独立发生的独特过程，但随着铁蛋白/钼铁蛋白比例增加，乙炔还原比H还原下降得更快，这表明当铁蛋白结合时，在钼铁蛋白的两个αβ半部分之间传递了一种反应以进行乙炔还原。由此推导了一个机制模型来研究这种行为。该模型预测，每个位点在2H还原为H的过程中独立发挥作用。对于乙炔还原，该模型预测在底物还原发生之前，乙炔与两个位点结合会产生正（同步）而非负协同效应。当该模型应用于被Cp2和修饰的Av2蛋白（L127∆）抑制的情况时，它们形成强的、不可解离的复合物，此时不存在正协同效应，每个位点独立起作用。这些结果为固氮酶催化过程中钼铁蛋白的催化功能提出了一种新的范式。
