Lanzilotta W N, Seefeldt L C
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Biochemistry. 1997 Oct 21;36(42):12976-83. doi: 10.1021/bi9715371.
All nitrogenase-catalyzed substrate reduction reactions require the transient association between the iron (Fe) protein component and the molybdenum-iron (MoFe) protein component with concomitant intercomponent electron transfer and MgATP hydrolysis. Understanding the effects of Fe protein-MoFe protein complex formation on the properties of the nitrogenase metal centers is thus essential to understanding the electron transfer reactions. This work presents evidence for significant shifts in midpoint potentials for two of the three nitrogenase metal centers as a result of Fe protein binding to the MoFe protein. The midpoint potentials for the three nitrogenase metal centers, namely the [4Fe-4S] cluster of the Fe protein, and the [8Fe-7S] (or P-) clusters and FeMo cofactors (or M-centers) of the MoFe protein, were determined within a nondissociating nitrogenase complex prepared with a site-specifically altered Fe protein (Leu at position 127 deleted, L127Delta). The midpoint potential for each metal center was determined by mediated redox titrations, with the redox state of each center being monitored by parallel and perpendicular mode EPR spectroscopy. The midpoint potential of the Fe protein [4Fe-4S]2+/1+ cluster couple was observed to change by -200 mV from -420 mV in the uncomplexed L127Delta Fe protein to -620 mV in the L127Delta Fe protein-MoFe protein complex. The midpoint potential of the two electron oxidized couple of the P-clusters (P2+/N) of the MoFe protein was observed to shift by -80 mV upon protein-protein complex formation. No significant change in the midpoint potential of an oxidized state of FeMoco (Mox/N) was observed upon complex formation. These results provide insights into the energetics of intercomponent electron transfer in nitrogenase, suggesting that the energy of protein-protein complex formation is coupled to an increase in the driving force for electron transfer. The results are interpreted in light of the expected changes in the protein environments of the metal centers within the nitrogenase complex.
所有固氮酶催化的底物还原反应都需要铁（Fe）蛋白组分与钼铁（MoFe）蛋白组分之间短暂结合，并伴随组分间电子转移和MgATP水解。因此，了解Fe蛋白-MoFe蛋白复合物形成对固氮酶金属中心性质的影响对于理解电子转移反应至关重要。这项工作提供了证据，表明由于Fe蛋白与MoFe蛋白结合，三种固氮酶金属中心中的两个的中点电位发生了显著变化。在使用位点特异性改变的Fe蛋白（第127位的亮氨酸缺失，L127Δ）制备的非解离固氮酶复合物中，测定了三种固氮酶金属中心的中点电位，即Fe蛋白的[4Fe-4S]簇，以及MoFe蛋白的[8Fe-7S]（或P-）簇和铁钼辅因子（或M-中心）。每个金属中心的中点电位通过介导的氧化还原滴定法测定，每个中心的氧化还原状态通过平行和垂直模式的EPR光谱监测。观察到Fe蛋白[4Fe-4S]2+/1+簇对的中点电位从未复合的L127Δ Fe蛋白中的-420 mV变化到L127Δ Fe蛋白-MoFe蛋白复合物中的-620 mV，变化了-200 mV。观察到MoFe蛋白的P-簇（P2+/N）的两个电子氧化对的中点电位在蛋白质-蛋白质复合物形成时移动了-80 mV。在复合物形成时，未观察到FeMoco（Mox/N）氧化态的中点电位有显著变化。这些结果为固氮酶中组分间电子转移的能量学提供了见解，表明蛋白质-蛋白质复合物形成的能量与电子转移驱动力的增加相关联。根据固氮酶复合物中金属中心蛋白质环境的预期变化对结果进行了解释。
