Peters J W, Fisher K, Dean D R
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
J Biol Chem. 1994 Nov 11;269(45):28076-83.
During nitrogenase catalysis the Fe protein and the MoFe protein associate and dissociate in a MgATP-dependent process involving electron transfer from the Fe protein to the MoFe protein. A docking model, based primarily on the crystal structures of the separate components from Azotobacter vinelandii, was previously proposed in which the 2-fold symmetric surface of the homodimeric Fe protein interacts with the exposed surface of a MoFe protein pseudosymmetric alpha beta-unit interface. In this model, a loop, which is included within residues 59 through 67 of the Fe protein primary sequence, is likely to interact with the MoFe protein during component protein docking. In the present study, evidence supporting the component protein docking model was obtained by construction of an A. vinelandii strain that produces a hybrid Fe protein for which residues 59 through 67 have been replaced by the corresponding residues from the Fe protein of Clostridium pasteurianum. Biochemical analyses of the hybrid Fe protein revealed the following features when compared with the unaltered Fe protein. First, the hybrid Fe protein exhibited half the maximum specific activity of the normal Fe protein and was insensitive to inhibition by low levels of NaCl. Second, the hybrid Fe protein activity was hypersensitive to a molar excess of MoFe protein, which also resulted in the uncoupling of MgATP hydrolysis from substrate reduction. Third, stopped-flow spectrophotometry experiments showed that during catalysis the hybrid Fe protein dissociates from the MoFe protein at only half the normal rate of Fe protein-MoFe protein dissociation. Thus, the salient feature of the hybrid Fe protein is that it appears to form a relatively tighter complex with the MoFe protein. This property is in line with previous biochemical reconstitution experiments where it was shown that a heterologous mixture of Fe protein from C. pasteurianum and MoFe protein from A. vinelandii form a tight, inactive complex and supports the proposal that a region defined by residues 59 through 67 within the Fe protein is involved in component protein interaction.
在固氮酶催化过程中，铁蛋白和钼铁蛋白在一个依赖于MgATP的过程中缔合和解离，该过程涉及电子从铁蛋白转移到钼铁蛋白。先前曾提出一种对接模型，该模型主要基于来自棕色固氮菌的各个组分的晶体结构，其中同二聚体铁蛋白的2倍对称表面与钼铁蛋白假对称αβ单元界面的暴露表面相互作用。在该模型中，铁蛋白一级序列中59至67位残基内的一个环在组分蛋白对接过程中可能与钼铁蛋白相互作用。在本研究中，通过构建一株棕色固氮菌菌株获得了支持组分蛋白对接模型的证据，该菌株产生一种杂合铁蛋白，其59至67位残基已被巴氏梭菌铁蛋白的相应残基所取代。与未改变的铁蛋白相比，对杂合铁蛋白的生化分析揭示了以下特征。首先，杂合铁蛋白的最大比活性为正常铁蛋白的一半，并且对低水平NaCl的抑制不敏感。其次，杂合铁蛋白活性对过量的钼铁蛋白高度敏感，这也导致MgATP水解与底物还原解偶联。第三，停流分光光度法实验表明，在催化过程中，杂合铁蛋白与钼铁蛋白解离的速率仅为铁蛋白 - 钼铁蛋白正常解离速率的一半。因此，杂合铁蛋白的显著特征是它似乎与钼铁蛋白形成相对紧密的复合物。这一特性与先前的生化重组实验一致，在该实验中表明，巴氏梭菌的铁蛋白和棕色固氮菌的钼铁蛋白的异源混合物形成紧密的无活性复合物，并支持以下提议，即铁蛋白中由59至67位残基定义的区域参与组分蛋白相互作用。
