Golinelli M P, Gagnon J, Meyer J
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble, France.
Biochemistry. 1997 Sep 30;36(39):11797-803. doi: 10.1021/bi970528p.
Putative physiological partners of the [2Fe-2S] ferredoxin from Clostridium pasteurianum have been searched by running crude soluble extracts of this bacterium through an affinity column to which the [2Fe-2S] ferredoxin had been covalently bound. Subsequent washing of the column with buffers of increasing ionic strength revealed a strong and specific interaction of the ferredoxin with the MoFe protein of nitrogenase. This interaction was further investigated by performing cross-linking experiments with mixtures of the two purified proteins in solution. Analysis of the reactions by SDS-polyacrylamide gel electrophoresis evidenced only two covalently linked products. These were identified by N-terminal sequencing as the alpha and beta subunits of the MoFe protein, each cross-linked to a single polypeptide chain of the ferredoxin. This result, taking into account the dimeric structure of the ferredoxin in solution, strongly suggests an interaction of the ferredoxin with the MoFe protein at a site contributed to by both subunits of the MoFe protein. The ionic strength dependence of the interaction evidenced by affinity chromatography was confirmed in the cross-linking reactions, and its specificity was assessed by showing that no cross-linking occurred when the [2Fe-2S] C. pasteurianum ferredoxin was denatured or replaced by spinach ferredoxin or by clostridial rubredoxin, or when the MoFe protein from C. pasteurianum was either inactivated or replaced by its counterpart from Azotobacter vinelandii. It has also been observed that the ferredoxin inhibits cross-linking between the nitrogenase Fe protein and the MoFe protein, which suggests overlapping binding sites of the ferredoxin and of the Fe protein on the MoFe protein. Cross-linking experiments implementing a number of molecular variants of the [2Fe-2S] C. pasteurianum ferredoxin demonstrated that glutamate residues 31, 34, and 38 are important contributors to the interaction with the MoFe protein.
通过将巴氏梭菌的粗可溶性提取物通过一个已共价结合了[2Fe-2S]铁氧还蛋白的亲和柱，来寻找巴氏梭菌[2Fe-2S]铁氧还蛋白可能的生理伙伴。随后用离子强度递增的缓冲液冲洗该柱，结果显示铁氧还蛋白与固氮酶的钼铁蛋白之间存在强烈且特异的相互作用。通过对溶液中两种纯化蛋白的混合物进行交联实验，进一步研究了这种相互作用。用SDS-聚丙烯酰胺凝胶电泳分析反应，结果仅显示出两种共价连接产物。通过N端测序鉴定这些产物为钼铁蛋白的α和β亚基，它们各自与铁氧还蛋白的一条多肽链交联。考虑到溶液中铁氧还蛋白的二聚体结构，这一结果有力地表明铁氧还蛋白与钼铁蛋白在由钼铁蛋白的两个亚基共同构成的位点上发生了相互作用。亲和色谱所证明的相互作用对离子强度的依赖性在交联反应中得到了证实，其特异性通过以下实验得以评估：当巴氏梭菌的[2Fe-2S]铁氧还蛋白变性、被菠菜铁氧还蛋白或梭菌红素替代时，或者当巴氏梭菌的钼铁蛋白失活或被来自棕色固氮菌的对应蛋白替代时，均未发生交联。还观察到铁氧还蛋白会抑制固氮酶铁蛋白与钼铁蛋白之间的交联，这表明铁氧还蛋白和铁蛋白在钼铁蛋白上的结合位点存在重叠。对巴氏梭菌[2Fe-2S]铁氧还蛋白的多个分子变体进行的交联实验表明，谷氨酸残基31、34和38是与钼铁蛋白相互作用的重要贡献位点。
