García-Sánchez M I, Gotor C, Jacquot J P, Stein M, Suzuki A, Vega J M
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC y Universidad de Sevilla, Centro de Investigaciones Científicas Isla de la Cartuja, Spain.
Eur J Biochem. 1997 Dec 1;250(2):364-8. doi: 10.1111/j.1432-1033.1997.0364a.x.
Incubation of wild-type ferredoxin (Fd) with Chlamydomonas reinhardtii crude extract in the presence of a carboxyl activator resulted in the formation of a unique 1:1 covalent complex with nitrite reductase. However, glutamate synthase was able to form two covalent complexes of Fd: GOGAT with 1:1 and 2:1 stoichiometries. These complexes were functional only when reduced methyl viologen was used as electron donor. Kinetic studies of complex formation suggested the presence of two Fd-binding domains with similar affinity for Fd in the glutamate synthase molecule. Using site-directed mutagenesis with recombinant Fd, we have shown that Fd-Glu91 is directly involved in Fd interaction with glutamate synthase and nitrite reductase. Moreover, a negative core of residues in the alpha1 helix of Fd was also critical in binding the enzymes. These data highlight the analogy in the Fd-binding sites of nitrite reductase and glutamate synthase, which may compete for the electrons coming from the photosynthetic chain.
在羧基激活剂存在的情况下，将野生型铁氧还蛋白（Fd）与莱茵衣藻粗提物一起温育，会导致其与亚硝酸还原酶形成独特的1:1共价复合物。然而，谷氨酸合酶能够形成两种Fd与谷氨酰胺合成酶（GOGAT）的共价复合物，其化学计量比分别为1:1和2:1。只有当使用还原型甲基紫精作为电子供体时，这些复合物才具有功能。复合物形成的动力学研究表明，谷氨酸合酶分子中存在两个对Fd具有相似亲和力的Fd结合结构域。通过对重组Fd进行定点诱变，我们发现Fd-Glu91直接参与Fd与谷氨酸合酶和亚硝酸还原酶的相互作用。此外，Fd的α1螺旋中一个由负电荷残基组成的核心区域在与这些酶的结合中也至关重要。这些数据突出了亚硝酸还原酶和谷氨酸合酶的Fd结合位点之间的相似性，它们可能会竞争来自光合链的电子。
