Differential stabilization of the three FMN redox forms by tyrosine 94 and tryptophan 57 in flavodoxin from Anabaena and its influence on the redox potentials?
Differential stabilization of the three FMN redox forms by tyrosine 94 and tryptophan 57 in flavodoxin from Anabaena and its influence on the redox potentials?
复制标题
DOI：
10.1021/bi971384h
复制
发表时间：
1997-11-25
期刊：
影响因子：
2.9
通讯作者：
Sancho, J
中科院分区：
文献类型：
--
作者：
Lostao, A;GomezMoreno, C;Sancho, J
黄素蛋白是一种电子转移蛋白，它携带一个非共价结合的黄素单核苷酸分子作为氧化还原活性中心。黄素核苷酸的氧化还原电位在与蛋白质相互作用后发生了深刻的变化。在鱼腥藻黄素氧还蛋白中，如同在许多黄素氧还蛋白中一样，黄素夹在两个芳香残基（Trp 57和Tyr 94）之间，这两个芳香残基被认为与氧化还原电位的改变有关。我们分别用其他芳香族残基，丙氨酸和亮氨酸取代了这两个残基。对于每个突变体，我们已经确定了氧化的FMN-apoflavodoxin复合物的氧化还原电位和结合能。从这些数据中，半还原和还原复合物的结合能已被计算。野生型和突变体的结合能在三个氧化还原状态的比较表明，Tyr 94和FMN之间的相互作用稳定的脱辅基黄素-FMN复合物在所有的氧化还原状态。然而，氧化和半还原复合物比还原复合物更稳定，使得黄氧还蛋白中的半醌/对苯二酚中点电位比未结合的FMN中的半醌/对苯二酚中点电位更负。Trp 57还稳定FMN的所有氧化还原形式，因此与Tyr 94在强FMN结合中协作。另一方面，Trp 57似乎稍微不稳定的半还原复合物相对于氧化的。最后，我们已经观察到，相对于野生型或缺乏Tyr 94的突变体，缺乏Trp 57的突变体的还原是缓慢的，这表明Trp 57可以在黄素氧还蛋白氧化还原反应的动力学中发挥作用。
Flavodoxins are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule as the redox-active center. The redox potentials of the flavin nucleotide an profoundly altered upon interaction with the protein. In Anabaena flavodoxin, as in many flavodoxins, the flavin is sandwiched between two aromatic residues (Trp57 and Tyr94) thought to be implicated in the alteration of the redox potentials. We have individually replaced these two residues by each of the other aromatic residues, by alanine and by leucine. For each mutant, we have determined the redox potentials and the binding energies of the oxidized FMN-apoflavodoxin complexes. From these data, the binding energies of the semireduced and reduced complexes have been calculated. Comparison of the binding energies of wild-type and mutant flavodoxins at the three redox states suggests that the interaction between Tyr94 and FMN stabilizes the apoflavodoxin-FMN complex in all redox states. The oxidized and semireduced complexes are, however, more strongly stabilized than the reduced complex, making the semiquinone/ hydroquinone midpoint potential more negative in flavodoxin than in unbound FMN. Trp57 also stabilizes all redox forms of FMN, thus cooperating with Tyr94 in strong FMN binding. On the other hand, Trp57 seems to slightly destabilize the semireduced complex relative to the oxidized one. Finally, we have observed that reduction of mutants lacking Trp57 is slow relative to that of wild-type or mutants lacking Tyr94, which suggests that Trp57 could play a role in the kinetics of flavodoxin redox reactions.