Aliverti A, Deng Z, Ravasi D, Piubelli L, Karplus P A, Zanetti G
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, I-20133 Milano, Italy.
J Biol Chem. 1998 Dec 18;273(51):34008-15. doi: 10.1074/jbc.273.51.34008.
Ferredoxin-NADP+ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP+. Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steady-state, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (approximately 1%), whereas unexpectedly the E312A reductase was 10-100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form.
铁氧化还原蛋白-NADP⁺还原酶是一大类结构相关的黄素酶的原型，它将铁氧化还原蛋白I携带的单个电子配对，并将它们作为氢化物转移到NADP⁺上。该酶的四个突变体，其中Glu-312被Asp、Gln、Leu和Ala取代，以探究该残基的电荷、大小和极性在酶活性中的作用，已通过稳态、快速动力学研究、配体结合实验以及X射线晶体学三维结构测定进行了异源表达、纯化和表征。E312L突变体是唯一几乎无活性的（约1%），而出乎意料的是，E312A还原酶对所测试的各种受体具有10%-100%的活性。快速动力学吸收光谱研究表明，突变体中NADPH对黄素的还原受到损害。此外，NADP(H)的结合也受到部分干扰。这些功能和结构研究使我们得出结论，Glu-312在催化过程中不充当质子供体的角色，但它是NADP(H)烟酰胺环正确结合所必需的。此外，其电荷调节黄素的两个单电子氧化还原电位以稳定半醌形式。
