Kimata-Ariga Yoko, Shinkoda Rina, Abe Ryuya
Department of Biological Chemistry, College of Agriculture, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yoshida, Yamaguchi 753-8515, Japan.
J Biochem. 2023 Sep 29;174(4):327-334. doi: 10.1093/jb/mvad046.
Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH. The affinity between FNR and Fd is weakened by the allosteric binding of NADP(H) on FNR, which is considered as a part of negative cooperativity. We have been investigating the molecular mechanism of this phenomenon and proposed that the NADP(H)-binding signal is transferred to the Fd-binding region across the two domains of FNR, NADP(H)-binding domain and FAD-binding domain. In this study, we analyzed the effect of altering the inter-domain interaction of FNR on the negative cooperativity. Four site-directed FNR mutants at the inter-domain region were prepared, and their NADPH-dependent changes in the Km for Fd and physical binding ability to Fd were investigated. Two mutants, in which an inter-domain hydrogen bond was changed to a disulfide bond (FNR D52C/S208C) and an inter-domain salt bridge was lost (FNR D104N), were shown to suppress the negative cooperativity by using kinetic analysis and Fd-affinity chromatography. These results showed that the inter-domain interaction of FNR is important for the negative cooperativity, suggesting that the allosteric NADP(H)-binding signal is transferred to Fd-binging region by conformational changes involving inter-domain interactions of FNR.
植物中的铁氧还蛋白 - NADP⁺还原酶（FNR）从铁氧还蛋白（Fd）接收电子，并将NADP⁺转化为NADPH。NADP（H）在FNR上的变构结合会削弱FNR与Fd之间的亲和力，这被认为是负协同效应的一部分。我们一直在研究这种现象的分子机制，并提出NADP（H）结合信号通过FNR的两个结构域，即NADP（H）结合结构域和FAD结合结构域，传递到Fd结合区域。在本研究中，我们分析了改变FNR的结构域间相互作用对负协同效应的影响。制备了四个位于结构域间区域的定点FNR突变体，并研究了它们对Fd的Km的NADPH依赖性变化以及与Fd的物理结合能力。通过动力学分析和Fd亲和色谱法表明，两个突变体，即结构域间氢键变为二硫键的突变体（FNR D52C/S208C）和失去结构域间盐桥的突变体（FNR D104N），能够抑制负协同效应。这些结果表明，FNR的结构域间相互作用对负协同效应很重要，这表明变构NADP（H）结合信号通过涉及FNR结构域间相互作用的构象变化传递到Fd结合区域。
