Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.
Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.
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DOI：
10.1021/acs.biochem.7b00025
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发表时间：
2017-10-17
期刊：
影响因子：
2.9
通讯作者：
Knaff DB
中科院分区：
文献类型：
--
作者：
Srivastava AP;Hardy EP;Allen JP;Vaccaro BJ;Johnson MK;Knaff DB
1：1铁氧还蛋白（Fd）/硝酸还原酶（NR）复合物的计算机模拟模型，使用集胞藻属PCC 6803 Fd的已知结构和集胞藻属PCC 7942 NR的计算机模拟模型，用于映射定义Fd和NR之间界面的相互作用位点。为了测试模型复合物预测的静电相互作用，五个带正电荷的NR氨基酸（Arg 43，Arg 46，Arg 197，Lys 201和Lys 614）和带负电荷的氨基酸（Glu 219）使用定点诱变改变，其特征在于活性测量，金属分析和电子顺磁共振（EPR）研究。所有电荷置换变体在还原甲基紫精（MV）的情况下保留野生型水平的活性，但当还原Fd充当电子供体时，观察到R43 Q、R46 Q、K201 Q和K614 Q变体的活性显著降低。EPR分析以及Fe和Mo分析表明，这些变体观察到的活性损失不是Mo中心或[4Fe-4S]簇的扰动的结果。因此，在这些变体中观察到的Fd相关比活性的丧失只能通过引起Arg 43、Arg 46、Lys 201和Lys 614在Fd结合中的作用来解释。与野生型NR相比，R43 Q、R46 Q、K201 Q和K614 Q NR变体也显示出对Fd的结合亲和力降低，这支持了这四个带正电荷的残基在Fd的生产性结合中的关键作用。
An in silico model for the 1:1 ferredoxin (Fd)/nitrate reductase (NR) complex, using the known structure of Synechocystis sp. PCC 6803 Fd and the in silico model of Synechococcus sp. PCC 7942 NR, is used to map the interaction sites that define the interface between Fd and NR. To test the electrostatic interactions predicted by the model complex, five positively charged NR amino acids (Arg43, Arg46, Arg197, Lys201, and Lys614) and a negatively charged amino acid (Glu219) were altered using site-directed mutagenesis and characterized by activity measurements, metal analysis, and electron paramagnetic resonance (EPR) studies. All of the charge replacement variants retained wild-type levels of activity with reduced methyl viologen (MV), but a significant decrease in activity was observed for the R43Q, R46Q, K201Q, and K614Q variants when reduced Fd served as the electron donor. EPR analysis as well as the Fe and Mo analyses showed that loss of activity observed with these variants was not the consequence of perturbation of the Mo center or [4Fe-4S] cluster. Therefore, the loss of the Fd-linked specific activity observed with these variants can be explained only by invoking a role for Arg43, Arg46, Lys201, and Lys614 in Fd binding. The R43Q, R46Q, K201Q, and K614Q NR variants also showed a decreased binding affinity for Fd, compared to that of wild-type NR, supporting a key role of these four positively charged residues in the productive binding of Fd.
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影响因子：
2.9
作者：
Kozakov, Dima;Hall, David R.;Beglov, Dmitri;Brenke, Ryan;Comeau, Stephen R.;Shen, Yang;Li, Keyong;Zheng, Jiefu;Vakili, Pirooz;Paschalidis, Ioannis Ch.;Vajda, Sandor
通讯作者：
Vajda, Sandor
DOI：
10.1038/nsb994
发表时间：
2003-11-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Arnoux, P;Sabaty, M;Pignol, D
通讯作者：
Pignol, D
影响因子：
56.9
作者：
Boyington, JC;Gladyshev, VN;Sun, PD
通讯作者：
Sun, PD
DOI：
10.1016/0005-2728(93)90070-v
发表时间：
1993-01-08
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
HIRASAWA, M;DEBEST, JH;KNAFF, DB
通讯作者：
KNAFF, DB
影响因子：
2.9
作者：
Dementin, Sebastien;Arnoux, Pascal;Pignol, David
通讯作者：
Pignol, David