X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin
X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin
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DOI：
10.1021/acs.biochem.5b00601
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发表时间：
2015-10-06
期刊：
影响因子：
2.9
通讯作者：
Kurisu, Genji
中科院分区：
文献类型：
--
作者：
Mutoh, Risa;Muraki, Norifumi;Kurisu, Genji
在叶绿体中，铁氧还蛋白（Fd）被光系统I（PSI）还原，并被参与NADP（+）还原的Fd-NADP（+）还原酶（FNR）氧化。为了了解通过Fd的电子转移反应的动力学和效率的结构基础，我们补充使用X射线晶体学和核磁共振（NMR）光谱。在与钯形成的电子转移复合物的核磁共振分析中，钯的[2 Fe-2S]簇的顺磁效应使我们无法检测到簇周围的核磁共振信号。为了解决这个问题，顺磁性铁硫簇被替换为抗磁性金属簇。我们确定的Ga取代的Fd（GaFd）从集胞藻属PCC 6803在1.62 A分辨率的晶体结构，并验证其功能互补使用亲和层析。利用高场核磁共振谱对GaFd与长高温聚球藻PSI（分子量约为1 MDa）和FNR的相互作用位点进行了分析。根据已发表的Anabaena sp. PCC 7119晶体结构数据，Fd与FNR和PSI在溶液中的相互作用位点可分为两类：（1）金属中心附近的核心疏水残基和（2）核心周围的亲水残基。前一个位点在Fd：FNR和Fd：PSI复合物中是共享的，而后一个位点是靶特异性的并且在残留水平上不保守。
In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used Xray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Pd, the paramagnetic effect of the [2Fe-2S] cluster of Pd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 A resolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of similar to 1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.