Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp PCC 6803
Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp PCC 6803
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DOI：
10.1074/jbc.m109.028795
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发表时间：
2009-12-25
影响因子：
4.8
通讯作者：
Appel, Jens
中科院分区：
文献类型：
--
作者：
Germer, Frauke;Zebger, Ingo;Appel, Jens
蓝细菌集胞藻属PCC 6803的双向[NiFe]氢化酶通过单一亲和层析步骤纯化至表观均一性，所述单一亲和层析步骤使用具有与HoxF的C末端融合的Strep-标签II的集胞藻属突变体。为了提高纯化酶的产量并测试其在集胞藻中的过表达能力，将psbA II启动子插入hoxE基因的上游。此外，来自念珠藻属PCC 7120的辅助基因（hypF、C、D、E、A和B）在psbA II启动子的控制下表达。与野生型相比，各菌株显示出更高的氢化酶活性。第一次傅里叶变换红外（FTIR）光谱表征的[NiFe]氢化酶从一个产氧光养生物，揭示了两个氰化物和一氧化碳协调的铁的活性位点。在可逆活化/失活过程中，检测到活性位点的至少四种不同的氧化还原状态。虽然这些国家出现类似的标准[NiFe]氢化酶中观察到的，没有顺磁性镍状态可以检测到在完全氧化和还原形式。电子顺磁共振光谱证实还原活化后存在几个铁硫簇。可以鉴定出一个[4Fe4S]（+）和至少一个[2Fe2S]（+）簇。催化量的NADH或NADPH足以激活这种酶与氢的反应。
The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S](+) and at least one [2Fe2S](+) cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.