Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae
Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae
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DOI：
10.1016/j.bbabio.2008.02.002
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发表时间：
2008-05-01
影响因子：
4.3
通讯作者：
Happe, Thomas
中科院分区：
文献类型：
--
作者：
Kamp, Christina;Silakov, Alexey;Happe, Thomas
莱茵衣藻的氢化酶表达可以通过厌氧适应人工诱导，也可以在缺硫条件下自然建立。与厌氧适应相比，无硫藻培养物的[FeFe]-氢化酶（HydA1）的表达率要高得多，并且体外氢化酶活性要高25倍。基于这种高效的诱导原理，我们建立了一种新的纯化方案，可以分离HydA1，也可以用于其他绿藻。从8升莱茵C菌培养液中分离出0.52 mg的HydA1，比活性为741 μ mol H-2 min(-1) mg（-1）。从海底绿球藻和莫氏衣单胞菌中也纯化出类似的量。极大的蛋白质产量使得首次对这些最小的[FeFe]氢化酶的活性位点进行光谱表征成为可能。EPR光谱初步分析显示，CO抑制形式的轴向EPR信号是[FeFe]-氢化酶活性位点H-ox-CO态的典型特征。然而，已经观察到的g张量分量的偏差表明，不同氢化酶之间的电子结构存在明显差异。在低温下，观察到EPR光谱的光诱导变化，并解释为抑制CO配体的光解离。(c) 2008 Elsevier B.V.版权所有
Hydrogenase expression in Chlamydomonas reinhardtii can be artificially induced by anaerobic adaptation or is naturally established under sulphur deprivation. In comparison to anaerobic adaptation, sulphur-deprived algal cultures show considerably higher expression rates of the [FeFe]-hydrogenase (HydA1) and develop a 25-fold higher in vitro hydrogenase activity. Based on this efficient induction principle we have established a novel purification protocol for the isolation of HydA1 that can also be used for other green algae. From an eight liter C reinhardtii culture 0.52 mg HydA1 with a specific activity of 741 mu mol H-2 min(-1) mg(-1) was isolated. Similar amounts were also purified from Chlorococcum submarinum and Chlamydomonas moewusii. The extraordinarily large yields of protein allowed a spectroscopic characterization of the active site of these smallest [FeFe]-hydrogenases for the first time. An initial analysis by EPR spectroscopy shows characteristic axial EPR signals of the CO inhibited forms that are typical for the H-ox-CO state of the active site from [FeFe]-hydrogenases. However, deviations in the g-tensor components have been observed that indicate distinct differences in the electronic structure between the various hydrogenases. At cryogenic temperatures, light-induced changes in the EPR spectra were observed and are interpreted as a photodissociation of the inhibiting CO ligand. (c) 2008 Elsevier B.V. All rights reserved.