Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site
Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site
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DOI：
10.1074/jbc.m503260200
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发表时间：
2005-06-24
影响因子：
4.8
通讯作者：
Friedrich, B
中科院分区：
文献类型：
--
作者：
Buhrke, T;Lenz, O;Friedrich, B
氢化酶是微生物界丰富的蛋白质，催化氢-2裂解为质子和电子或通过质子还原使氢-2进化。氢代谢主要发生在由氢化酶介导的缺氧环境中，氢化酶对氧的抑制很敏感。那些在含氧环境中茁壮成长的微生物含有在氧气存在下起作用的氢化酶。我们选择了富营养化Ralstonia eutropha H16的h -2传感调控[NiFe]氢化酶来研究其氧耐受性的分子背景。有证据表明，导致调控氢化酶活性位点的分子内疏水空腔的形状和大小对氧不敏感至关重要。通过位点定向诱变扩大假定的气体通道，产生对氧抑制敏感的突变衍生物，可能是因为活性位点已经可以被氧气进入。突变蛋白显示了典型的标准[NiFe]氢化酶的特征，如对Desulfovibrio gigas和Allochromatium vinosum的描述。这些数据为如何设计用于生物技术应用的耐氧氢化酶提供了新的策略。
Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H-2 into protons and electrons or the evolution of H-2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H-2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [ NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.