Oxygen-tolerant H2 Oxidation by Membrane-bound [NiFe] Hydrogenases of Ralstonia Species
Oxygen-tolerant H2 Oxidation by Membrane-bound [NiFe] Hydrogenases of Ralstonia Species
复制标题
Ralstonia 种膜结合 [NiFe] 氢化酶的耐氧 H2 氧化
DOI：
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发表时间：
2009
影响因子：
4.8
通讯作者：
O. Lenz
中科院分区：
文献类型：
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作者：
M. Ludwig;J. Cracknell;K. Vincent;F. Armstrong;O. Lenz
Knallgas细菌，如某些Ralstonia sp .能够利用O2作为终端电子受体，通过氧化微量的H2来获得代谢能量。这些生物产生的[NiFe]氢化酶在O2存在下氧化H2的能力是不寻常的，O2是一种通过攻击活性位点而使大多数氢化酶失活的强有力的灭活剂。为了探究这种不同寻常的氧耐受性的起源，我们研究了富营养拉尔斯顿菌H16和其密切相关的金属拉尔斯顿菌CH34的膜结合氢化酶，这两种酶是用一种新的异种过量生产系统纯化的。采用直接电化学方法测定O2对H2氧化的表观抑制常数（atchmode documentclass[fleqn,10pt,legalpaper]{article} uspackage {amssymb} uspackage {amsfonts} uspackage {amsmath} pagestyle{empty} egin{document} (K_{I(mathrm{app})}^{mathrm{O}_{2}}) end{document}）。这些数值比“标准”[NiFe]氢化酶的数值至少高出2个数量级。研究人员将富营养酵母H16膜结合氢化酶中靠近活性位点的氨基酸与标准氢化酶中的氨基酸交换，以探讨单个残基在赋予氧敏感性中的作用。确定了H2 （atchmode documentclass[fleqn,10pt,legalpaper]{article} uspackage {amssymb} uspackage {amsfonts} uspackage {amsmath} pagestyle{empty} egin{document} (K_{M}^{mathrm{H}_{2}}) end{document}）的Michaelis常数，其中一些突变体的Michaelis常数相对于外型增加了20倍以上。突变导致膜结合氢酶与atchmode documentclass[fleqn，10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} egin{document} （K_{M}^{mathrm{H}_{2}}) end{document}或atchmode documentclass[fleqn，10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} egin{document} (K_{I(mathrm{app})}^{mathrm{O}_{2}}) end{document}）值升高相关在高浓度氧气环境下，细胞的自养生长受到损害。
Knallgas bacteria such as certain Ralstonia spp. are able to obtain metabolic energy by oxidizing trace levels of H2 using O2 as the terminal electron acceptor. The [NiFe] hydrogenases produced by these organisms are unusual in their ability to oxidize H2 in the presence of O2, which is a potent inactivator of most hydrogenases through attack at the active site. To probe the origin of this unusual O2 tolerance, we conducted a study on the membrane-bound hydrogenase from Ralstonia eutropha H16 and that of the closely related organism Ralstonia metallidurans CH34, which was purified using a new heterologous overproduction system. Direct electrochemical methods were used to determine apparent inhibition constants for O2 inhibition of H2 oxidation (atchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} egin{document} (K_{I(mathrm{app})}^{mathrm{O}_{2}}) end{document}) for each enzyme. These values were at least 2 orders of magnitude higher than those of “standard” [NiFe] hydrogenases. Amino acids close to the active site were exchanged in the membrane-bound hydrogenase of R. eutropha H16 for those from standard hydrogenases to probe the role of individual residues in conferring O2 sensitivity. Michaelis constants for H2 (atchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} egin{document} (K_{M}^{mathrm{H}_{2}}) end{document}) were determined, and for some mutants these were increased more than 20-fold relative to the wild type. Mutations resulting in membrane-bound hydrogenase enzymes with increased atchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} egin{document} (K_{M}^{mathrm{H}_{2}}) end{document} or decreased atchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} egin{document} (K_{I(mathrm{app})}^{mathrm{O}_{2}}) end{document}) values were associated with impaired lithoautotrophic growth in the presence of high O2 concentrations.