Activation mechanism of the Active Site of [NiFe] hydrogenas
[NiFe]氢活性位点的激活机制
基本信息
- 批准号:16074214
- 负责人:
- 金额:$ 6.91万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research on Priority Areas
- 财政年份:2004
- 资助国家:日本
- 起止时间:2004 至 2007
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
[NiFe] hydrogenase is composed of two subunits and has a Ni-Fe active site in the large subunit. Ni is coordinated by four cysteine sulfurs (two of them form a bridge between Fe and Ni). Fe has additional non-protein diatomic ligands, and a third bridge in the oxidized form. The as-purified (inactive-oxidized), Ni-C (active-reduced) and CO-inhibited forms of the [NiFe] hydrogenase from D. v. Miyazaki F were already reported. Recently, it was found that the oxidized form is a mixture of Ni-A and Ni-B, and the enzyme is activated from Ni-A to Ni-C through Ni-B.1. In this project, we have discovered the protocol to prepare pure Ni-A from Ni-B by using 50 mM Na_2S and exposure to O_2, and elucidated the crystal structures of Ni-A and Ni-B. We found that Ni-B has a monatomic non-protein bridging ligand (X_<B1>), whereas the Ni-A has a diatomic species (X_<A1>-X_<A2>). In addition, the sulfurs of cysteines are found to have a modified atomic species (X_<546>) in both Ni-A and Ni-B.2. In orde … More r to clarify the activation mechanism of H_2 at the active site, we have succeeded in preparing the large single crystal (1.0 mm^3) of the enzyme in D_2O solution. Neutron diffraction experiments showed that- the crystal diffract about 10 A. The crystallization condition for the larger crystals is being improved.3. The Ni-Fe active site is matured by a series of the proteins coded in the hyp operon. HypE is involved in the biosynthesis of CN which is coordinated to Fe. We determined the crystal structures of HypE in the absence and presence of ATP at 2.0 and 2.6 A resolution, respectively. Comparison of the structures reveals that the binding of ATP does not entail an overall structural change. The residue Cys341 at the C-terminus, whose thiol group is supposed to be carbamoylated prior to the nitrile group synthesis, is completely buried within the protein, and is located in the vicinity of the ・-phosphate group of the bound ATP. The obtained structure suggests that the catalytic reaction occurs in this configuration but that a conformational change is required for the carbamoylation of Cys341.4. CooA is a transcription factor, and is responsible for the expression of CO-tolerant hydrogenases in some bacteria. We have determined the crystal structure of an imidazole (Im)-bound CooA from C. hydrogenoformans (Ch-CooA) at 2.2 A. The structure of Ch-CooA reveals that Im binds to the heme Fe, and replaces the N-terminus, as does CO. Even though the ligand exchange, Im-bound Ch-CooA remains in the inactive form. These results indicate that the release of the N-terminus resulting from Im-binding is not sufficient to activate CooA. The structure provides new insights into the structural changes required to achieve activation. Less
[NiFe]氢化酶由两个亚基组成,大亚基中有一个Ni-Fe活性位点。Ni由四个半胱氨酸硫配位(其中两个在Fe和Ni之间形成桥)。Fe具有额外的非蛋白质双原子配体,以及氧化形式的第三桥。本文研究了D.已经报告了宫崎F案。近年来研究发现,该酶的氧化态为Ni-A和Ni-B的混合物,酶的活性由Ni-A经Ni-B活化为Ni-C。在本项目中,我们发现了用50 mM Na_2S和暴露于O_2从Ni-B制备纯Ni-A的方案,并阐明了Ni-A和Ni-B的晶体结构。发现Ni-B具有单原子的非蛋白质桥连配体(X_<B1>),而Ni-A具有双原子的非蛋白质桥连配体(X_<A1>-X_<A2>)。此外,半胱氨酸的硫在Ni-A和Ni-B中均存在修饰的原子物种(X_<546>)。依序 ...更多信息 为了阐明H_2在活性中心的活化机理,我们在D_2O溶液中成功地制备了该酶的大单晶(1.0mm^3)。中子衍射实验表明,晶体的衍射率约为10.较大晶体的结晶条件正在改善.超操纵子中编码的一系列蛋白质使Ni-Fe活性位点成熟。HypE参与CN的生物合成,CN与Fe配位。我们确定了HypE的晶体结构,在ATP的存在和不存在下,在2.0和2.6 A的分辨率,分别。结构的比较表明,ATP的结合并不需要一个整体的结构变化。在C-末端的残基Cys 341,其硫醇基被认为在腈基合成之前被氨甲酰化,被完全掩埋在蛋白质内,并且位于结合ATP的·-磷酸基附近。所获得的结构表明,催化反应发生在这种配置,但构象变化所需的氨基甲酰化的Cys341.4。CooA是一种转录因子,在某些细菌中负责CO耐受氢化酶的表达。我们测定了C.在2.2A下,Ch-CooA的结构揭示了Im与血红素Fe结合,并取代了N-末端,CO也是如此。即使配体交换,Im结合的Ch-CooA仍保持非活性形式。这些结果表明,由Im结合引起的N-末端的释放不足以激活CooA。该结构为实现激活所需的结构变化提供了新的见解。少
项目成果
期刊论文数量(116)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F
普通脱硫弧菌 Miyazaki F 的 [NiFe] 氢化酶氧化态的单晶 ENDOR 和密度泛函理论研究
- DOI:
- 发表时间:2006
- 期刊:
- 影响因子:0
- 作者:M.van Gastel;M.Stein;M.Brecht;O.Schroder;F.Lendzian;R.Bittl;H.Ogata;Y.Higuchi;W.Lubitz
- 通讯作者:W.Lubitz
Redox-Dependent Interaction of Cytochrome c_3 with [NiFe] Hydrogenase from D. vulgaris Miyazaki F
细胞色素 c_3 与 D. vulgaris Miyazaki F 的 [NiFe] 氢化酶的氧化还原依赖性相互作用
- DOI:
- 发表时间:2006
- 期刊:
- 影响因子:0
- 作者:N.Yahata;K.Ozawa;A.Nakahara;H.Ogata;Y.Higuchi;H.Akutsu
- 通讯作者:H.Akutsu
6-アミノカプロン酸環状2量体加水分解酵素(EI)・基質複合体のX線結晶構造解析
6-氨基己酸环状二聚体水解酶 (EI)/底物复合物的 X 射线晶体结构分析
- DOI:
- 发表时间:2007
- 期刊:
- 影响因子:0
- 作者:安平 健吾;柴田 直樹;門上 剛;樋口 芳樹;加藤 太一郎;武尾 正弘;根来 誠司
- 通讯作者:根来 誠司
Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: Conversion of the ready to the unready state
- DOI:10.1016/j.str.2005.07.018
- 发表时间:2005-11-01
- 期刊:
- 影响因子:5.7
- 作者:Ogata, H;Hirota, S;Higuchi, Y
- 通讯作者:Higuchi, Y
The mechanism of CO sensing in the heme-based CO sensor CooArevealed by the crystal structure of the exogenous ligand bound form
血红素基 CO 传感器 Coo 中 CO 传感的机制通过外源配体结合形式的晶体结构揭示
- DOI:
- 发表时间:2007
- 期刊:
- 影响因子:0
- 作者:Yamashita H;Yagi T;西村 宗十;小島利之;Yamamoto M;下川 千寿;烏山一;峯岸克行;Yamashita H;青野 重利;向井香織;Yagi T;Shigetoshi Aono
- 通讯作者:Shigetoshi Aono
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HIGUCHI Yoshiki其他文献
HIGUCHI Yoshiki的其他文献
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{{ truncateString('HIGUCHI Yoshiki', 18)}}的其他基金
Study of support of the seniors living alone by estimating of living life log from the power consumption
从用电量估算生活日志对独居老人的支持研究
- 批准号:
26870541 - 财政年份:2014
- 资助金额:
$ 6.91万 - 项目类别:
Grant-in-Aid for Young Scientists (B)
Reaction Mechansim of [NiFe]-hydrogenase by Neutron Structural Chmeistry
[NiFe]-氢化酶的中子结构化学反应机理
- 批准号:
24657077 - 财政年份:2012
- 资助金额:
$ 6.91万 - 项目类别:
Grant-in-Aid for Challenging Exploratory Research
Study of Catalytic Mechanism of Hydrogen Activation by Hydrogenase using Neutron Crystal Structure Analysis
中子晶体结构分析研究氢化酶氢活化催化机制
- 批准号:
22657031 - 财政年份:2010
- 资助金额:
$ 6.91万 - 项目类别:
Grant-in-Aid for Challenging Exploratory Research
Structural Study of the Mechanism of Hydrogen Activation on Ni-enzymes
镍酶氢活化机制的结构研究
- 批准号:
22370061 - 财政年份:2010
- 资助金额:
$ 6.91万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Structural study of the activation mechanism of dihydrogen at the Ni-Fe active site of [NiFe] hydrogenase
[NiFe]氢化酶Ni-Fe活性位点氢气激活机制的结构研究
- 批准号:
14380317 - 财政年份:2002
- 资助金额:
$ 6.91万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Structural study of [NiFe]hydrogenase
[NiFe]氢化酶的结构研究
- 批准号:
12680654 - 财政年份:2000
- 资助金额:
$ 6.91万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Structural Study of Hydrogenase
氢化酶的结构研究
- 批准号:
08458209 - 财政年份:1996
- 资助金额:
$ 6.91万 - 项目类别:
Grant-in-Aid for Scientific Research (B)